Diabetes Ebook:Antioxidants in diabetes management

Views:
 
Category: Others/ Misc
     
 

Presentation Description

This volume summarizes current understanding of the pathogenic role of oxidative stress in the onset and progression of diabetes and its complications, and presents results of studies aimed at regulating oxidatively induced complications through the use of antioxidants. Examines the presence of impaired microcirculation, capillary hypoxia, and ischemia syndrome in diabetic complications! Designed to stimulate scientific discussion and curiosity about the causes of diabetes, with contributions from nearly 65 clinicians and researchers who cite more than 1300 sources, Antioxidants in Diabetes Management

Comments

Presentation Transcript

slide 1:

www.ebook3000.com

slide 2:

1n Antioxidanu I Diabete manaement Want To Diabetes Free Life Click Here edite d b IJ LESTER PRC KER University of California Berkeley. California PETER ROSER Diabetes Research Institute Heinrich Heine University Diisseldorf. Germany HRilS J. TRITSCHLER ASTA Medico AWD GmbH Frankfurt. Germany GEORGE l. KIIlG Harvard Medical School Joslin Diabetes Center Boston Massachusetts RR GELO R 2 2 I University of Bern Bern Switzerland mm UNESCO-MCBN 8 Global Network of Molecular Cell Biology Oxygen Club of California 98 Wo Co o n rg ld ress Sponsored Workshop n MARCEL . . - MARCEL DEKKER. INC. NEW YORK. BASEL

slide 3:

DEKKER

slide 4:

ISBN: 0-8247-8844-3 This book is printed on acid-free paper. Headquarters Marcel Dekker Inc. 270 Madison Avenue New York NY 10016 tel: 212-696-9000 fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4 Postfach 812 CH-4001 Basel Switzerland tel: 41-61-261-8482 fax: 41-61-261-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information write to Special Sales/Professional Marketing at the headquarters address above. Copyrigh 2000 by Marcel Dekker Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying microfilming and re• cording or by any information storage and retrieval system without permission in writing from the publisher. Current printing last digit: 10 9 8 7 6 5 4 3 2 PRINTED IN THE UNITED STATES OF AMERICA

slide 5:

Series Introduction Click Here If You Also Want To Be Free From Diabetes Oxygen is a dangerous friend. Overwhelming evidence indicates that oxidative stress can lead to cell and tissue injury. However the same free radicals that are generated during oxidative stress are produced during normal metabolism and thus are involved in both human health and disease. Free radicals are molecules with an odd number of electrons. The odd or unpaired electron is highly reactive as it seeks to pair with another free electron. Free radicals are generated during oxidative metabolism and energy pro• duction in the body. Free radicals are involved in: Enzyme-catalyzed reactions Electron transport in mitochondria Signal transduction and gene expression Activation of nuclear transcription factors Oxidative damage to molecules cells and tissues Antimicrobial action of neutrophils and macrophages Aging and disease Normal metabolism is dependent upon oxgyen a free radical. Through evolution oxygen was chosen as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same direction thus oxygen is a biradical but is not a very dangerous free radical. Other oxygen-derived free radical species such as superoxide or hydroxyl radicals formed during metabolism or by ionizing radiation are stronger oxidants and are therefore more dangerous.

slide 6:

In addition to research on the biological effects of these reactive oxygen species research on reactive nitrogen species has been gathering momentum. iii

slide 7:

iv Series Introduction NO or nitrogen monoxide nitric oxide is a free radical generated by NO synthase NOS. This enzyme modulates physiological responses such as vasodilation or signaling in the brain. However during inflammation synthe• sis of NOS iNOS is induced. This iNOS can result in the overproduction of NO causing damage. More worrisome however is the fact that excess NO can react with superoxide to produce the very toxic product peroxynitrite. Oxidation of lipids proteins and DNA can result thereby increasing the likeli• hood of tissue injury. Both reactive oxygen and nitrogen species are involved in normal cell regulation in which oxidants and redox status are important in signal transduc• tion. Oxidative stress is increasingly seen as a major upstream component in the signaling cascade involved in inflammatory responses stimulating ad• hesion molecule and chemoattractant production. Hydrogen peroxide which breaks down to produce hydroxyl radicals can also activate NF-KBa transcrip• tion factor involved in stimulating inflammatory responses. Excess production of these reactive species is toxic exerting cytostatic effects causing membrane damage and activating pathways of cell death apoptosis and/or necrosis. Virtually all diseases thus far examined involve free radicals. In most cases free radicals are secondary to the disease process but in some instances free radicals are causal. Thus there is a delicate balance between oxidants and antioxidants in health and disease. Their proper balance is essential for ensuring healthy aging. The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by exposure to oxidants. The redox status is thus depen• dent upon the degree to which a cells components are in the oxidized state. In general the reducing environment inside cells helps to prevent oxidative damage. In this reducing environment disulfide bonds S-S do not sponta• neously form because sulfhydryl groups kept in the reduced state SH prevent protein misfolding or aggregation. This reducing environment is maintained by oxidative metabolism and by the action of antioxidant enzymes and sub• stances such as glutathione thioredoxin vitamins E and C and enzymes such as superoxide dismutase SOD catalase and the selenium-dependent gluta• thione and thioredoxin hydroperoxidases which serve to remove reactive oxy• gen species. Changes in the redox status and depletion of antioxidants occur during oxidative stress. The thiol redox status is a useful index of oxidative stress mainly because metabolism and NADPH-dependent enzymes maintain cell glutathione GSH almost completely in its reduced state. Oxidized glutathi• one glutathione disulfide GSSG accumulates under conditions of oxidant exposure and this changes the ratio of oxidized to reduced glutathione and

slide 8:

Series Introduction v increased ratio indicates oxidative stress. Many tissues contain large amounts of glutathione 2-4 mM in erythrocytes or neural tissues and up to 8 mM in hepatic tissues. Reactive oxygen and nitrogen species can directly react with glutathione to lower the levels of this substance the cells primary preventa• tive antioxidant. Current hypotheses favor the idea that lowering oxidative stress can have a clinical benefit. Free radicals can be overproduced or the natural antioxidant system defenses weakened first resulting in oxidative stress and then leading to oxidative injury and disease. Examples of this process include heart disease and cancer. Oxidation of human low-density lipoproteins is considered the first step in the progression and eventual development of atherosclerosis leading to cardiovascular disease. Oxidative DNA damage initiates carcinogenesis. Compelling support for the involvement of free radicals in disease devel• opment comes from epidemiological studies showing that an enhanced anti• oxidant status is associated with reduced risk of several diseases. Vitamin E and prevention of cardiovascular disease is a notable example. Elevated antioxidant status is also associated with decreased incidence of cataracts and cancer and some recent reports have suggested an inverse correlation between antioxidant status and occurrence of rheumatoid arthritis and diabetes mellitus. Indeed the number of indications in which antioxidants may be useful in the prevention and/or the treatment of disease is increasing. Oxidative stress rather than being the primary cause of disease is more often a secondary complication in many disorders. Oxidative stress diseases include inflammatory bowel disease retinal ischemia cardiovascular disease and restenosis AIDS ARDS and neurodegenerative diseases such as stroke Parkinsons disease and Alzheimers disease. Such indications may prove amenable to antioxidant treatment because there is a clear involvement of oxidative injury in these disorders. In this new series of books the importance of oxidative stress in diseases associated with organ systems of the body will be highlighted by exploring the scientific evidence and the medical applications of this knowledge. The series will also highlight the major natural antioxidant enzymes and antioxi• dant substances such as vitamins E A and C flavonoids polyphenols carot• enoids lipoic acid and other nutrients present in food and beverages. Oxidative stress is an underlying factor in health and disease. More and more evidence is accumulating that a proper balance between oxidants and antioxidants is involved in maintaining health and longevity and that altering this balance in favor of oxidants may result in pathological responses causing functional disorders and disease. This series is intended for researchers in the basic biomedical sciences and clinicians. The potential for healthy aging and

slide 9:

vi Series Introduction disease prevention necessitates gaining further knowledge about how oxidants and antioxidants affect biological systems. Diabetes both type I and type II has been found to be associated with indices of oxidative damage. This suggests that oxidative stress is a contribut• ing factor in these disorders. Aging itself is known to involve the deleterious effects of oxygen and glucose which albeit essential for energy may also lead to oxidative stress. Indeed insulin insufficiency is thought to be one of the underlying factors in accelerating aging. Thus it is logical that natural antioxidants may have beneficial implications for diabetes management and healthy aging. Vitamin E and lipoic acid are two such substances receiving attention in this regard. Therefore Antioxidants in Diabetes Management is an appropriate addition to the entire series Oxidant Stress and Disease since this volume provides a comprehensive and up-to-date evaluation of the role of oxidants and antioxidants in diabetes. Chapters focusing on biochemistry molecular biology cell and organ physiology as well as human clinical studies of diabetes management are included. Lester Packer Enrique Cadenas

slide 10:

Preface To Stop Diabetes In Few Days Click Here Diabetes and its complications present a serious medical and socioeconomic problem. Diabetes mellitus is a chronic derangement of insulin action and carbohydrate metabolism. Its major distinguishing diagnostic feature is hyper• glycemia in which blood glucose rises above 200 mg/dL within 2 h of in• gestion of 75 g oral glucose. There are two major classifications: insulin• dependent diabetes IDDM also known as type I or juvenile-onset diabetes which accounts for about 20 of cases and non-insulin-dependent diabetes NIDDM also known as type II or adult-onset diabetes which accounts for the rest. IDDM is characterized by lack of insulin secretion whereas NIDDM patients generally exhibit normal or elevated insulin levels but their peripheral tissues lack insulin sensitivity. NIDDM presently affects 30 to 50 of the elderly and is also characterized by the loss of insulin-triggered glucose uptake from the bloodstream to the insulin-sensitive tissues thus leading to elevated blood glucose levels. The management of diabetes has changed significantly in the past half century. Diabetic coma due to uncontrolled hyperglycemia was the medical challenge 50 years ago but with greater availability of increasingly effective insulin e.g. recombinant human insulin the focus of medical care has shifted toward the management of diabetic complications. Polyneuropathy one of the most common of these occurs in 50 of those patients who have had diabetes for 25 or more years and leads to pain and decreased mobility and function. Nephropathy and retinopathy are also common complications which can re• sult in kidney dysfunction and blindness respectively. The major cause of death among diabetic patients cardiovascular disease is two to four times more common in diabetic than in nondiabetic populations. In addition periph• eral vascular disease and neuropathy can cause ischemia and in some cases gangrene of the lower limbs leading to amputation.

slide 11:

The medical and socioeconomic impact of these complications is enor- vii

slide 12:

viii Preface mous: diabetes is the leading cause of adult blindness dialysis kidney trans• plantation and foot amputations. Diabetes afflicts approximately 6 of the population and is expected to rise to over 10. Globally there will be about 150 million diabetic patients by the year 2000 and 215 million by the year 2015. In the United States alone the cost of diabetes mellitus and its complica• tions increased from 20 billion in 1987 to 90 billion in 1997. The current strategy to combat diabetes focuses on increasingly stringent control of hyperglycemia to prevent or modify the onset and progression of the disease and it complications. The Diabetes Control and Complication Trial DCCT demonstrated that intensified insulin treatment with an improvement in blood sugar control in type I diabetic patients reduced the rate of develop• ment and progression of some diabetic micro- and neurovascular complica• tions. This clinical study supported the hypothesis that hyperglycemia is a major risk factor in the development of diabetic complications. Research over the last 10 years has shown that there are direct potentially damaging effects of diabetic hyperglycemia including glucose-induced vascular abnormalities that are relevant to many diabetic complications. Cardiovascular risk how• ever was not diminished by the intensified insulin treatment used in the study. Only a minority of diabetic patients can achieve this kind of strict blood sugar control over several years in order to reach the preventive effects of hypoglycemia therapy on late diabetic complications. This is especially true for type II diabetic patients. The UKPDS study has shown that only 30 of type II diabetic patients can achieve the required glycemic control levels necessary to prevent late diabetic complications after 3 years and only 10 can do so after 9 years. Because of the therapeutic limitations of hypoglycemic therapy in practice further interventional strategies must be developed. Recent research indicates another promising area for inquiry and ther• apy-glucose AGE-induced vascular abnormalities which are of relevance for all or most diabetic complications. Impaired microcirculation capillary hypoxia and ischemia syndrome are present in most diabetic complications. In addition to elevated blood glucose levels increased production of reactive oxygen species free radicals which are known to exhibit direct tissue• damaging properties may contribute to a number of diabetic complications and to the development of insulin resistance itself. These deleterious species can be neutralized by endogenous and exogenous antioxidants such as vitamin E vitamin C and thioctic lipoic acid. Compared to control subjects NIDDM patients have lower plasma antioxidant vitamins E and C and double the lipid hydroperoxides a measure of oxidative damage. IDDM patients also have low serum total antioxidant activity. Increased oxidative stress in diabetic patients appears to be related to

slide 13:

Preface ix the underlying metabolic abnormalities and is also an early stage in the disease pathology that may contribute to the development of complications. The im• paired microcirculation capillary hypoxia and ischemia syndrome present in most diabetic complications are associated with the production of reactive oxygen species. In addition to control of blood sugar control of oxidative stress offers another avenue for the treatment of the disease. This volume summarizes the current knowledge of the pathogenic role of oxidative stress in the onset and progression of diabetes and its complica• tions and presents results of studies aimed at modulating oxidatively induced complications through the use of antioxidants. Chapters in this volume focus on 1 basic research on oxidative stress in the development of diabetes and diabetic complications 2 studies aimed at specific complications such as cardiovascular disease and polyneuropathy and 3 clinical trials of antioxi• dants in diabetic subjects. An overall understanding of free radical pathology and its modulation by antioxidants is central to basic research into the relationships between diabetes oxidative stress and antioxidants. The concept of oxidative stress and antioxi• dant protection is explored with emphasis on the potential synergistic effects of an interlinked antioxidant network. The significance of oxidative stress markers in diabetes and the evidence for and against an oxidative component in the genesis of diabetes is an ongoing controversy. Oxidative stress appears to play a role not only in complications arising from the disease but in the development of insulin resistance in NIDDM. The possibility of its modulation by the antioxidant o-lipoic acid is explored. A related report focuses on oxida• tive stress and antioxidant treatment in animal models of both IDDM and NIDDM. The nonobese diabetic mouse model is often used in diabetic re• search providing the basis for respective inquiries on basic research into dia• betic mechanisms and prevention of NIDDM by antioxidant therapy. Recently much interest has been expressed in the possible interactions of oxidative stress induced by diabetes and cell signaling molecules in the development of diabetic complications. Studies concentrate on the tissue• damaging effects of free radicals and also on oxidative-stress-sensitive molec• ular factors such as IRS-I Pl-3 PKC and NF-KB which are known to contrib• ute to insulin resistance and diabetic complications. All of these molecular factors are redox-sensitive which means that an imbalance between oxidative stress and antioxidant dysfunction can convert them to pathogenic factors. If one accepts the concept of oxidative stress described here as an important risk factor for diabetes and its complications one has to consider the possible therapeutic value of antioxidants in treating this disorder. Research is pre• sented in this volume that examines in particular the relationship between

slide 14:

x Preface oxidative stress NF-KB activation and late diabetic complications as well as the effects of o-tocopherol vitamin E on protein kinase C and its implications for diabetes. Polyneuropathy one of the most common painful and disabling com• plications of diabetes also comes into focus. The hypothesis that ischemic reperfusion a mechanism that induces oxidative stress is the primary cause of diabetic polyneuropathy is explored as is the potential for treatment with antioxidants in synergistic combination with essential fatty acids. This concept is further examined in an experimental model of polyneuropathy using a thi• octic acid u-Iipoic acid-gamma-linolenic acid conjugate for protection. As mentioned cardiovascular complications represent the most common cause of death among diabetics as well as the most frequent reason for limb amputation. Potential mechanisms for antioxidant intervention are discussed. Myocardial infarction is a leading cause of death among diabetics and oxida• tion of low-density lipoprotein LDL is now a well-established causal factor in this pathology. One chapter links protein kinase C activation with the devel• opment of diabetic vascular complications and also suggests a role for vitamin E in their prevention. Another way in which oxidative stress may be involved in vascular complications especially microangiopathy is explored by one group through effects on cell adhesion molecules and the related potentially protective effect of antioxidants. Antioxidant vitamins especially vitamin E may reduce LDL oxidation in diabetes. The ultimate goal of research on oxidative stress and diabetes is to intro• duce new therapeutic possibilities and to establish the efficacy of various thera• peutic regimens. Therefore several contributions to this volume relate to clini• cal trials of therapeutic effects of antioxidants such as vitamin E and lipoic acid. These include an overview of clinical trials of antioxidants to reduce isulin resistance an evaluation of the clinical evidence on antioxidants in the treatment of diabetic polyneuropathy and a report on the clinical status of antioxidants in the treatment of diabetic vascular abnormalities. Thus research in oxidative stress antioxidants and diabetes may be achieving progress in attacking the basic mechanisms of the disease and in ameliorating some of its most common complications. The medical and socioeconomic burden of diabetes and its complica• tions requires a successful therapeutic concept. We hope that this selection of preclinical and clinical studies will stimulate scientific discussion of the possi• ble pathogenic role of oxidative stress in diabetes and its complications and will help to illustrate the therapeutic potential of antioxidants for treatment of the disease. This volume is the result of two recent workshops "Oxidative Stress

slide 15:

Preface xi in Diabetes and Its Complications: Implications of Antioxidant Treatment" held in Leipzig as a satellite of the German Diabetes Association annual meet• ing and in Santa Barbara at the Oxygen Club of California World Congress. We would like to acknowledge the support of the following sponsors: UN• ESCO-MCBN Global Network for Molecular and Cell Biology the Ameri• can Diabetes Association the Henkel Nutrition and Health Group and ASTA Medica AWD GmbH. Lester Packer Peter Rosen Hans J. Tritsch/er George L. King Angelo Azzi

slide 16:

This Page Intentionally Left Blank

slide 17:

Contents To Cure Diabetes Naturally Click Here Series Introduction Lester Packer and Enrique Cadenas iii Preface vii Contributors xvii 1. Oxidative Stress and Antioxidants: The Antioxidant Net- work rx-Lipoic Acid and Diabetes Lester Packer 2. Oxidative Stress in Diabetes: Why Does Hyperglycemia Induce the Formation of Reactive Oxygen Species 17 Peter Rosen Xueliang Du and Guang-Zhi Sui 3. Oxidative Stress Markers in Human Disease: Application to Diabetes and to Evaluation of the Effects of Antioxidants 33 Barry Halliwell 4. Plasma Lipid Hydroperoxide and Vitamin E Profiles in Patients with Diabetes Mellitus 53 Jaffar Nourooz-Zadeh 5. Concentrations of Antioxidative Vitamins in Plasma and Low-Density Lipoprotein of Diabetic Patients 65 Wolfgang Leonhardt 6. Oxidative Stress in Diabetes 77 John W. Baynes and Suzanne R. Thorpe

slide 18:

xiii

slide 19:

xiv Contents 7. Antioxidative Defense in Diabetic Peripheral Nerve: Effects of m-rz-Lipoic Acid Aldose Reductase Inhibitor and Sorbi- tol Dehydrogenase Inhibitor 93 Irina G. Obrosova Douglas A. Greene and Hans-Jochen Lang 8. Pathways of Glucose-Mediated Oxidative Stress in Diabetic Neuropathy 111 Douglas A. Greene Irina G. Obrosova Martin J. Stevens and Eva L. Feldman 9. Experimental Diabetic Neuropathy: Oxidative Stress and Antioxidant Therapy 121 Hans J. Tritsch/er James D. Schmelzer Yutaka Kishi Yoshiyuki Mitsui Masaaki Nagamatsu Kim K. Nickander Paula . Zollman and Phillip A. Low 10. Antioxidants in the Treatment of Diabetic Polyneuropathy: Synergy with Essential Fatty Acids 129 Norman E. Cameron and Mary A. Cotter 11. A Thioctic Acid-Gamma-Linolenic Acid Conjugate Protects Neurotrophic Support in Experimental Diabetic Neuropathy 155 Luke Hounsom and David R. Tomlinson 12. Clinical Trials of n-Lipcic Acid in Diabetic Polyneuropathy and Cardiac Autonomic Neuropathy 173 Dan Ziegler 13. Oxidative Stress NF-KB Activation and Late Diabetic Complications 185 Peter P. Nawroth Valentin Borcea Angelika Bierhaus Martina Joswig Stephan Schiekofer and Hans J. Tritschler 14. Role of Oxidative Stress and Antioxidants on Adhesion Molecules and Diabetic Microangiopathy 205 Klaus Kusterer Jorg Bojunga Gerald Bayer Thomas Konrad Eva Haak Thomas Haak Klaus H. Usadel and Hans J. Tritsch/er

slide 20:

Contents xv 15. Molecular Basis of n-Tocopherol Action and Its Protective Role Against Diabetic Complications 219 Angelo Azzi Roberta Ricciarelli Sophie Clement and Nesrin Ozer 16. Protein Kinase C Activation Development of Diabetic Vascular Complications and Role of Vitamin E in Pre- venting These Abnormalities 241 Sven-Erik Bursell and George L. King 17. Oxidative Stress and Pancreatic P-Cell Destruction in Insulin-Dependent Diabetes Mellitus 265 Mizuo Hotta Eiji Yamato and Jun-ichi Miyazaki 18. Interrelationship Between Oxidative Stress and Insulin Resistance 275 Karen Yaworsky Rome Somwar and Amira Klip 19. Oxidative Stress and Antioxidant Treatment: Effects on Muscle Glucose Transport in Animal Models of Type I and Type 2 Diabetes 303 Erik J. Henriksen 20. Oxidative Stress and Insulin Action: A Role for Antioxidants 319 Stephan Jacob Rainer Lehmann Kristian Rett and Hans-Ulrich Haring Index 339

slide 21:

This Page Intentionally Left Blank

slide 22:

Contributors To Get Best Natural Diabetes Treatment Click Here Angelo Azzi M.D. Institute of Biochemistry and Molecular Biology Uni• versity of Bern Bern Switzerland Gerald Bayer University of Frankfurt Frankfurt Germany John W. Baynes Ph.D. Department of Chemistry and Biochemistry Uni• versity of South Carolina Columbia South Carolina Angelika Bierhaus University of Heidelberg Heidelberg Germany Jorg Bojunga University of Frankfurt Frankfurt Germany Valentin Borcea University of Heidelberg Heidelberg Germany Sven-Erik Bursell Harvard Medical School Beetham Eye Institute Eye Research Boston Massachusetts Norman E. Cameron D.Phil. Institute of Medical Sciences University of Aberdeen Aberdeen Scotland Sophie Clement Ph.D. Institute of Biochemistry and Molecular Biology University of Bern Bern Switzerland Mary A. Cotter Ph.D. Department of Biomedical Sciences University of Aberdeen Aberdeen Scotland

slide 23:

xvii

slide 24:

xviii Contributors Xueliang Du Ph.D. Department of Clinical Biochemistry Diabetes Re• search Institute Heinrich-Heine-University Dilsseldorf Germany Eva L. Feldman University of Michigan Medical Center Ann Arbor Michigan Douglas A. Greene Division of Endocrinology and Metabolism University of Michigan Medical Center Ann Arbor Michigan Eva Haak University of Frankfurt Frankfurt Germany Thomas Haak University of Frankfurt Frankfurt Germany BarryHalliwell Department of Biochemistry National University of S inga• pore Singapore Hans-Ulrich Haring M.D. Department of Endocrinology Metabolism and Pathobiochemistry University of Ti.ibingen Tubingen Germany Erik J. Henriksen Ph.D. Department of Physiology University of Ari• zona Tucson Arizona Mizuo Botta M.D. Ph.D. Department of Nutrition and Physiological Chemistry Osaka University Medical School Osaka Japan Luke Hounsom B.Sc. Hons Department of Pharmacology Queen Mary and Westfield College London England Stephan Jacob M.D. Department of Endocrinology Metabolism and Pathobiochemistry University of Tubingen Tubingen Germany MartinaJoswig University of Heidelberg Heidelberg Germany George L. King Joslin Diabetes Center Harvard Medical School Boston Massachusetts Yutaka Kishi Department of Neurology Mayo Foundation Rochester Minnesota Amira Klip Ph .D. Cell Biology Programme The Hospital for Sick Chil• dren and University of Toronto Toronto Ontario Canada

slide 25:

Contributors xix Thomas Konrad University of Frankfurt Frankfurt Germany Klaus Kusterer University of Frankfurt Frankfurt Germany Hans-Jochen Lang Hoechst Marion Roussel Frankfurt Germany Rainer Lehmann Ph.D. Department of Endocrinology Metabolism and Pathobiochemistry University of Tiibingen Tubingen Germany Wolfgang Leonhardt Technical University Dresden Germany Phillip A. Low Department of Neurology Mayo Foundation Rochester Minnesota Yoshiyuki Mitsui Department of Neurology Mayo Foundation Rochester Minnesota Jun-ichi Miyazaki M.D. Ph.D. Professor Department of Nutrition and Physiological Chemistry Osaka University Medical School Osaka Japan Masaaki Nagamatsu Department of Neurology Mayo Foundation Roch• ester Minnesota PeterP. Nawroth Department of Internal Medicine I University of Heidel• berg Heidelberg Germany Kim K. Nickander Department of Neurology Mayo Foundation Roches• ter Minnesota JaffarNourooz-Zadeh Ph.D. Department of Medicine University College London London England Irina G. Obrosova Division of Endocrinology and Metabolism University of Michigan Medical Center Ann Arbor Michigan Nesrin Ozer Ph.D. Department of Biochemistry Marmara University Is• tanbul Turkey LesterPackerPh.D. Department of Molecular and Cell Biology Univer• sity of California Berkeley California

slide 26:

xx Contributors Kristian Rett M.D. Department of Endocrinology Metabolism and Pathobiochemistry University of Ti.ibingen Ti.ibingen Germany RobertaRicciarelli Ph.D. Institute of Biochemistry and Molecular Biol• ogy University of Bern Bern Switzerland Peter Rosen Prof.Dr. Department of Clinical Biochemistry Diabetes Re• search Institute Heinrich-Heine-University Diisseldorf Germany Stephan Schiekofer University of Heidelberg Heidelberg Germany James D. Schmelzer Department of Neurology Mayo Foundation Roches• ter Minnesota Romel Somwar The Hospital for Sick Children and University of Toronto Toronto Ontario Canada Martin J. Stevens University of Michigan Medical Center Ann Arbor Michigan Guang-Zhi Sui Ph.D. Department of Clinical Biochemistry Diabetes Re• search Institute Heinrich-Heine-University Diisseldorf Germany Suzanne R. Thorpe Department of Chemistry and Biochemistry University of South Carolina Columbia South Carolina David R. Tomlinson Ph.D. D.Sc. University of Manchester Manchester England Hans J. Tritschler ASTA Medica Aktiengesellschaft Frankfurt Germany Klaus H. Usadel University of Frankfurt Frankfurt Germany Eiji Yamato M.D. Ph.D. Associate Professor Department of Nutrition and Physiological Chemistry Osaka University Medical School Osaka Japan Karen Yaworsky The Hospital for Sick Children and University of To• ronto Toronto Ontario Canada

slide 27:

Contributors xxi Dan Ziegler M.D. Professor Diabetes Research Institute Heinrich Heine University Dusseldorf Germany PaulaJ. Zollman Department of Neurology Mayo Foundation Rochester Minnesota

slide 28:

1 Oxidative Stress and Antioxidants: The Antioxidant Network o-Lipoic Acid and Diabetes To Cure Diabetes Naturally Click Here Lester Packer University of California Berkeley California In this introductory chapter oxidative stress in diabetes and implications of antioxidant treatment are considered. It is thought that free radicals may play a major role in aging and disease. Free radicals arise from radiation environ• mental chemicals cigarette smoke and various other environmental sources. In addition all through our life we have a fire burning inside of us-our own body metabolism which generates free radicals. Finally many environmental substances as well as drugs and alcohol are metabolized in our body generat• ing free radicals through cytochrome P450-mediated oxidations. Many free radicals can be cytotoxic. However free radical reactions are also essential. They are essential for enzymes and for host defense mechanisms such as neutrophils macrophages and other cells of the immune system. Free radicals are important in the activa• tion of transcription factors and in cell signal transduction and gene expres• sion. But if free radicals are overproduced they also can create oxidative stress and damage to molecules cells and tissues. So what then is oxidative stress Oxidative stress is an upset in the balance between oxidants and antioxidants. It was defined by Helmut Sies I in the following way: "Oxidative stress is a change in the pro-oxidant/ antioxidant balance in the favor of the former potentially leading to biological

slide 29:

damage." The result is molecular damage products which are markers of oxidative stress. 1

slide 30:

2 Packer I. DEFINITION OF AN ANTIOXIDANT What is an antioxidant To find a definition we went to the dictionary. Dor• lands Medical Dictionary reports 2 "An antioxidant is one of many widely used synthetic or natural substances added to a product to prevent or delay deterioration by action of oxygen in the air." Examples of such products to which antioxidants may be added are rubber paint vegetable oils and so on. But there are many other definitions of an antioxidant. For example Halliwell and Gutteridge 3 defined an antioxidant as any substance that when present at low concentrations compared to those of an oxidizable substrate signifi• cantly delays or inhibits oxidation of that substrate." Another definition of an antioxidant and the one I favor is that of a metabolic antioxidant 45: An antioxidant is a substance which protects biological tissues from free radical damage which is able to be recycled or regenerated by biological re• ductants. Thus metabolic antioxidants have something similar to a catalytic activity as long as they are connected directly or indirectly to biological reductants. So what then is the antioxidant network If antioxidants can be recycled and regenerated then there must be some sort of coordinated network connect• ing them to one another and to cellular metabolic processes. The antioxidant network consists of a series of proteins and substances that provide these con• nections. Among the proteins that are most important in antioxidant defense are superoxide dismutases to remove superoxide enzymes that catalyze the re• moval of hydroperoxides reduced thioredoxin and a number of proteins like transferrin and ceruloplasmin that bind transition metals like iron and copper in such a way that they are not able to catalyze free radical reactions. Another vital antioxidant enzyme is methionine sulfoxide reductase which repairs sulf• hydryl groups of methionine residues thus protecting cysteine residues which are critical for biological protection against oxidation. The group of antioxi• dant substances all of which are phytonutrients is rather small: vitamin C also known as ascorbate vitamin E a family of eight compounds-four to• copherols and four tocotrienols carotenes of which 500 different varieties may exist in nature and flavonoids and polyphenols of which there may be 4000-5000 different varieties. Of course few of the carotenoids and flavo• noids are common in the diet. n-Lipoic acid is another antioxidant compound naturally occurring in foods and also produced by the body. Others are metals that are covalently bound to the antioxidant defense proteins to assist with the proteins catalytic functions.

slide 31:

Oxidative Stress and Antioxidants 3 Lipoic acid is a good example of a metabolic antioxidant. It is an ana• logue of octanoic acid and has a dithiolane ring in its oxidized form but the ring can be broken by reduction to form dihydrolipoic acid. Both lipoic acid and dihydrolipoic acid have unique antioxidant profiles 6. The reduced form of lipoate has somewhat more antioxidant properties than the oxidized form in that the reduced form can scavenge superoxide and peroxyl radicals. Lipoic acid was found some years ago in work from Helmut Siess laboratory to be taken up by the fatty acid carrier in isolated mammalian hepatocytes 7. Thus lipoic acid can readily be taken up by cells. Three different enzymes have been identified as contributing to the reduction of lipoic acid so far: glutathione reductase 89 and thioredoxin reductase 10 which are NADPH• dependent enzymes and the more abundant lipoamide dehydrogenase an NADH-dependent enzyme. Lipoamide dehydrogenase is the E-3 component common to the n-keto acid dehydrogenase complexes that exist only in the mitochondria of animal cells. After reduction lipoic acid can be released to the extracellular compartment so there can be a cycle of lipoic acid reduction inside the cell its release to the outside where it is oxidized its reuptake into cells as the oxidized form and its reduction again as the cycle continues 11 . II. THE ANTIOXIDANT NETWORK The antioxidant network is composed of redox-sensitive antioxidant sub• stances. I like to say that the hub of the antioxidant network is vitamin C. The antioxidant network usually gets activated by vitamin E 12. After vitamin E is oxidized by oxidants or lipid free radicals then the vitamin E free radical is formed which in tum activates vitamin C to regenerate vitamin E nonenzy• matically. Vitamin C itself becomes a radical the vitamin C radical in this process. Glutathione with the aid of enzymes can reduce the vitamin C radical or dehydroascorbate the completely reduced form of vitamin C. The oxi• dized glutathione thus produced can be reduced through enzymatic reactions that draw on cellular reducing power. There are also substances that we can obtain in our diet or that we can supplement-like flavonoids polyphenols and lipoic acid-that can also act in the antioxidant network 13 14. An ex• ample of how the antioxidant network works with respect to vitamin C vita• min E and thiol antioxidants is shown in Figure l. If vitamin E is made into a radical by reacting with a lipid peroxyl radical the chromanol of vitamin E becomes a chromanoxyl radical and a lipid hydroperoxide forms. If this process is induced in human low-density

slide 32:

V NADPH Glutathione Ascorbate + H• ls ls Ascorbate Glutathione 4 Packer Vitamin E Chromanoxyl Radical itamin E Cycle ROCH ROH Vitamin E Tocophero Tocotrieno ROO• RO• PUFA Radical lipoic acid 02-- Other Radicals UVA UVB Ozone Cigarette Smoke Figure 1 Oxidative stress activates network antioxidants. lipoproteins there is enough vitamin E present in these lipoproteins to follow these reactions by detecting the electron spin resonance ESR signal of the vitamin E radical. Once vitamin E is made into a radical it is more reactive of course. It can react with itself or other radicals in a chain-breaking reaction. It is a slowly reacting radical because the free electron is delocalized around the chromanol ring of vitamin E. Thus it exists for a sufficient time for ascorbic acid to react with most of the vitamin E radicals and convert them back to vitamin E thus sparing vitamin E 15 16. When ascorbic acid does that it becomes an ascorbyl radical Fig. 2. The vitamin C radical can be regenerated by glutathione with the aid of enzymes or nonenzymatically by lipoic acid or certain flavonoids. Lipoic acid is unique in this regard because it has a redox potential - 320 mV that is even lower 17 than the glutathione system -280 m V thus in its reduced form it can nonenzymatically regenerate vitamin C which in tum can regenerate vitamin E. A dramatic example of this effect was recently observed by Podda et al. 18. When animals were placed on a vitamin E-deficient diet they lost weight and eventually died. But when 1.65 g lipoic acid/kg of diet was fed to these animals they did not develop the symptoms of vitamin E deficiency• weight loss and motor discoordination. Hence lipoic acid was obviously able to take over some functions of vitamin E in these animals presumably either

slide 33:

+"" r Oxidative Stress and Antioxidants 5 Formation of Chromanoxyl Radical ROO• + Chr-OH --- Decay to Non-Radical Products Chr-0• + RO• ------... Ohr-Os + ROO• ---• Chr--0• + Chr--0• ---• products products . products ······--- Regeneration-Recycling by Adding Vitamin c Chr-0• + Ascorbic - Chr-OH · · · ........ Acid Ascl+ Semiascorbyl Radical AscHr When added vitamin C becomes oxidized Asc2- A A The vitamin E radical reappears · · · · · · · · · · · · · · · · · · · · - V Figure 2 Vitamin E radical reactions during lipid peroxidation. through regenerating vitamin E as described above or through directly substi• tuting for vitamin E as an antioxidant or some combination of both. The vitamin E radical slowly decomposes as a result of reacting with itself or with other radicals. When ascorbic acid is added to a system in which vitamin E radical is being generated the vitamin E radical disappears as it is reduced back to vitamin E by reaction with ascorbate and the semiascorbyl radical appears. But the vitamin E radical eventually returns as the the semi• ascorbyl radical disappears. From this experiment the time that it takes for the vitamin E radical to reappear or the lag period can be determined. The time of the lag period is directly related to the vitamin C concentration. How• ever if dihydrolipoic acid is added to the reaction mixture as well as vitamin C and the same experiment is performed one now observes that it takes a much longer time for the vitamin E radical to return. In this experiment the lag period time is directly related to the concentration of the reduced lipoic acid. Reduced lipoic acid recycles the vitamin C radical 13. There are other ways in which lipoic acid can react with and thus recycle other antioxidants. After Jipoic acid is reduced it can regenerate oxidized thioredoxin glutathione disulfide or dehydroascorbate. Also reduced lipoic acid has been reported to regenerate the semiquinone of ubiquinone coenzyme Q 10 in membranes H.

slide 34:

I + l 6 Packer Noh Laboratory Vienna. Thus the entire antioxidant defense system can be affected by the presence of reduced lipoic acid. Ill. OXIDATIVE STRESS THE ANTIOXIDANT NETWORK AND DIABETES Why are oxidative stress antioxidants and the antioxidant network important in diabetes Hyperglycemia causes as a result of stimulation of the sorbitol dehydrogenase pathway accumulation of NADH and an increase in the lactate/pyruvate ratio. This is accompanied by decreased glycolysis increased reactive oxygen species formation increased protein kinase C activity and decreased Na+/K+ ATPase among other effects as shown in Figure 3. It is reasoned that the reductive imbalance that occurs in hyperglycemia and which may also occur in ischemia/hypoxia injury 20-22 might be reversed by Diabetes/Hyperglycemia stimulation of sorbitol pathway Diabetes/Hyperglycemia I ----- stimulation of sorbitol pathway dihydrolipoate/lipoate lschemia/Hypoxia impaired mitochondrial electron transport scavenges lree radicals recycles antioxidants increases cellular GSH lschemia/Hypoxia impaired mitochondrial electron transport N":ate l Pyruvate I Lactate i- Dihydrolipoate NAD+ Pyruvate - Lipoate NADH ..1

slide 35:

Glycolysis O I Roso / I Roso\ Glycolysis Respiration 0•2· 0 Fatty Acid Oxidation l PKC Activity i Na+K+-ATPase \ + R-a-lipoate I Figure 3 Proposed mechanism of lipoate-rnediated reversal of the reductive imbal• ance in hyperglycemia.

slide 36:

0 Oxidative Stress and Antio xidants 7 lipoic acid. Roy et al. 19 performed experiments to study if lipoic acid could affect the metabolic situation in hyperglycemia. If R-lipoic acid is added to cells it should as a result of its reduction by mitochondrial dihydrolipoamide dehydrogenase activity reverse the reductive imbalance in hyperglycemia and perhaps normalize the imbalance of overpro• duction of NADH and change the NADH/NAD+ ratio toward normal. Using human T lymphocytes as a model system we performed experiments to deter• mine if this was the case 19. Indeed treating these human T cells with R• lipoic acid but not S-lipoic acid normalized the redox status Fig. 4. Lipoate treatment caused the NADH/NAD+ ratio to be reversed in hy• perglycemia the ATP/ ADP ratio which had fallen was increased and the imbalance of the pyruvate/lactate ratio was also reversed. Furthermore the uptake of glucose by these cells was stimulated. With lipoic acid treatment even at I 00-µM concentrations significant increases in the uptake of glucose by these cells were observed 19. Of course one may wish to know how relevant these observations from a cellular system are to diabetes. We have proposed two models to link the ideas presented above and the possible therapeutic effects of lipoic acid in diabetes: 40 change 20 I I -20 • 40 - NADH/NAD ATP/ADP pyruvate/lactate • p0.05 0.5 mM R-llpoate treatment for 24 hours Figure 4 Effect of R-lipoate treatment on redox status and ATP/ADP ratio in Wurz• burg T cells.

slide 37:

I GSH TR reductase Lipoamide dehydrogenase - Fatty Acid transporter plasma I extr :»: 8 . . . a-LA Packer human leukocytes erythrocytes Reduced Lipoic Acid increases the Ascorbate:Dehydroascorbate ratio. This relieves inhibition of glucose uptake by overcoming competitive inhibition by dehydroascorbate which enters the cell by the glucose transporter. acellular space where vitamin C concentrations are high This may contribute to the hypoglycemic effect of Lipoate Ascorbate Lipoate Figure 5 Proposed mechanism whereby o-Iipoic acid stimulates glucose uptake. 1. Lipoic acid when it became reduced would be exported from the cells as dihydrolipoic acid this in tum could regenerate vitamin C radicals or dehydroascorbate because vitamin C in the plasma becomes oxidized after reacting with radicals that are produced by neutrophils or other cells of the immune system. This maintains plasma ascorbate in its reduced form. This is important because dehydroascorbate competes with glucose for uptake by the "fast track" glucose GLUT transport system present in most cells. Hence the inhibition of glucose uptake by dehydroascorbate would be overcome 23 as shown schematically in Figure 5. 2. There also may be a direct effect of lipoate on the uptake of glucose by the glucose GLUT transport system and thus an effect on the insulin-dependent stimulation of glucose uptake. It was of interest to investigate some of those parameters using the skeletal muscle• derived L6 myotube cell culture system. When L6 myoblasts differ• entiate into myotubes they gain the ability to take up glucose. Amira Klips laboratory has reported extensively on this system

slide 38:

Oxidative Stress and Antioxidants 9 75 2-Deoxy-Glucose Uptake change compared with basal rate in non• treated cells 50 - - - 25 - - t-- - t-- 0 I I I I IOOµM 250µM 500µM IOOOµM R-LA 30 minutes in DMSO Figure 6 Dose-dependent effect of lipoate on glucose uptake by skeletal muscle L6 myotubes. From Sen CK Khanna S Loukianoff S Roy S Packer L unpublished data. 2425. Using similar conditions my colleagues Chandan Sen Sav• ita Khanna Sonia Loukianoff and Sashwati Roy have measured the cellular uptake of deoxy-d-glucose from a buffer system by following the uptake of the radiolabeled deoxy-glucose. A dose• dependent effect of lipoate on glucose uptake by L6 myotubes showed that under our conditions even 100 µM lipoic acid was suf• ficient to markedly stimulate glucose uptake. At higher concentra• tions it continuously increased glucose uptake in a dose-dependent manner Fig. 6. In further experiments pretreatment with 250 µM lipoic acid for 30 min was used after such treatment a 30-40 stimulation of glucose uptake was usually observed. This is about the same extent of stimulation that has been observed under the same conditions with insulin treatment. If insulin and lipoic acid are added together an additive not a synergistic effect is observed as has been reported previously 2425. It was of interest to determine whether the effect of lipoic acid on stimu• lating glucose uptake was due to the fatty acid molecular structure of lipoic

slide 39:

10 Packer acid. Octanoic acid an analogue of lipoic acid had no effect on glucose uptake stimulated by 250 µM lipoic acid indicating that the fatty acid structure is not the cause of the stimulation. Lipoic acid is a thiol antioxidant. Hence it was of interest to know whether other thiol antioxidants or thiol reagents can mimic the effect of lipoic acid one of which is the ability to increase glutathione levels or maintain levels under oxidative stress conditions 2627. Therefore we tested pyrroli• dine dithiocarbamate PDTC 28 a thiol reagent which upregulates gluta• thione levels in cells diamide which oxidizes thiol residues and thioredoxin which can reduce thiol residues. None of these reagents or treatments had any stimulatory effects on the lipoate-induced uptake of glucose by L6 myotubes. Next we wanted to determine if the intracellular glutathione level which is the cells primary preventive antioxidant was important for glucose uptake. To find out whether modulations in the internal glutathione level was responsi• ble for promoting glucose uptake we treated the L6 myotubes with the inhibi• tor of a glutathione synthesis butamine sulfoxamine BSO 29. This reagent inhibits cell glutathione synthesis. After treating cells for 24 h with BSO glutathione levels in cells fall to very low levels. The effect of lipoic acid in stimulating glucose uptake in the presence of BSO was unchanged. So modu• lation of the internal glutathione level is not responsible for the stimulation of glucose uptake by lipoic acid. Confirming this PDTC the thiol reagent known to upregulate glutathione also does not prevent lipoic acid from stimu• lating glucose uptake. What was regulating the Iipoate-dependent glucose uptake Because cal• cium is an important factor in cell regulation 30 we investigated the effect of calcium-binding reagents. Two types of calcium chelators were used: EGTA which is membrane impermeable and the esterified form of EGTA which is known to permeate to the inside of cells. Both reagents when added to the L6 myotubes inhibited the lipoic acid-stimulated glucose uptake. Fur• ther evidence was obtained from the effects of calcium channel blockers like verapamil and nifedipine both of these reagents inhibited lipoic acid• stimulated glucose uptake. To prove that one of the effects of lipoic acid was stimulating calcium uptake we directly followed the uptake of radiolabeled 45C by L6 myotubes. After 30 min 250 µM lipoic acid markedly stimulated - 30 45Ca2+ uptake. It is known that a ryanodine-sensitive receptor is involved in calcium entry. Indeed 25 µM ryanodine inhibited the lipoic acid-stimulated uptake of glucose. Moreover by adding 250 µM 4-chloro-m-cresol it was possible to mimic the effect of lipoic acid. 4-Chloro-m-cresol is known to stimulate the

slide 40:

Oxidative Stress and Antioxidants 11 ryanodine receptor 3132. Stimulating the ryanodine gives almost the same effect as the lipoic acid suggesting that this receptor is involved. IV. LIPOATE- AND INSULIN-DEPENDENT CELL SIGNALING PATHWAYS Insulin activates numerous metabolic and mitogenic effects by first binding to its specific transmembrane glycoprotein receptor which has intrinsic tyrosine kinase activity. Tyrosine phosphorylation of various other substrates particu• larly the insulin receptor substrate IRS proteins then induces formation of a network of docking proteins that mediate insulin action of gene expression involved in its anabolic and catabolic effects 33. From the results of the present study it would appear that the action of lipoate in stimulating glucose uptake may also be through protein kinase activation likely mediated by transient increases in cytosolic calcium which is essential for the Wortrnanin-sensitive Pl-3 kinase-dependent lipoate• stimulated glucose uptake in the L6 myotube system 2425. After upregula• tion of glucose transport after mobilizing GLUT transporters from the cyto• solic to the plasma membrane domain lipoate like insulin may recruit a broad array of kinases in target cells to activate its numerous metabolic actions. V. LIPOIC ACID DIABETIC POLYNEUROPATHY AND DIABETES Lipoic acid has been used successfully as a therapeutic agent in the treatment of diabetic polyneuropathy both in animal models and in human clinical trials 34-36. Diabetes is considered as an oxidative stress disease evidence indi• cates that both insulin-dependent and noninsulin-dependent diabetes exhibit molecular markers indicative of oxidative stress. Thus it could be anticipated that one of the most potent metabolic antioxidants known in biological sys• tems free cc-Iipoicacid should be effective in treating diabetic complications. In particular R-lipoic acid is recognized by the mitochondrial lipoamide dehy• drogenase that reduces it to dihydrolipoate a powerful reductant that is capa• ble of direct scavenging of radicals regenerating vitamins E and C increasing the potency of the entire redox antioxidant network upregulating cellular lev• els of glutathione affecting important cell regulatory activities such as nuclear factor-KBtranscriptional activation regulating free cytosolic calcium and re-

slide 41:

/ 12 Packer dihydrolipoamide Li oate + NADPH + H+ dehy_drogenase p glutath1one reductase Dihyrloate + NADP+ thioredoxin reductase " / / Redox regulation of / -"----- reductive stress: / / Redox regulation: high NADH/NAO+ " / Ca 2+1 in hyperglycemia and " / / NF-KB activation hypoxia is reversed " / CAM expression ----------- " / / Apoptosis Direct scavenging " I of free radicals GSH synthesis e.g. •OH 02._ Enhancing activity of other cysteine availability LOO• NO• antioxidants Low negative redox potential Ascorbate Glutathione Thioredoxin Figure 7 Redox regulation of cell functions by o-lipoate: biochemical and molecular aspects. From Ref. 37. versing the reductive imbalance in diabetes resulting from hyperglycemic con• ditions. These various effects of lipoic acid have been described previously 37 and are shown schematically in Figure 7. These properties may have therapeutic effects in oxidative stress diseases and aging. Importantly lipoic acid has also been demonstrated at higher concentra• tions to have hypoglycemic effects. It exhibits effects on glucose disposal an important function of skeletal muscle as demonstrated in animal models by Henriksen et al. 38 and in human clinical studies. It is therefore to be expected that lipoic acid somehow has a profound effect on the mechanism of glucose uptake and disposal and in regulating the glucose-dependent metabolic changes that ensue. The evidence summarized in this chapter provides new and interesting findings relevant to these questions. The many molecular effects of lipoate and dihydrolipoate on receptor• mediated activity cell signaling transcriptional activation and gene expres• sion remain to be elucidated. Important among these considerations for diabe• tes is how it modulates insulin-dependent cell signaling system pathways. Because the effects of insulin and lipoate in the L6 myotube experiments are additive it is reasonable to suggest that the pathways of insulin-stimulated glucose uptake and utilization and that of lipoate-stimulated glucose uptake differ from one another. Lipoate may affect protein kinases and phosphatases

slide 42:

Oxidative Stress and Antioxidants 13 which will modulate phosphorylation systems and at some point may have common actions with the insulin-signaling pathways. These pathways re• main to be elucidated particularly the mechanism whereby lipoate stimulates calcium-dependent signaling pathways related to glucose transport. VI. SUMMARY Oxidative stress antioxidants and the antioxidant network can be relevant to diabetes because diabetes appears to involve oxidative stress. One antioxidant that may have particular relevance to diabetes is lipoic acid. Reduced lipoic acid powers the antioxidant network after being taken up by cells. Lipoic acid reverses the reductive imbalance that occurs in hyperglycemia. Plausible mechanisms for this effect are as follows. First cell reduction of lipoic acid is released into the extracellular space and maintains reduced plasma ascor• bate. It can thus relieve the competitive inhibition of glucose uptake by dehy• droascorbate. Second lipoic acid stimulates glucose uptake in skeletal muscle the main tissue responsible for glucose disposal. In L6 myotubes used as a model system for glucose disposal this stimulation apparently occurred by a calcium-dependent mechanism. REFERENCES I. Sies H ed. Oxidative Stress: Oxidants and Antioxidants. London: Academic Press 1991. 2. Dorlands Illustrated Medical Dictionary. 25th ed. Philadelphia: W.B. Saunders 1974:111. 3. Halliwell B Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford: Clarendon Press 1985. 4. Packer L Tritschler HJ. Alpha-lipoic acid-the metabolic antioxidant. Free Rad Biol Med 1996 20:625-626. 5. Packer L Roy S Sen CK. Alpha-lipoic acid: metabolic antioxidant and potential redox modulator of transcription. Adv Pharmacol 1996 38:79-101. 6. Packer L Witt EH Tritschler HJ. Alpha-lipoic acid as a biological antioxidant. Free Rad Biol Med 1995 19:227-250. 7. Peinado J Sies H Akerboom TP. Hepatic lipoate uptake. Arch Biochem Biophys 1989 273:389-395. 8. Haramaki N Han D Handelman GJ Tritschler H-J Packer L. Cytosolic and mitochondrial systems for NADH and NADPH dependent reduction of a-lipoic acid. Free Rad Biol Med 1997 22:535-542.

slide 43:

14 Packer 9. Pick U Haramaki N Constantinescu A Handelman GJ Tritschler H-J Packer L. Glutathione reductase and lipoamide dehydrogenase have opposite stereospec• ificities for o:-lipoic acid enantiomers. Biochem Biophys Res Commun 1995 206:724- 730. l 0. Marcocci L Flohe L Packer L. Evidence for a functional relevance of the seleno• cysteineresidue in mammalianthioredoxinreductase. BioFactors 19976:351-358. 11. Handelman GJ Han D Tritschler H-J Packer L. Alpha-lipoic acid reduction by mammalian cells to the dithiol form and release into the culture medium. Bio• chem Pharmacol 1994 47:1725-1730. 12. Packer L. Vitamin Eis natures master antioxidant. Sci Am Sci Med 1994 I: 54-63. 13. Packer L. Antioxidant defenses in biological systems: an overview. In: Packer L Traber M Xin W eds. Proceedings of the International Symposium on Natural Antioxidants: Molecular Mechanisms and Health Effects. Champaign: AOCS Press 1996:9-23. 14. Packer L Witt EH Tritschler HJ. Antioxidant properties and clinical implica• tions of alpha-lipoic acid and dihydrolipoic acid. In: Cadenas E Packer L eds. Handbook of Antioxidants. Vol. 3. New York: Marcel Dekker 1996:545-591. 15. Kagan VE Serbinova EA Forte T Scita G Packer L. Recycling of vitamin E in human low density lipoproteins. J Lipid Res 1992 33:385-397. 16. Kagan VE Shvedova A Serbinova E Khan S Swanson C Powell R Packer L. Dihydrolipoic acid-a universal antioxidant both in the membrane and in the aqueous phase. Reduction of peroxyl ascorbyl and chromanoxyl radicals. Biochem Pharmacol 1992 44:1637-1649. 17. Jocelyn PC. The standard redox potential of cysteine-cystine from the thiol• disulphide exchange reaction with gluatathione and lipoic acid. Eur J Biochem 1967 2:327-331. 18. Podda M Tritschler H-J Ulrich H and Packer L. Alpha-Iipoic acid supplemen• tation prevents symptoms of vitamin E deficiency. Biochem Biophys Res Com• mun 1994 204:98-104. 19. Roy S Sen CK Tritschler H-J Packer L. Modulation of cellular reducing equiva• lent homeostasis by o:-lipoic acid: mechanisms and implications for diabetes and ischemic injury. Biochem Pharmacol 1997 53:393-399. 20. Williamson JR Chang K Frangos M Hasan KS Ida Y Kawamura T Nyen• gaard JR van den Enden M Kilo C Tilton RG. Hypoglycemic pseudohypoxia and diabetic complications. Diabetes 1993 42:801-813. 21. Dawson TL Gotes GJ Nieminen AL Herman B Lemasters JJ. Mitochondria as a source of reactive oxygen species during reactive stress in rat hepatocytes. Am J Physiol 1993 264:C961-C967. 22. Jaeschke H Kleinwaechter C Wendel A. NADH-dependent reductive stress and territin-bound iron in ally alcohol induced lipid peroxidation in vivo: the protec• tive effect of vitamin E. Chem Biol Interact 1992 81 :57-68. 23. Packer L. The role of anti-oxidative treatment in diabetes mellitus. Diabetologia 1993 36:1212-1213.

slide 44:

Oxidative Stress and Antioxidants 15 24. Estrada E Ewart HS Tsakiridis T Volchuk A Ramlal T Tritschler H Klip A. Stimulation of glucose uptake by the natural coenzyme u-lipoic acid/thioctic acid: participation of element of the insulin signaling pathway. Diabetes 1996 45:1798-1804. 25. Han D Handelman G Marcocci L Sen CK Roy S Kobuchi H Tritschler H-J Flohe L Packer L. Lipoic acid increases de wvo synthesis of cellular gluta• thione by improving cystine utilization. BioFactors 1997 6:321-338. 26. Panigrahi M Sadguna Y Shivakumar BR Kolluri YR Roy S Packer L Ravin• dranath V. Alpha-lipoic acid protects against reperfusion injury following cere• bral ischemia in rats. Brain Res 1996 7 I 7: I 84-188. 27. Packer L Tritschler JJ Wessel K. Neuroprotection by the metabolic antioxidant n-lipoic acid. Free Rad Biol Med 1997 22:359-378. 28. Sen CK Khanna S Reznick A Roy S Packer L. Glutathione regulation of tumor necrosis factor-ex-induced NF-KB activation in skeletal muscle-derived L6 cells. Biochem Biophys Res Commun 1997 237:645-649. 29. Maitra I Serbinova E Trischler H Packer L. Alpha-lipoic acid prevents buthio• nine sulfoximine-induced cataract formation in newborn rats. Free Rad Biol Med 1995 18:823-829. 30. Sen CK Packer L. Antioxidant regulation of gene transcription. FASEB J 1996 10:709-720. 31. Herrmannfrank A Richter M Sarkozi S Mohr U Lehmann-Horn F. 4-Chloro- 111-cresol a potent and specific activator of the skeletal muscle ryanodine recep• tor. Biochim Biophys Acta I 996 1289:31-40. 32. Zorzato F Scutari E. Tegazzin V Clementi E. Treves S. Chlorocresol: an activa• tor of ryanodine receptor-mediated Ca1+ release. Mo Pharmacol I 993 44: I 192- 1201. 33. Avruch J. Insulin signal transduction through protein kinase cascades. Mol Cell Biochem I 998 I 82:3 I -48. 34. Ziegler D. Hanefeid M. Ruhnau KJ. MeiBner HP Lobisch M Schutte K Gries FA. Treatment of symptomatic diabetic peripheral neuropathy with the antioxi• dant rz-lipoic acid. Diabetologia 1995 38:1425-1433. 35. Strodter D Lehmann E Lehmann U Tritschler H-J Bretzel RG Rederlin K. The influence of thioctic acid on metabolism and function of diabetic heart Dia• betes Res Clin Pract 1995 29: I 9-26. 36. Ziegler D. Gries FA. Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy. Diabetes 1997 46suppl 2:62-66. 37. Roy S Packer L. Rcdox regulation of cell functions by n-lipoate: biochemical and molecular aspects. BioFactors 1998 7:263-267. 38. Henriksen E Jacob S Streeper R Fogt D Hokama J Tritschler H. Stimulation by alpha-lipoic acid of glucose transport activity in skeletal muscle of lean and obese Zucker rats. Life Sci 1997 61:805-812.

slide 45:

This Page Intentionally Left Blank

slide 46:

2 Oxidative Stress in Diabetes: Why Does Hyperglycemia Induce the Formation of Reactive Oxygen Species To Get Best Natural Diabetes Treatment Click Here Peter Rosen Xueliang Du and Guang-Zhi Sui Diabetes Research Institute Heinrich-Heine-University Diisseldort Germany There is much evidence that the formation of various markers of oxidative stress are increased in diabetes: In the plasma of diabetic patients the concen• trations of lipid hydroperoxides isoprostanes malonic dialdehyde and oxi• dized lipoproteins are elevated 1-6. The intracellular levels of antioxidants such as tocopherol and glutathione are reduced whereas the enzymatic activity of antioxidative acting enzymes is at least partly increased 7-12. Similarly there are many reports about the consequences of an imbalance between pro• and antioxidant actions in the cells "oxidative stress" and the importance of disturbances in the intracellular antioxidant network for the development of vascular complications in hypertensive or hypercholesterolemic patients. Such a pathophysiological link between oxidative stress and vascular compli• cations is in line with many experimental observations with large epidemio• logical studies and to a lesser extent with recent clinical investigations 13- 22. There is increasing evidence that the generation of reactive oxygen inter• mediates is also of major importance for the development of vascular compli•

slide 47:

cations in diabetes 23-29. However neither the mechanisms that specifically lead to the generation of reactive oxygen intermediates ROI in hyperglyce- 17

slide 48:

18 Rosen et al. mic states nor the cascade of reactions linking the formation of ROI with the pathophysiological event are well understood. Here we present evidence that the vasculature is an important source for the formation of reactive oxygen species that high glucose activates an endo• thelial NADPH-oxidase and thereby causes the release of superoxide anions that the superoxide anions are able to react with nitric oxide leading to the for• mation of peroxynitrite and that the formation of peroxynitrite is responsible for the impaired endothelium-dependentvasodilatation and variety of cytotoxic effects on the vasculature observed in hyperglycemia such as activation of the nuclear transcription factor kappa-B NF-KB and induction of a apoptosis. In addition peroxynitrite has been shown to accelerate the oxidation of low-density lipoproteins and to activate metalloproteinases. Thus it is intri• guing to suggest that the generation of ROI induced by hyperglycemia is one of the major causes for the transformation of endothelium into a proinflamma• tory and thrombogenic state as observed in diabetes. We assume that this endo• thelial activation or dysfunction is the basis for the enhancement of atheroscle• rosis and the development of other vascular complications in diabetes and may contribute to a destabilization of established plaques that has been shown to be one of the most decisive events for induction of myocardial infarction angina pectoris and cardiac death 3031 . I. IS THE VASCULATURE A SOURCE FOR ROls We have already shown 29 that the endothelium-dependent increase in coro• nary flow is disturbed in isolated perfused hearts of diabetic rats. The dose• response curve for the increased coronary flow in response to 5-hydroxytryp• tamin is shifted to higher concentrations in diabetes whereas the maximum coronary flow is not altered under these conditions. This defect could be pre• vented in vivo by treatment of the animals with high concentrations of vitamin E 1000 U/kg/day and more interestingly under the aspect of mechanisms by the addition of superoxide dismutase to the perfusion medium Fig. I. This observation suggests that the vasculature of diabetic rats releases superox• ide anions spontaneously and continuously into the perfusion medium and that the generated superoxide anions are the cause for the disturbed endothelium• dependent flow regulation. We assume that nitric oxide NO as the main mediator of endothelium-dependent vasodilatation becomes inactivated by the simultaneously released superoxide anions. SOD 2 ONoo- 2 02- H202 + 02 2 NO m+

slide 49:

100 50 0 Oxidative Stress in Diabetes 19 / QI 0 . E s 0 Ul . w C DB +SOD +Vit E Figure 1 Impairment of the endothelium-dependent increase in coronary flow and its prevention by superoxide dismutase SOD and vitamin E. Diabetes was induced in rats by streptozotocin. After a diabetes duration of 16 weeks the stimulation of coronary flow by 5-hydroxytryptamin was measured in the isolated heart preparation as described 29. The half-maximal concentration EC-50 was determined and repre• sents a measure for the sensitivity of endothelium to dilate the coronary vasculature. As can be seen in diabetes DB the sensitivity of endothelium is impaired as com• pared with healthy controls C but perfusion with SOD 50 µU/mL or pretreatment of the animals with n-tocopherol 1000 U/kg body weight were able to improve or to restore the endothelium-dependent vasodilatation in diabetes. From Ref. 23. Such an interaction between superoxide anions with NO has already been described. In a diffusion controlled reaction both compounds react with each other under the formation of peroxynitrite 32-34. Direct evidence for this conclusion is derived from experiments using isolated aortas from streptozotocin diabetic rats. This model enables us to directly measure the formation of superoxide anions by standard techniques as the reduction of cytochrome c 35. When aortas from diabetic and control animals were perfused under normoglycemic conditions vessels from diabetic rats released significantly more superoxide anions than those from controls. In addition the generation of superoxide anions was stimulated in both types of aortas by hyperglycemic buffers 10-30 mM glucose. The increased gener• ation of superoxide anions could be totally reduced to control values when the endothelium was removed from the intact aortas by mechanical disruption Rosen 1998 unpublished data. It is interesting to note that an endothelial production of ROI has also been reported for vessels isolated from hypercho-

slide 50:

20 Rosen et al. lesterolemic and hypertonic animals 13-15. Thus the stimulus for activation of endothelium is different in these various pathophysiological conditions but the consequences seem to be comparable. These experimental observations lead to the conclusion that endothelium is an important source of ROI and identify hyperglycemia as a stimulus for the formation of superoxide anions. Furthermore the disturbed endothelium• dependent vasomotion in diabetes is an immediate pathophysiological conse• quence of the release of superoxide anions by the vasculature. II. WHICH MECHANISMS CONTRIBUTE TO THE ENDOTHELIAL FORMATION OF SUPEROXIDE ANIONS IN DIABETES To study the mechanisms of ROI generation induced by hyperglycemia in more detail we used human umbilical vein endothelial cells HUVECs. To identify the generation of ROI HUVECs were loaded with dichlorodihy• drofluorescin ester DCF 36 which is taken up by the cells and then rapidly hydrolyzed. DCF reacts with superoxide anions but presumably also other ROI under the emission of fluorescence light so that the formation of ROI can be determined in a time- and concentration-dependent manner. Incubation of DCF-loaded cells with increasing concentrations of glu• cose 5-30 mM leads to a time- and glucose-dependent increase in fluores• cence Fig. 2. A comparable increase in fluorescence was also observed if the cells were incubated with 3-0-methyl-o-glucose 30 mM a glucose deriv• ative which is taken by the cells but not metabolized by glycolysis. These data indicate that the formation of ROI is dependent on high glucose in the culture medium but not on the synthesis of diacyl-glycerol and a glucose• dependent activation of protein kinase C. In line with this conclusion we did not observe an alteration in DCF fluorescence by treating the cells with an inhibitor bisindolylmaleimide BIM or activator phorbol 12-myristate 13-ace• tate PhA of protein kinase C 23. The formation of ROI by endothelial cells incubated with high glucose was completely inhibited by antioxidants u-tocopherol 10 µg/mL and thioctic acid 0.5 µM and by diphenyliodonium DPI l µM a selective inhibitor of flavoprotein containing NADPH oxidases 37. The inhibitory effect of DPI is consistent with the assumption that NADPH oxidases are the major source of ROI in HUVECs cultivated in hyperglycemic glucose. DPI was also re• ported to inhibit the NADH-dependent production of superoxide anions in bovine coronary endothelial 38.

slide 51:

Oxidative Stress in Diabetes 21 0 5 10 15 20 25 30 35 0-Glucose mM Figure 2 Increase in the formation of ROls by human endothelial cells in depen• dence of glucose. HUVECs were preloaded with the DCF I µM and dichlorodihy• drofluorescin 10 µM for 45 min. After washing the cells were incubated with n• glucose 5-30 mM. For control cells were incubated with mannitol and L-glucose 25 + 5 mM. After a 15-min incubation 37°C the fluorescence intensity as a parame• ter of the ROI generation was analyzed by fluorescent microscopy and quantified. Although cyclooxygenases and lipoxygenases may also be sources of superoxide anion generation in endothelium 3538 our data do not link these enzymes to the production of superoxide anions induced by hyperglycemia because indomethacin and nordihydroguaretic acid did not inhibit the release of superoxide anions. Similar observations have already been reported for por• cine endothelial cells 35. Surprisingly the DCF fluorescence was also prevented by inhibitors of NO synthase t-nitroarginine I 00 µM and a chelator of intracellular calcium 12-bis 2-Aminoprenoxyjethane-NNN N-tetraacetic acid BAPTA. These observations indicate that the mobilization of intracellular calcium and an acti• vation of NO synthesis are necessary steps for the formation of DCF fluores• cence by hypoglycemia. In line with this conclusion the release of nitrite as parameter of NO synthesis by HUVECs was stimulated by glucose Fig. 3. Thus under hyperglycemic conditions both NADPH oxidase and NO synthase become activated and both steps are a precondition for the formation of DCF fluorescence by HUVECs in hyperglycemia. This synergistic actions of NADPH oxidase and NO synthase suggest that DCF fluorescence does not

slide 52:

1000 800 600 400 200 - z - 22 Rosen et al. i a i 0 E e a l: 5 mM 30 mM 30 mM D·Glucose L·Glucose Figure 3 Glucose stimulates the formation of NO by human endothelial cells. HUVECs were incubated with glucose 5-30 mM for 24 h. The formation of nitrite in the supernatant was analyzed by the Gries reaction. For control cells were incubated with i.-glucose 25 + 5 mM. specifically reflect the generation of superoxide anions but rather the reaction product of both NO and superoxide anions presumably peroxynitrite. Because peroxynitrite has been reported to react with tyrosine residues in proteins leading to the formation of o-nitrotyrosylated proteins o-nitroty• rosylation has been suggested as a long-term parameter for an enhanced forma• tion of peroxynitrite and oxidative stress 32-34. Demonstration of nitroty• rosylated proteins in the vasculature would represent direct evidence of a preceding formation of peroxynitrite and oxidative stress. Endothelial cells were therefore incubated with high glucose and the proteins were extracted separated by gel electrophoresis and stained by an antibody specifically recog• nizing o-nitrotyrosylated proteins. As expected hyperglycemia results in a dose-dependent formation of o-nitrotyrosylated proteins data not shown. There are at least two other pathways that might contribute to the genera• tion of ROI. Giardino et al. 39 showed that the intracellular formation of advanced glycation endproducts AGE products is closely associated with the generation of ROI determined by DCF fluorescence and lipid peroxidation. Inhibition of lipid peroxidation also prevented the formation of AGE products

slide 53:

endothelial cells smooth muscle cells ox-LDL macrophages monocytes Tcells Oxidative Stress in Diabetes 23 TNFa IL-1 NF-KB-activation M-CSF GM-CSF c-myc proliferation MCP-1 Chemotaxis VCAM-1 ICAM-1 Adhesion TF Thrombogenesis Figure 4 NF-KB-mediated pathways leading to a thrombogenic transformation of the vessel wall. suggesting that the ROI generation is necessary for the synthesis of AGE prod• ucts. On the other hand there is some evidence that endothelium starts to produce ROI as soon as the receptor for AGE products becomes occupied 4041. Although the exact intracellular signaling is not yet known there is some evidence that binding of AGE products to its receptors causes an activa• tion of NADPH oxidases 41. This AGE-mediated production of ROI is pre• vented by antioxidants and inhibitors of NADPH oxidases but not inhibitors of NO synthases cyclo- and lipoxygenases or xanthin oxidase. Thus the available evidence suggests that activation of NADPH oxidase by glucose or AGE is a key step for the generation of ROI by endothelium. Whether the generated ROI are transformed to peroxynitrite depends on the type of cell and the concomitant reactions. If as in HUVECs NO-synthase becomes activated simultaneously peroxynitrite may be formed and represent the key mediator for the subsequent transformation of endothelium. In the absence of NO synthase activation or insufficient amounts of NO superoxide anions may directly act as signal mediators and exert the deleterious cytotoxic effects of hyperglycemia and AGE on endothelium and other vascular cells. There are two open questions. What are the mechanisms for the hyper• glycemia-mediated increase in intracellular calcium The formation of vascu• lar endothelial growth factor VEGF as a consequence of the generation of superoxide anions is one interesting mechanism especially because it has been shown that AGE are able to induce the expression of VEGF 42. Do changes

slide 54:

24 Rosen et al. in the cytosolic NADH/NAD ratio contribute to the formation of ROI in addi• tion to the activation of NADPH oxidases It has been suggested that the NADH/NADPH ratio is elevated in diabetes because more glucose is metabo• lized by the sorbitol pathway and that the increased NADH/NAD ratio causes the formation of superoxide anions by various mechanisms 43. We do not believe that this mechanism is working in endothelium because the formation of ROI was not inhibited by inhibitors of the sorbitol dehydrogenase either in HUVEC or in porcine endothelial cells 35. In summary there is good evidence that the vasculature and more spe• cifically the endothelium is one important source for the generation of ROis. The formation of ROis is specifically related to the diabetic state because it is stimulated by glucose and advanced glycation end products in a dose• dependent manner. It is interesting to note that similar observations have been reported for the vasculature in hypertension and hypercholesterolemia sug• gesting that the initial processes and stimuli might be different but that the three pathophysiological conditions finally result in an enhanced oxidative stress. Ill. WHAT ARE THE CONSEQUENCES OF OXIDANT STRESS IN DIABETES There is a lot of evidence that ROI and especially peroxynitrite are involved in activation of transcription factors such as NF-KB. NF-KB is responsible for a variety of reactions contributing to the thrombogenic transformation of endothelium Fig. 4 44-48: release of tumor necrosis factor ex. and interleu• kin I proinflammatory release of growth factors M-CSF monocyte col• ony stimulating factor GM-CSF granulocyte-monocyte colony stimulating factor c-myc activation of the monocyte chemoattractant protein MCP-1 chemotaxis expression of adhesion proteins VCAM-1 vascular cell adhe• sion molecule-I ICAM-1 intercellular adhesion molecule-l j and expres• sion of tissue factor thrombogenesis. We used two different approaches to test whether hyperglycemia causes an activation of NF-KB: the electromobility shift assay EMSA measuring the DNA-binding activity of nuclear proteins to an NF-KB-specificoligonucle• otide 48 and a histochemical approach using a fluorescence-labeled antibody coupled to the NF-KB-specificoligonucleotide 49. Using both methods we can show that hyperglycemia causes a dose- and time-dependent activation of NF-KB.The maximum of activation by high glucose is achieved after 4 h after 10-12 h NF-KB is again completely inactivated Fig. 5. This activation is inhibited by antioxidants tocopherol 10 µg/mL thioctic acid 0.5 µM and

slide 55:

---·----· - o Oxidative Stress in Diabetes 25 100 ...-----------------. 5 80 iii c: Q .5 60 Q o c: Q UI 40 Q ... . 0 ::I U:: 20 ....._ . .... 0 4 8 12 Time hrs Figure 5 Time dependence of NF-KB activation by hyperglycemia in human endo• thelial cells. HUVECs were incubated with low glucose 5 mM high glucose 30 mM and 3-0-methyl-D-glucose 3-0MG 25 + 5 mM glucose. After a 2- 4- 6- and I 2-h incubation 37 °C the cells were fixed with paraformaldehyde and stained by the specific fluorescein isothiocyanate FITC-labeled consensus sequence for NF• KB as described. Osmotic controls 25 mM mannitol + 5 mM glucose did not show staining above the background. e Controls•. high glucose . 3-0MG. by the NO synthase inhibitor t-nitroarginine 100 µM whereas the modula• tion of protein kinase C was without any influence. These data suggest that the short-term activation of NF-KB by hyperglycemia is caused by peroxynitrite. It is an open question whether the recently reported long-term activation of NF• KB by AGE products 48 was caused by a similar mechanism. In any case hyperglycemia seems to cause an activation of NF-KB by different signaling cascades: High glucose leads to a short-term activation which might be impor• tant for the transformation of immediate and short-term variations in blood glucose into vascular reactions. Such a mechanism would also explain why not only the long-term elevation of blood glucose is cytotoxic for the vessel wall but also the spikes in blood glucose that are often observed even in pa• tients with an overall near normoglycemic metabolic control. Activation of NF-KB by AGE products on the other hand would induce a long-term modu• lation of vascular functions and might be especially of importance for angio• genesis.

slide 56:

26 Rosen et al. In addition to activation of NF-KB the formation of peroxynitrite may have several other consequences that may contribute to the development of vascular complications in diabetes. First the induction of apoptosis. We have recently reported 50 that high glucose induces the programmed cell death in HUVECs. This process was inhibited by antioxidants thioctic acid and tocopherol but also by inhibitors of NO synthases. The underlying mecha• nism is not yet fully understood at this time but there are several lines of evidence that the induction of apoptosis is independent of the activation of NF• KB. The induction of apoptosis can be understood as an indicator of damage of endothelium by high glucose as an attempt of the vasculature to get rid of damaged endothelial cells. Such a loss of endothelium would be associated with induction of angiogenesis a process typically observed in the eye and the kidney of many diabetic patients 5152. On the other hand Joss of endo• thelium would lead to an exposition of thrombogenic structures subendothel• ial matrix to the bloodstream and thereby cause an increased thrombotic risk. Another consequence is the oxidation of low-density lipoproteins. It has been reported that peroxynitrite is a strong prooxidant and accelerates the oxi• dation of low-density lipoproteins 53. Because oxidized low-density lipopro• teins are themselves cytotoxic for endothelium the formation of peroxynitrite would reinforce the oxidant stress by constituting a deleterious vicious cycle. Finally the activation of metalloproteinases has been reported to become acti• vated by peroxynitrite in vivo and in vitro 54. Such an activation of metallo• proteinases at the edge of an atherosclerotic plaque is assumed to cause a destabilization of the plaque enhance plaque rupture and finally a thrombotic event that is the most common cause for myocardial infarction angina pecto• ris and cardiac death 3031 . IV. CONCLUSIONS Taken together there is good evidence that short- and long-term hyperglyce• mia cause an activation of NADPH oxidase and the formation of ROI. The endothelium has been demonstrated as one of the major sources of ROI gen• eration. Experimental data from in vitro and in vivo studies clearly show that these ROis are able to induce a thrombogenic transformation of the vessel wall and to be the cause for the endothelial dysfunction observed in diabetes but also in hypertension and hypercholesterolemia. Whether these cytotoxic effects are exerted by superoxide anions directly or are mediated by peroxynitrite depends on the local environment and the type of vascula• ture regarded. Our current knowledge is summarized in the hypothesis shown

slide 57:

VEGF I NADH • -Oxidase Oxidative Stress in Diabetes 27 Gluco • se I AGE --t-- NO-Synthase I Apoptosis NF-KB ox-LDL MMP Figure 6 Current hypothesis: formation of ROI by hyperglycemia and the effects of ROI on the vessel wall as cause for the development of vascular complications in diabetes. in Fig. 6. It is important to recognize that the consequences of these ROI• induced vascular dysfunctions might be different depending on the size of the vasculature affected: In small vessels the oxidative stress induced by hypergly• cemia might be one important factor for the stimulation of proliferation of endothelium and the formation of new partially malfunctional vessels and thereby contribute to the development of small vessel disease retinopathy nephropathy. In coronary vasculature the generation of ROI might be more important for the destabilization of atherosclerotic plaques causing an in• creased cardiac risk in diabetic patients. The reestablishment of the antioxida• tive network might therefore be a useful approach to protect the vasculature in diabetes. ACKNOWLEDGMENTS Supported by the Ministerium fiir Frauen Farnilie und Gesundheit der Bundes• republik Deutscbland and the Wissenschaftsministerium des Landes NRW the Deutsche Forschungsgemeinschaft Bonn and the Klinische Zellbiologie und Biophysik" e.V. Diisseldorf.

slide 58:

28 Rosen et al. REFERENCES 1. Nourooz-Zadeh J Tajaddini-Sarrnadi J McCarthy S Betteridge DJ Wolff SP. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: I 054-1058. 2. Nishigaka 1 Hagihara M Tsunekawa H Maseki M Yagi K. Lipid peroxide levels of serum lipoprotein fractions of diabetic patients. Biochem Med 1981 25:373-378. 3. Gopaul NK Anggard EE Mallet AI Betteridge DJ Wolff SP Nourooz-Zadeh J. Plasma 8-epi-PGFa levels are elevated in individuals with NIDDM. FEBS Lett 1995 368:225-229. 4. Davi G Mezzetti A Vitacolonna E Costantini F Pennese E Falco A Ciabattoni G Patrono C Consoli A. In vivo formation of 8-epiprostaglandin F2a in diabetes mellitus. Effects of tight control and vitamin E supplementation. Diabetes 1997 46suppl 1 : l 3A. 5. Jain SK Mc Vie R Duett J Herbst JJ. Erythrocyte membrane lipid peroxidation and glycosylated hemoglobin in diabetes. Diabetes 1989 38:1539-1543. 6. Bellomo G Maggi E Polli M Agosta FG Bollati P Finardi G. Autoantibodies against oxidatively modified low density lipoproteins in NIDDM. Diabetes 1991 44:60-66. 7. Nourooz-Zadeh J Halliwell B Tritschler HJ Betteridge DJ. Decreased lipid standardised plasma n-tocopherol in non-insulin-dependent diabetes mellitus. Diabetes 1997 6suppl 2:20-23. 8. Leonhardt W Hanefeld M Lattke P Jarob W. Vitamin E Mangel und Oxidier• barkeit der Low-Density-Lipoproteine bei Typ I und Typ II Diabetes: Einfiuj der Qualilat der Stoffwechselkontrolle. Diabetes 1997 6suppl 2:24-28. 9. Simon-Schnass I Rosak C Tritschler HJ Roesen P. Alpha-Tocopherolaufnahme und -zufuhr bei Typ 11 Diabetikern. Diabetes 1997 6suppl 2:16-19. 10. Maxwell SR Thomason H Sandler D Leguen C Baxter MA Thorpe GH Jones AF Barnett AH. Antioxidant status in patients with uncomplicated insulin• dependent and non-insulin-dependent diabetes mellitus. Eur J Clin Invest 1997 27:484-490. 11. Kashiwagi A Asahina T Ikebuchi M Tanaka Y Takagi Y Nishio Y Kikkawa R Shigeta Y. Abnormal glutathione metabolism and increased cytotoxicity caused by H202 in human umbilical vein endothelial cells cultured in high glu• cose. Diabetologia 1994 37:264-269. 12. Bellomo G. Maggi E Palladini G Perugini Seccia M. Oxidation of low density lipoproteins and vitamin E status in non insulin dependent diabetes rnellitus NIDDM. Diabetes 1997 6suppl 2: 29-33. 13. Minor RL Myers PR Guerra R Bates JN Harrison DG. Diet-induced athero• sclerosis increases the release of vascular relaxing factor. J Clin Invest 1990 86:2109-2116. 14. Ohara Y Peterson TE Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 1993 91 :2546-2551.

slide 59:

Oxidative Stress in Diabetes 29 15. Harrison DG. Endothelial function and oxidant stress. Clin Cardiol 1997 20suppl 2:Il-11-II-l 7. 16. Riemersma RA Wood DA Macintyre CC Elton RA Gey KF Oliver MF. Risk of angina pectoris and plasma concentrations of vitamins A C and E and caro• tene. Lancet 1991 337: 1-5. 17. Gey KF Puska P Jordan P Moser UK. Inverse correlation between plasma vita• min E and mortality from ischaemic heart disease in crossculturalepidemiology. Am J Clin Nutr 1991 53:326S-334S. 18. Stampfer MJ Hennekens CH Marrison JE Colditz GA Rosner B Willet WC. Vitamin E consumption and the risk of coronary disease in woman. N Engl J Med 1993 328:1444-1449. 19. Rimm EB Stampfer MJ Ascherio A Giovannuci E Colditz GA Willet WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993 328:1450-1456. 20. Stephens NG Parsons A Schofield PM Kelly F Cheeseman K Mitchinson M Brown Ml. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study CHAOS. Lancet 1996 347:781- 786. 21. Hodis HN Mack WJ LaBree L Cashin-Hemphill L Sevanian A Johnson R Azen SP. Serial coronary angiographic evidence that antioxidant vitamin intake reduced progression of coronary artery atherosclerosis. JAMA 1995 273: I 849- 1854. 22. DeMaio SJ King SB Lembo NJ Roubin GS Heam JA Bliagavan HN Sgoutas DS. Vitamin E supplementation plasma lipids and incidence of restenosis after percutaneous transluminal coronary angioplasty PTCA. J Am Coll Nutr 1992 11:68-73. 23. Rosen P Du XL Tschope D. Role of oxygen derived radicals for vascular dys• function in the diabetic heart: prevention by o-tocopherol Mol Cell Biol 1998 188: I 03-111. 24. Roesen P Tchoepe D. Vitamin E and diabetes. Fat Sci Technol 1991 11 :425- 431. 25. Lyons TJ. Oxidised low density lipoproteins: a role in the pathogenesis of athero• sclerosis in diabetes Diabetes Med 1991 8:411-419. 26. Pieper GM Gross GJ. Oxygen free radicals abolish endothelium dependent re• laxation in diabetic rat aorta. Am J Phyiol 1988 255:H825-H833. 27. Tesfamariam B. Free radicals in diabetic endothelial cell dysfunction. Free Rad Biol Med 1994 16:383-391. 28. Cohen RA. Dysfunction of vascular endothelium in diabetes mellitus. Circulation 1993 87suppl V:V67-V76. 29. Rosen P Ballhausen Th Bloch W Addicks K. Endothelial relaxation is disturbed in the diabetic rat by oxidative stress: the influence of tocopherol as antioxidant. Diabetologia 1995 38:1157-1168. 30. Falk E Shah PK Fuster V. Coronary plaque disruption. Circulation 1995 92: 657-671.

slide 60:

30 Rosenet al. 31. Constantinides P. Plaque hemorrhages their genesis and therir role in supra• plaque thrombosis and atherogenesis. In: Glagov S Newman WPI Schaffer SA eds. Pathobiology of the Human Atherosclerotic Plaque. New York: Springer• Verlag 1989:392-412. 32. Huie RE Padmaja S. The reaction of NO with superoxide. Free Rad Res Com• mun 1993 18:195-199. 33. Beckman JS Koppenol WH. Nitric oxide superoxide and peroxynitrite: the good the bad and the ugly. Am J Physiol 1996 271:Cl424-Cl437. 34. Beckman JS Ye YZ Anderson PG Chen J Accavitti MA Tarpey MM White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe-Seyler 1994 375:81-88. 35. Graier WF Simecek S Kukovetz WR Kostner DM. High n-glucose-induced changes in endothelial Ca2+ /EDRF signalling are due to generation of superoxide anions. Diabetes 1996 45: 1386-1396. 36. Posse H Noack H Augustin W Keilhoff G Wolf G. 27-Dihydrodichloro-Huo• rescein diacetate as a fluorescent marker for peroxinitrite formation. FEBS Lett 1997 416:175-178. 37. Cross AR. Jones OTG. Enzymatic mechanisms of superoxide production. Bio• chim Biophys Acta 1991 1057:291-298. 38. Mohazzab-H KM Kaminski PM Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol 1994 266:H2568-H2572. 39. Giardino I Edelstein D Brownlee M. BCL-2 expression or antioxidants prevent hyperglycemia induced formation of intracellular advanced glycation endpro• ducts in bovine endothelial cells. J Clin Invest 1996 97: 1422-1428. 40. Yan SI Schmidt AM Anderson GM Zhang J Brett J Zou YS Pinsky D Stern D. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem 1994 269:9889- 9897. 41. Chappey 0 Dosquet C Wautier MP Wautier JL. Advanced glycation end products oxidant stress and vascular lesions. Eur J Clin Invest 1997 27:97- 108. 42. Tilton RG Kawamura T Chang KC do Y Bjercke RJ Stephan CC Brock TA Williamson JR. Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth actor. J Clin Invest 1997 99: 2192-2202. 43. Williamson JR Chang K Frangos M. Hasan KS ldo Y Kawamuea T. Nyen• gaaard JR van den Enden M Kilo C Tilton RG. Hyperglycernic pseudohypoxia and diabetic complications. Diabetes 1993 42:1497-1505. 44. Brand K Page S Rogler G Bartch A Brandl R Knuechel R Page M Kaltsch• midt C Baeuerle PA Neumeier D. Activated transcription factor-kappa B is present in atherosclerosis lesion. J Clin Invest 1996 97: 1715-1722. 45. Lenardo MJ Baltimore D. NF-KB: a pleiotropic mediator of inducible and tissue• specific gene control. Cell 1989 58:227-229.

slide 61:

Oxidative Stress in Diabetes 31 46. Baeuerle PA. The inducible transcription factor NF-KB: regulation by distinct protein subunits. Biochim Biophys Acta 1991 1972:63-80. 47. Powis G Gasdaska JR Baker A. Redox signaling and the control of cell growth and death. In: Sics H ed. Antioxidants in Disease: Mechanisms and Therapy. New York: Academic Press 1997:329-359. 48. Bierhaus A Chevion S Chevion M Hofmann M Quehenberger P Illmer T Luther T Berentshtein E Tritschler H Miiller M Wahl P Ziegler R. Nawroth P. Advanced glycation end product-induced activation of NF-KB is suppressed by ce-lipoicacid in cultured endothelial cells. Diabetes 1997 46:1481-1490. 49. Du XL Stockklauser-Farber K Rosen P. Generation of reactive oxygen interme• diates activation of NF-KB and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide Free Rad Res Med Biol in press. 50. Du X Guang-Zhi Sui Stockklauser K Wei J Zink S. Schwippert B Qi-Xia Wu Tschope D Rosen P. Induction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species. Diabetologia 1998 41 :249-256. 51. Klein R. Retinopathy and other ocular complications in diabetes. In: Porte D Sherwin RS eds. Ellenberg Rifkins Diabetes Mellitus. Stamford: Appleton Lange 1997:931-970. 52. DeFronzo RA. Diabetic renal disease. In: Porte D Sherwin RS eds. Ellenberg Rifkins Diabetes Mellitus Stamford: Appleton Lange 1997:971-1008. 53. Hogg N Darley-Usmar VM Wilson MT Moncada S. The oxidation of a• tocopherol in human low density lipoproteins by the simultaneous generation of superoxide amd nitric oxide. FEBS Lett 1993 326: 199-203. 54. Rajagopalan S Meng XP Ramasamy S Harrison DG Galis ZS. Reactive oxy• gen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. J Clin Invest 1996 98:2572-2579.

slide 62:

This Page Intentionally Left Blank

slide 63:

3 Oxidative Stress Markers in Human Disease: Application to Diabetes and to Evaluation of the Effects of Antioxidants To Get Rid Of Diabetes Permanently Click Here Barry Halliwell National University of Singapore Singapore The biomedical literature contains multiple claims that "free radicals" and other "reactive species" are involved in different human diseases. They have been implicated in over 100 disorders ranging from rheumatoid arthritis hem• orrhagic shock and ulcerative colitis to gastrointestinal damage by Helico• bacter pylori and acquired immunodeficiency syndrome reviewed in Refs. 1-4. Indeed their importance in diabetes is widely proposed 56. This wide range of disorders implies that free radicals are not something esoteric but that their increased formation accompanies tissue injury in most or all human diseases 1-8 for the reasons summarized in Figure 1. Sometimes they make a significant contribution to the disease pathology at other times they may not 78. Reasons for such differences are summarized in Figure 2. Establish• ing the real importance of reactive oxygen nitrogen and chlorine species ROS/RNS/RCS requires specific assays for their formation and the damage that they do in vivo. The lack of such assays in the past has impeded progress in our understanding of the role played by reactive species in normal physiol• ogy and in human disease. Thus as summarized in Table 1 demonstrating

slide 64:

that reactive species are important in diabetes or indeed any other disease involves much more than a mere demonstration of their formation. Table 2 lists some of the reactive species to be considered. 33

slide 65:

34 Halliwell IN.JllRY Infection Radial ion Exogenous toxins Elevated levels of endogenous toxins e.g .glycoxidation Exercise 10 excess Freezing Trauma lschcmia/rcpcrfusion • Phagocyte recruitment and activation makes 0 -. 1201 NO. HOC • Arachidonic acid release. cnzymic peroxide formation by activation of lipoxygcnase. cyclooxygenase enzymes Decomposition of peroxides to pcroxyl/alkoxyl radicals can spread damage to other lipids/proteins • Metal ion release from storage sites Fe2· Cu stimulating conversion of HO and perhaps HOC to OH and lipid peroxide breakdown to RO/RO • Heme protein release myoglobin. hemoglobin cytochromcs. heme proteins react with peroxides to stimulate free-radical damage and if peroxide is in excess to release Fe and haem. which can decompose peroxides to RO and RO • lntcrfercnce with antioxidant defence systems. cg GSH loss from cells • Conversion ofxamhinc dchydrogcnase to oxidase in certain tissues. possible release of oxidase from damaged cells to cause systemic damage. Increased hypoxanthine levels due to disrupted energy metabolism • Mitochondrial damage. increased leakage of electrons to form O. •. H.O. • Raised intracellular a1·. stimulating calpains c·a·- -dependent nuclcases and Ca1 /calmodulin-depcndent nitric oxide synthase giving more NO and increased risk of0\00- formation. -- OXIDATIVE STRESS Figure 1 Some reasons why tissue injury causes oxidative stress. I. OXIDATIVE STRESS IN DISEASE: DOES IT MATTER The term oxidative stress is widely used in the literature but rarely defined. In essence it refers to the situation of a serious imbalance between production of ROS/RNS/RCS and antioxidant defense. Sies who introduced the term from the title of the book he edited in 1985 introduced a somewhat vague definition in 1991 in the introduction to the second edition 9 as a disturbance in the pro-oxidant-antioxidant balance in favor of the former leading to poten• tial damage. In principle oxidative stress can result from two mechanisms: I. Diminished antioxidants for example mutations affecting antioxi• dant defense enzymes such as CuZnSOD MnSOD and glutathione peroxi• dase or toxins that deplete such defenses. For example many xenobiotics are metabolized by conjugation with GSH high doses can deplete GSH and cause

slide 66:

f Oxidative Stress Markers in Diabetes 35 I Tissue injury l 1 Induction o antioxidant and other defense systems l Neutralization of Late stage in tissue injury accompanying cell death \ Early stage in tissue injury antioxidant defense nduction i oxidative stress \ Necrotic death of some cells spreads injury to others e.g. by releasing Fe/Cu/heme proteins inadequate or nonexi stent No significant contribution to disease pathology Aggravates disease Amenable to antioxidant intervention Figure 2 Why reactive species may be important or not important in human disease.

slide 67:

36 Halliwell Table 1 Criteria for Implicating Reactive Oxygen/Nitrogen/Chlorine Species or Any Other Agent as a Significant Mechanism of Tissue Injury in Human Disease I. The agent should always be present at the site of injury. 2. Its time course of formation should be consistent with the time course of tissue injury. 3. Direct application of the agent to the tissue at concentrations within the range found in vivo should reproduce most or all of the damage observed. 4. Removing the agent or inhibiting its formation should diminish the injury to an extent related to the degree of removal of the agent or inhibition of its formation. oxidative stress even if the xenobiotic is not itself a generator of ROS or RNS. Depletions of dietary antioxidants and other essential dietary constituents can also lead to oxidative stress. 2. Increased production of ROS/RNS/RCS for example by exposure to elevated 02 the presence of toxins that are themselves reactive species e.g. N02 or are metabolized to generate ROS/RNS/RCS or excessive activation of "natural" ROS/RNS/RCS-producing systems e.g. inappropriate activa• tion of phagocytic cells in chronic inflammatory diseases such as rheumatoid arthritis and ulcerative colitis. Mechanism 2 is usually thought to be more relevant to human diseases and is frequently the target of attempted therapeutic intervention but rarely is much attention paid to the antioxidant nutritional status of sick patients e.g. Ref. 10. For example calculations show that diabetic patients on fat-restricted diets may sometimes have a suboptimal intake of vitamin E I 1 . Prolonged oxidative stress can lead to depletion of essential antioxidants. For example subnormal plasma ascorbate levels are well known in diabetics 12 13. There are conflicting views on whether diabetic patients show depleted plasma vita• min E levels but data of Nourooz-Zadeh et al. 14 show a clear decrease in lipid standardized o-tocopherol levels in diabetic patients Table 3 although there is considerable variability between patients as indicated by the ranges in Table 3. It is possible that variations in dietary intake can account for some of the different results reported in the literature. In principle the onset of oxidative stress can result in adaptation tissue injury or cell death. Adaptation most often by upregulation of defense sys• tems may completely protect against damage protect against damage but not completely or "overprotect" for example the cell is then resistant to higher levels of oxidative stress imposed subsequently. As an example of not com• pletely protecting against damage if adult rats are gradually acclimatized to

slide 68:

Oxidative Stress Markers in Diabetes 37 Table 2 Reactive Species Radicals Nonradicals ROS Superoxide 02·• Hydroxyl OH" Peroxyl RO Alkoxyl RO" Hydroperoxyl HO RNS Nitric oxide nitrogen monoxide NO" Nitrogen dioxide NO RCS Atomic chlorine Cl" Hydrogen peroxide H202 Hypochlorous acid HOC Hypobromous acid HOBr Ozone 03 Singlet oxygen 1g Nitrous acid HN02 Nitrosyl cation No+ Nitroxyl anion No• Dinitrogen tetroxide N204 Dinitrogen trioxide N203 Peroxynitrite ONoo• Peroxynitrous acid ONOOH Nitronium nitryl cation N02 + e.g. as nitryl chloride N02Cl Alkyl peroxynitrites ROONO Hypochlorous acid HOC Chlorine Cl2 Nitronium nitryl chloride N02Cl ROS is a collective term that includes both oxygen radicals and certain nonradicals that are oxidiz• ing agents and/or are easily converted into radicals HOCI 03 ONoo-. 102 H202. RNS is also a collective term including nitric oxide and nitrogen dioxide radicals and such nonradicals as HNO and N204• ONoo- is often classified as both an RNS and an ROS. "Reactive" is not always an appropriate term: H02 NO" and o- react quickly with few molecules whereas Off reacts quickly with almost everything. RO RO" HOCI No· ONoo- and 01 have intermediate reactivities. HOCI and N02CI can also be classified as "reactive chlorine species" HOBr as a "reactive bromine species." elevated 02 they can tolerate pure 02 for much longer than control rats appar• ently due to increased synthesis of antioxidant defense enzymes and of GSH in the lung. However the damage is merely slowed not prevented 15. As an example of overprotection treatment of Escherichia coli with low levels of H202 increases transcription of genes regulated by the oxyR protein and renders the bacteria resistant to higher H202 levels 16. However few exam• ples of this type of "overadaptation" have been reported in animals. Oxidative stress can cause tissue injury to all molecular targets: DNA proteins and lipids lipid peroxidation. Often it is not clear which is the first

slide 69:

38 Halliwell Table 3 Parameters of Oxidative Stress in Healthy and Diabetic NIDDM Subjects Variables Healthy NIDDM /1 Total cholesterol 5.0 :::: I. I 6.0 :::: 1.3 0.002 mmol/L Fasting glucose mmol/L 4.9 :::: 0.4 12.1 :::: 5.1 0.0005 HbA1 11.0 :::: 2.4 ROOH µmol/L 4.1 :::: 2.2 9.4 :::: 3.3 0.0005 n-Tocopherol 23.8 :::: 8.3 10.6-47.0 19.6 :::: 7.5 8.6-44.3 0.05 umol/L a-Tocopherol/ 5.1 :::: 2.3 1.9-13.0 3.3 :::: 1.0 1.5-6.2 0.0005 cholesterol µmol/L/11111101/L ROOH/a- 0.9 :::: 0.6 0.1-2.7 3.2 :::: 1.6 0.7-8.3 0.0005 tocopherol/ cholesterol Values arc means :t SD with ranges in parentheses. Measured by FOX assay. Source: Ref. 14. point of attack because injury mechanisms are interrelated in a complex way l 17. Indeed depending on the tissue under study and the type of reactive species causing the insult the primary cellular target of oxidative stress can vary 18. For example DNA is an important early target of damage when H202 is added to many mammalian cells increased DNA damage occurs be• fore detectable lipid peroxidation or oxidative protein damage 19. Cell death can result from multiple different insults 20. Excessive acti• vation of poly ADP ribose polymerase can so deplete intracellular NAO+ I NADH levels that the cell cannot make ATP and dies. This effect has some• times been called a suicide response because DNA repair is not completely efficient a cell with extensively damaged DNA may "commit suicide" to avoid the risk of becoming an initiated cell. Cells can die from rupture of membrane blebs occurring as a result of uncontrolled increases in intracellular "free" Ca2+ concentrations 20. Cell death can be described by essentially two mechanisms necrosis and apoptosis both can result from oxidative stress 20. It is increasingly realized however that cell death can sometimes have features of both pro• cesses. In essence during necrotic cell death the cell swells and ruptures

slide 70:

Oxidative Stress Markers in Diabetes 39 releasing its contents into the surrounding area and affecting adjacent cells. Contents can include antioxidants such as catalase or GSH and pro-oxidants such as copper and iron ions Fig. I. Hence even if a cell dies by mechanisms not involving oxidative stress necrotic cell death can impose oxidative stress on the surrounding tissues Figs. 1 and 2. In apoptosis the cells own intrinsic "suicide mechanism" is activated apoptosing cells do not release their con• tents and so apoptosis does not in general cause disruption to surrounding cells. Apoptotic cell death may be accelerated by oxidative stress and added antioxidants often delay or prevent apoptosis induced by a range of insults 20- 22. The caspases essential to apoptosis have active site cysteine residues and so they can be inhibited by ROS/RNS/RCS at high levels 2324. Hence oxidative stress can induce apoptosis but high levels of such stress can halt the apoptotic process and cause cells to die by necrosis 23. II. NEED FOR "BIOMARKERS" To assess the importance of oxidative damage in human disease accurate methods to measure it are essential. Before clinical trials of putative "antioxi• dant agents take place it is important to establish whether or not the planned treatment really can decrease oxidative damage in vivo. In principle one can measure markers of oxidative damage in humans and examine how they are affected by intake of putative therapeutic antioxidants. The optimal dosage could then be determined. The same approach can be used to show not only that an antioxidant drug really is acting as an antioxidant in vivo but also to study nutritional antioxidants 25. It is important to assess all major molecular targets of damage by ROS/RNS/RCS DNA proteins lipids because an anti• oxidant that protects one target may fail to protect or even exacerbate injury to another 25. Hence measurements of oxidative damage must accompany clinical tests of the effects of antioxidants Fig. 3 to show that they actually did or did not affect the ROS/RNS/RCS. Ill. WHAT BIOMARKERS ARE AVAILABLE A. DNA Reactive species-mediated DNA damage can lead to cell death and even worse might produce initiated cells and thus facilitate cancer development 26-28. The chemistry of DNA damage by several reactive species has been well characterized in vitro 29-38 although further studies are needed with

slide 71:

EJ B 40 Halliwell SPECIFIC MARKERS show that oxidative dama e has occurred peroxides oxysterols chlorinated/nitrated lipids E oxidized bases in cells and urine LOOH MDA-HPLC other aldehydes isoprostanes nitrated/deaminated bases in cells and urine aldehyde-base adducts in cells and urine -SH oxidation carbonyls aldehyde adducts oxidized tyr trp his met lys leu ileu val nitrated/chlorinated tyrosine trp protein peroxides/hydroxides ANTIOXIDANT DEPLETION Does not prove oxidative damage only that the defence system is working Total AOX potential multiple methods e.g. TRAP FRAP ABTS· ORAC Depletion of specific AOX Measurement of AOX-derived species Ie.g. ascorbate radical urate oxidation products INDUCTION OF AOX ENZYMES Does not prove oxidative damage only that the defence system is working ROS/RNS/RCS TRAPPING ROS/RNS/RCS formation does not imply their importance. If they are important and the traps are efficient the traps should be protective. For example L-phe used as a trap for OH protected against myocardial stunning at the same time as it detected OH 251 PBN/other spin traps Aromatic probes e.g. salicylate phenylalanine other detectors Figure 3 Some biomarkers of oxidative stress used to study human disease. Based on Ref. 25.

slide 72:

Oxidative Stress Markers in Diabetes 41 RO/ RO" and 03• Nitric oxide NO" probably via products derived from it N02" HN02 ONoo- N203 etc. can cause nitrosation and deamination of amino groups on DNA bases 343940. Whereas 0/- and H202 appear not to react with DNA bases OH" gener• ates a multiplicity of products from all four DNA bases 31 . By contrast 102 appears selective for attack upon guanine 3233. The most common base lesion and the one most often measured as an index of oxidative DNA dam• age is the nucleoside 8-hydroxy-2-deoxyguanosine 80HdG 38. Measure• ment of 80HdG alone can give misleading results under certain circumstances reviewed in Ref. 40 although it is still widely used. In principle there are two types of measurement of oxidative DNA damage. Steady-state damage can be measured when DNA is isolated from human cells and tissues and analyzed for base damage products it presumably reflects the balance between damage and DNA repair. Hence a rise in steady-state oxidative DNA damage could be due to increased damage and/or decreased repair. It is worth men• tioning that the measurement of baseline levels of oxidatively modified DNA bases does not provide information as to whether this damage is in active genes or quiescent DNA. However it is important also to have an index of total DNA damage in the human body i.e. the "input" side of the steady-state equation. The most common approach has been to assess the "output" side i.e. trying to estimate the rate of repair of oxidized DNA. This is usually achieved by measuring urinary excretion of DNA base damage products. Several DNA base damage products are excreted in human urine including 80HdG 8-hydroxyguanine 8-hydroxyadenine and 7-methyl-8-hydroxy-guanine 394142 but the one most exploited is 80HdG usually measured by a method involving high• performance liquid chromatography HPLC with electrochemical detection 38. For example in one study of 169 humans the average 80HdG excretion was 200-300 pmol/kg per 24 h corresponding to 140-200 oxidative modifica• tions of guanine per cell per day 4243. Furthermore smokers excrete 50 more 80HdG than nonsmokers on average suggestive of a mean 50 in• creased rate of oxidative DNA damage from smoking 42. Gas chromatogra• phy-mass spectroscopy GC-MS has also been used to measure 80HdG in urine and the limit of detection was 1.8 pmol corresponding to a level of 80HdG in urine of 35 nM 44. The validity of these urinary measurements of oxidative DNA damage must be considered. The level of 80HdG in urine is presumably unaffected by the diet because nucleosides are thought not to be absorbed from the gut. However this question and the question as to whether any 80HdG is metabo• lized to other products in humans has not been rigorously addressed in the

slide 73:

42 Halliwell literature. In addition it is possible that some or all of the 80HdG excreted in urine may arise not from DNA but from dGTP in the DNA precursor pool of nucleotides. An enzyme has been described that hydrolyzes dGTP containing oxidized guanine presumably to prevent its incorporation into DNA 45. J. Relevance to Diabetes Both steady-state levels of 80HdG in blood monocytes 46 and urinary excre• tion of 80HdG 47 have been reported as elevated in diabetic patients. Gas chromatography/mass spectrometry has recently shown elevated levels of a wide range of base oxidation products in DNA from white blood cells of diabetic patients. The pattern of base damage was diagnostic of increased OH formation in vivo 48. B. Lipid Peroxidation Lipid peroxidation is important in vivo for several reasons in particular be• cause it contributes to the development of atherosclerosis 49-51 a process known to be accelerated in diabetic patients 6. Lipid peroxides and other end products of the peroxidation process may be toxic to vascular endothelium in diabetics 652. Many assays are available to measure lipid peroxidation but the simpler ones such as the TBA test and diene conjugation are notori• ously unreliable when applied to human tissues and body fluids reviewed in Ref. 53 although the TBA test can be improved by linking it to HPLC and adding antioxidants with the TBA reagents to prevent peroxidation during the assay procedure 54. Levels of lipid peroxides in human plasma as measured by reliable analytical methods 54-59 seem to be low usually 0.1 µM. Human body fluids also contain low levels of Frisoprostanes compounds isomeric to prostaglandins that are thought to arise by free radical oxidation of phospholipids containing arachidonic acid 60. It has been suggested that one of the Fj-isoprostanes 8-epiPGF2o: can be generated by cyclooxygenase in human platelets 61 although this does not appear to be a significant con• tributor to total body production of 8-epiPGF2o: 62. Isoprostanes appear to exist in human plasma largely esterified to phospholipids rather than "free" and sensitive assays to measure them have been described 60-65. Families of F3- and F4-isoprostanes derived from eicosapentaenoic and docosahexae• noic acids respectively have recently been described 66. J. Relevance to Diabetes There seems to be general agreement that lipid peroxidation is elevated in diabetes although its relationship to disease progression is uncertain. Some

slide 74:

Oxidative Stress Markers in Diabetes 43 investigators have reported an association between plasma or serum TBA• reactive substances TBARS or diene conjugates and diabetic complications whereas others have not 67- 70. However TBARS and diene conjugation assays should be interpreted with caution 53. MacRury et al. 70 compared different methods conjugated dienes TBARS and chemiluminescence of assessing free radical activities in diabetic subjects. In each case diabetes was associated with elevated levels of different indirect measurements of lipid peroxidation. However they did not find a relationship between diabetic com• plications and plasma measures of oxidative stress. More convincingly ele• vated levels of plasma 8-epi PGFa have been reported in diabetics although its association with disease progression was not discussed 71 . Another study using the ferrous oxidation with xylenol orange FOX assay to measure lipid peroxides found higher lipid-standardized peroxides in plasma from diabetic patients Table 3. This elevated level was not influenced by sex age smoking habit or diabetic complications and was taken to suggest that the elevated levels of plasma hydroperoxide in patients are associated with the diabetes itself rather than consequent tissue injury e.g. nephropathy neuropathy. The reliability of the FOX assay as a measure of lipid peroxides in human plasma remains to be established however. As Table 3 indicates "basal" peroxide levels in plasma from healthy subjects seem higher than the 0.1 µM or less measured by more chemically robust assays see above. 2. Measuring "Total" Lipid Peroxidation in the Human Body Peroxide levels in body fluids or tissues represent a balance between peroxide formation and peroxide metabolism or decomposition i.e. they are essentially a "steady-state" measurement. Can some measure of total body lipid peroxi• dation be obtained This has most often been attempted by measuring hydrocarbon gases ethane pentane in exhaled air 72 and urinary excretion of MDA more properly called TBA-reactive material 73. The latter assay is probably con• founded by diet: Most lipid-related TBARS appearing in urine seems to arise from lipid peroxides or aldehydes in ingested food which are presumably largely generated during cooking 7475. For example Brown et al. 75 showed that a diet rich in cooked meat promoted urinary TBARS excretion to an extent depending on the temperature at which the meat was cooked. Hence urinary TBARS is not a suitable assay to assess whole body lipid peroxidation although it could theoretically be used to look at effects of antioxidant supple• mentation of people on a controlled diet 74. In any case HPLC must be used to separate the real TBA2MDA adduct much TBARS in urine is not even lipid derived 76 or arises from aldehydes other than MDA 77.

slide 75:

44 Halliwell Breath excretion of ethane and pentane minor end products of lipid peroxidation is difficult but not impossible 78 to measure in humans be• cause of the problem of contamination of the atmosphere by these gases lead• ing to their partitioning into body fat stores 79. Particular problems with pentane include the fact that it is metabolized by cytochromes P450 7880 and that GC columns frequently used to separate "pentane" for measurement can fail to separate it from isoprene a hydrocarbon also excreted in exhaled air 798182. Indeed the levels of excreted real pentane seem close to zero in most humans 798182. Perhaps further evaluation of the technique of hydrocarbon gas exhalation should focus on ethane 72 but in general the technique would seem difficult to use reliably in human studies except where subjects are confined to controlled environments breathing air of minimal hydrocarbon content. The possible effect of dietary changes on hydrocarbon gas production by gut flora is another potential confounding factor. lsoprostanes and their metabolites can be measured in human urine 606264 and this may prove to be a valuable assay of whole body lipid peroxidation if a confounding effect of diet can be ruled out. C. Protein Damage Damage to proteins may be important in vivo both in its own right affecting the function of receptors enzymes transport proteins etc. and perhaps generating new antigens that provoke immune responses 83 and also because it can con• tribute to secondary damage to other biomolecules e.g. by inactivation of anti• oxidant defense enzymes or repair enzymes. Attack of various RNS ONOo- N02" N02Cl and possibly some other species upon tyrosine both free and within proteins leads to production of 3-nitrotyrosine which can be measured immunologically or by HPLC or GC-MS techniques reviewed in Refs. 84 and 85. RCS can produce chlorinated products e.g. 3-chlorotyrosine and these have been detected in human atherosclerotic lesions 50. The chemical reactions resulting from attack of ROS/RNS/RCS on pro• teins are complex. Free radical attack can generate protein peroxides which may decompose to generate free radicals reviewed in Ref. 86. Several assays to measure damage to specific amino acid residues in proteins by ROS/RNS/ RCS have been developed. They include assays of L-DOPA produced by tyrosine hydroxylation valine hydroxides produced from valine hydroperox• ides tryptophan hydroxylation and ring-opening products 8-oxohistidine di• tyrosine and ortho- and meta-tyrosines products of attack of Off upon phe• nylalanine 86. The levels of any one or preferably of more than one of these products in proteins could in principle be used to assess the balance

slide 76:

Oxidative Stress Markers in Diabetes 45 between oxidative protein damage and the removal of damaged proteins. The only products exploited to date have been the hydroxylated phenylalanines. For example levels of ortho- tyrosine and dityrosine in human lens proteins have been reported in relation to age 87. These products were also measured in hair from "Alpine Man" Homo tirolensis 88. 1. Carbonyl Assay More use has been made of the carbonyl assay a general assay of oxidative protein damage 89 to assess steady-state levels of such damage in human tissues and body fluids. The carbonyl assay is based on the ability of several ROS to attack amino acid residues in proteins particularly histidine arginine lysine and proline to produce carbonyl functions that can be measured after reaction with 24-dinitrophenylhydrazine 8990. The carbonyl assay has be• come widely used and many laboratories have developed individual protocols for it. Sometimes the assay procedures used in a particular laboratory are not specified precisely in published papers and often differ from those used origi• nally by the group of Stadtman et al. This point is important because there is a considerable variation in the "baseline" levels of protein carbonyls in certain human tissues depending on how the assay is performed reviewed in Ref. 91. By contrast most groups seem to obtain broadly comparable values for protein carbonyls in human plasma of l nmol/mg protein so plasma protein carbonyls should be a useful assay of oxidative protein damage. However protein glycation and covalent binding of certain aldehyde end products of lipid peroxidation to proteins can also generate carbonyls 89. 2. Relevance to Diabetes Glycoxidation seems to play an important role in the vascular endothelial dys• function detected in diabetic patients 9293 and perhaps in the nephropathy 94 . Elevated glucose may cause increased generation of o. by endothelium antagonizing the action of NO and perhaps forming peroxynitrite ONoo• 9293. All these effects should be amenable to treatment by appropriate anti• oxidants 92. IV. CONCLUSION Despite the arguments that can be raised about the validity of some individual biomarkers the sum of evidence from biomarkers reporting oxidative damage shows that such damage is increased in diabetes affecting DNA lipids and

slide 77:

46 Halliwell proteins glycoxidation supporting the concept of increased oxidative stress in diabetes. Indeed the newly introduced drug troglitazone may exert some of its protective effects by its antioxidant capacity 95. Further work is re• quired using modern biomarkers to evaluate the extent to which agents bene• ficial in the treatment of diabetes including lipoic acid see other chapters in this volume act by suppressing oxidative stress. Another exciting area is the prevention of the teratogenic effects of hyperglycemia: Studies in rats have shown that vitamin E 96 and overexpression of CuZnSOD 97 can be bene• ficial. REFERENCES I. Halliwell B Gutteridge JMC. Free Radicals in Biology and Medicine. 3rd ed. Oxford England: Clarendon Press 1999. 2. Halliwell 8 Cross CE. Reactive oxygen species antioxidants and acquired im• munodeficiency syndrome. Arch Intern Med 1991 157:29-31. 3. Parks DA. Oxygen radicals: mediators of gastrointestinal pathophysiology. Gut 1989 30:293-298. 4. Southorn PA. Free radicals in medicine. II. Involvement in human disease. Mayo Clin Proc 1988 63:390-408. 5. Jones AF Winkles JW Jennings PE ct al. Serum antioxidant activity in diabetes mellitus. Diabetes Res 1988 7:89-92. 6. Tesfamariam 8. Free radicals in diabetic endothelial cell dysfunction. Free Rad Biol Med 1994 16:383-391. 7. Halliwell 8 Gutteridge JMC. Lipid peroxidation oxygen radicals cell damage and antioxidant therapy. Lancet 1984 1:1396-1398. 8. Halliwell 8 Cross CE Gutteridge JMC. Free radicals antioxidants and human disease: where are we now J Lab Clin Med 1992 119:598-620. 9. Sies H. ed. Oxidative Stress: Oxidants and Antioxidants. New York: Academic Press 1991. 10. Scorah CJ Downing C Piripitsi A Gallivan L Al-Hazaa AH Sanderson MJ Bodenham A. Total vitamin C ascorbic acid and dehydroascorbic acid con• centrations in plasma of critically ill patients. Am J Clin Nutr 1996 63:760- 765. 11. Rosak C Sirnon-Schnass I Rosen P Tritscher HJ Halliwell 8. Possible dietary induced u-tocopherol deficiency in type II diabetic patients. In preparation. 12. Sinclair AJ Lunec J. Free radicals oxidative stress and diabetes mellitus. In: Winyard P Blake DR eds. lmmunopharmacology of Free Radical Species. Lon• don: Academic Press 1995:183-198. 13. Maxwell SRJ Thomason H Sandler D Lcguen C Baxter MA Thorpe GHG Jones AF Barnett AH. Antioxidant status in patients with uncomplicated insulin-

slide 78:

Oxidative Stress Markers in Diabetes 47 dependent and non-insulin-dependent diabetes mellitus. Eur J Clin Invest 1997 27:484-490. 14. Nourooz-Zadeh J Rahimi A Tajaddini-Sarmadi J Tritschler H Rosen P Halli• well B Betteridge DJ. Relationship between plasma measures of oxidative stress and metabolic control in NlDDM. Diabetologia 1997 40:647-653. 15. Kinnula VL Crapo JD Raivio KO. Generation and disposal of reactive oxygen metabolites in the lung. Lab Invest 1995 73:3-19. 16. Kullik I Stevens J Teledano MB Storz G. Mutational analysis of the redox• sensitive transcriptional regulator oxyR: regions important for DNA binding and multimerization. J Bacteriol 1995 177: 1285-1291. 17. Hyslop PA Hinshaw DB Halsey WA Jr et al. Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J Biol Chem 1988 263:1665-1675. 18. Halliwell B. Antioxidant characterization: methodology and mechanisms. Bio• chem Pharmacol 1995 49: 1341-1348. 19. Spencer JPE Jenner A Chime K Aruoma OJ Cross CE Wu R Halliwell B. DNA strand breakage and base modification induced by hydrogen peroxide treat• ment of human respiratory tract epithelial cells. FEBS Lett 1995 374:233-236. 20. Nicotera P. Orrenius S. Molecular mechanisms of toxic cell death: an overview. Methods Toxicol 1994 18:23-28. 21. Kohno T Yamada Y Hata T Mori H Yamamura M Tomonaga M Urata Y Goto S Kondo T. Relation of oxidative stress and glutathione synthesis to 095 Fas/ APO- I mediated apoptosis of adult T cell leukemia cells. J lmmunol 1996 156:4722-4728. 22. Johnson TM Yu ZX Ferrans VJ Lowenstein RA Finkel T. Reactive oxygen species are downstream mediators of p53-dependent apoptosis. Proc Natl Acad Sci USA 1996 93:11848-11852. 23. Hampton MB Orrenius S. Dual regulation of caspase activity by hydrogen per• oxide: implications for apotosis. FEBS Lett 1997 414:552-556. 24. Mohr S Zech B Lapetina EG. Brune B. Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide. Biochem Biophys Res Commun 1997 238: 387-391. 25. Halliwell B. Oxidative stress. nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Rad Res 1996 25:57-74. 26. Wiseman H Halliwell B. Carcinogenic antioxidants: diethylstilboestrol hexoes• trol and 17a-ethynyl-oestradiol. FEBS Lett 1993 322:159-163. 27. Totter JR. Spontaneous cancer and its possible relationship to oxygen metabo• lism. Proc Natl Acad Sci USA 1980: 77:1763-1767. 28. Ames BN Shigenaga MK Hagen TM. Oxidants antioxidants and the degenera• tive diseases of aging. Proc Natl Acad Sci USA 1993 90:7915- 7922. 29. von Sonntag C. The Chemical Basis of Radiation Biology. London: Taylor and Francis 1987.

slide 79:

48 Halliwell 30. Steenken S. Purine bases nucleosides and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and e- and OH· adducts. Chem Rev 1989 89:503-520. 31. Dizdaroglu M. Chemistry of free radical damage to DNA and nucleoproteins. In: Halliwell B Aruoma 01 eds. DNA and Free Radicals. Chichester: Ellis Hor• wood UK 1993:19-39. 32. Epe B. DNA damage induced by photosensitization. In: Halliwell B Aruoma OI eds. DNA and Free Radicals. Chichester: Ellis Horwood UK 1993:41-65. 33. Cadet J Ravanat JL Buchko GW et al. Singlet oxygen DNA damage: chromato• graphic and mass spectrometric analysis of damage products. Methods Enzymol 1994 234:79-88. 34. Spencer JPE Wong J Jenner A Aruoma 01 Cross CE Halliwell B. Base modi• fication and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholino• sydnonimine. Chem Res Tox 1996 9:1152-1158. 35. Whiteman M Jenner A Halliwell B. Hypochlorous acid-induced base modifica• tion in isolated calf thymus DNA. Chem Res Tox 1997 10:1240-1246. 36. de Rojas-Walker T Tamir S Ji H Wishnock JS Tannenbaum SR. Nitric oxide induces oxidative damage in addition to deamination in macrophage DNA. Chem Res Toxicol 1995 8:473-477. 37. Yermilov V Rubio J Oshima H. Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal by depurination. FEBS Lett 1995 376:207-210. 38. Floyd RA Watson JJ Wong PK et al. Hydroxy-free radical adduct of deoxygua• nosine: sensitive detection and mechanisms of formation. Free Rad Res Commun 1986 1:163-172. 39. Stillwell WG Xu HX Adkins JA et al. Analysis of methylated and oxidized purines in urine by capillary gas chromatography-mass spectrometry. Chem Res Toxicol 1989 2:94-99. 40. Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reac• tive oxygen chlorine and nitrogen species: measurement mechanism and the effects of nutrition. Mutat Res 1999 443:37-52. 41. Ames BN. Endogenous oxidative DNA damage aging and cancer. Free Rad Res Commun 1989 7:121-128. 42. Loft S Vistisen K Ewertz M et al. Oxidative DNA-damage estimated by 8- hydroxydeoxyguanosine excretion in man: influence of smoking gender and body mass index. Carcinogenesis 1992 13 :2241-224 7. 43. Loft S Fischer-Nielsen A Jeding JB et al. 8-Hydroxydeoxyguanosine as a uri• nary marker of oxidative DNA damage. J Toxicol Environ Health 1993 40:391- 404. 44. Teixeira AJR Gornmers-Ampt JH van de Werken G et al. Method for the analy• sis of oxidized nucleosides by gas chromatography/mass spectrometry. Anal Biochem 1993 214:474-483. 45. Sakumi K Furuichi M Tsuzuki T et al. Cloning and expression of cDNA for

slide 80:

Oxidative Stress Markers in Diabetes 49 a human enzyme that hydrolyzes 8-oxo-dGTP a mutagenic substrate for DNA synthesis J Biol Chem 1993 268:23524-23530. 46. Dandona P Thusu K Cook S Snyder B Makowski J Armstrong D Nicotera T. Oxidative damage to DNA in diabetes mellitus. Lancet 1996 347:444-445. 47. Leinonen J Lehtimaki T Toyokuni S Okada K Tanaka T Hiai H Ochi H Laippala P Rantalaiho V Wirta 0 Pasternack A Alho H. New biomarker evi• dence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus. FEBS Lett 1997 417:150-152. 48. Rehman A Nourooz-Zadeh J Moller W et al. Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus. FEBS Lett 1999 448: 120-122. 49. Steinberg D Parthasarathy S Carew TE et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989 320:915-924. 50. Heinecke JW. Mechanisms of oxidative damage of low density lipoprotein in atherosclerosis. Curr Opin Lipidol 1997 8:268-274. 51. Esterbauer H Gebicki J Puhl H Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992 13: 341-390. 52. Pieper GM Siebeneich W. Diabetes-induced endothelial dysfunction is pre• vented by long-term treatment with the modified iron chelator hydroxyethyl starch conjugated-deferoxamine J Cardiovasc Pharm 1997 30:734-738. 53. Halliwell B Chirico S. Lipid peroxidation: its mechanism measurement and significance. Am J Clin Nutr 1993 57:715S- 725S. 54. Chirico S Smith C Marchant C et al. Lipid peroxidation in hyperlipidaemic patients. A study of plasma using an HPLC-based thiobarbituric acid test. Free Rad Res Commun 1993 19:51-57. 55. Akasaka K. Ohata A Ohrui H Meguro H. Automatic determination of hydroper• oxides of phosphatidylcholine and phosphatidylethanolamine in human plasma. J Chromatogr 1995 8665:37-43. 56. Frei B Yamamoto Y Niclas D Ames BN. Evaluation of an isoluminol chemilu• minescence assay for the detection of hydroperoxides in human blood plasma. Anal Biochem 1988 175: 120-130. 57. Holley AE Slater TF. Measurement of lipid hydroperoxides in normal human blood plasma using HPLC-chemiluminescence linked to a diode array detector for measuring conjugated dienes. Free Rad Res Commun 1991 15:51-63. 58. Wilson R Smith R Wilson P Shepherd MJ Riemersma RA. Quantitative gas chromatography-mass spectrometry isomer-specific measurement of hydroxy fatty acids in biological samples and food as a marker of lipid peroxidation Anal Biochem 1997 248:76-85. 59. Yasuda M Narita S. Simultaneous determination of phospholipid hydroperox• ides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection. J Chromatogr 1997 693:211-217.

slide 81:

50 Halliwell 60. Morrow JD Roberts LJ II. Mass spectrometry of prostanoids: Fj-isoprostanes produced by non-cyclooxygenase free radical-catalyzed mechanism. Methods Enzymol 1994 233:163-174. 61. Pratico D Lawson JA Fitzgerald GA. Cyclooxygenase-dependent formation of the isoprostane 8-epiprostaglandin F2a. J Biol Chem 1995 270:9800-9808. 62. Wang Z Ciabattoni G Creminon C et al. Immunological characterization of urinary 8-epi-prostaglandin F2a excretion in man. J Pharmacol Exp Ther 1995 275:94-100. 63. Nourooz-Zadeh J Gopaul NK Barrow S et al. Analysis of Fy-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography• mass spectrometry: development of a solid-phase extraction procedure. J Chro• matogr 1995 8667:199-208. 64. Morrow JD Frei B Longmire AW et al. Increase in circulating products of lipid peroxidation Fj-isoprostanes in smokers. N Engl J Med 1995 332: 1198-1203. 65. Gopaul NK Nourooz-Zadeh J Mallet Al Anggard EE. Formation of Fj-isopros• tanes during aortic endothelial cell-mediated oxidation of low density lipopro• tein. FEBS Lett 1994 348:297-300. 66. Nourooz-Zadeh J Liu EHC A.nggiird EE Halliwell B. Fi-isoprostanes: a novel class of prostanoids formed during peroxidation of docosahexaenoic acid DHA. Biochem Biophys Res Commun 1998 242:338-344. 67. Griesmacher A Kinder Hauser M Andert S et al. Enhanced serum levels of TSARS in diabetes mellitus. Am J Med 1995 98:469-475. 68. Sundaram RK Bhaskar A Vijayalingam S Viswanatthan M Mohan R Shanmu• gasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes with and without complications. Clin Sci 1996 90:255-260. 69. Velazques E Winocour PH Kesteven P. Alberti KGMM Laker MF. Relation of lipid peroxides to macrovascular disease in type 2 diabetes. Diabet Med 1991 8:752-758. 70. MacRury SM Gordon D Wilson R et al. A comparison of different methods of assessing free radical activity in type 2 diabetes and peripheral vascular dis• ease. Diabet Med 1993 10:331-335. 71. Gopaul NK Anggard EE Mallet Al Betteridge DJ Wolff SP Nourooz-Zadeh J. Plasma 8-epi-PGFa levels are elevated in individuals with non-insulin dependent diabetes mellitus. FEBS Lett 1995 368:225-229. 72. Burk RF Ludden TM. Exhaled alkanes as indices of in vivo lipid peroxidation. Biochem Pharmacol 1989 38: I 029-1032. 73. McGiJT LG Hadley M Draper HH. Identification of N"-acetyl-£-2-propenal lysine as a urinary metabolite of malondialdehyde. J Biol Chem 1985 260: 15427-15431. 74. Dhanakoti SN Draper HH. Response of urinary malondialdehyde to factors that stimulate lipid peroxidation in vivo. Lipids 1987 22:643-646. 75. Brown ED. Morris VC Rhodes DG et al. Urinary excretion of malondialdehyde in subjects fed meat cooked at high or low temperatures. Lipids 1995 30: 1053- 1056.

slide 82:

Oxidative Stress Markers in Diabetes 51 76. Gutteridge JMC. Tickner TR. The characterisation of thiobarbituric acid reactiv• ity in human plasma and urine. Anal Biochem 1978 91:250-257. 77. Kosugi H Kojima T Kikugawa K. Characteristics of the thiobarbituric acid reac• tivity of human urine as a possible consequence of lipid peroxidation. Lipids 1993 28:337-343. 78. Kneepkens CMF Lepage G Roy CC. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Rad Biol Med 1994 17:127- 160. 79. Springfield JR Levitt MD. Pitfalls in the use of breath pentane measurements to assess lipid peroxidation. J Lipid Res 1994 35:1497-1504. 80. Wade CR van Rij AM. In vivo lipid peroxidation in Man as measured by the respiratory excretion of ethane pentane and other low-molecular-weight hydro• carbons. Anal Biochem 1985 150:1-7. 81. Kohlmi.iller D Kochen W. Is »-pentane really an index of lipid peroxidation in humans and animals A methodological reevaluation. Anal Biochem 1993 210: 268-276. 82. Phillips M Greenberg J Sabas M. Alveolar gradient of pentane in normal human breath. Free Rad Res 1994 20:333-337. 83. Aruoma OJ Halliwell B Butler J Hoey BM. Apparent inactivation of a1-antipro• teinase by sulphur-containing radicals derived from penicillamine. Biochem Pharmacol 1989 38:4353-4357. 84. Beckman JS Chen J. lschiropoulos H. Crow JP. Oxidative chemistry of peroxy• nitrite. Methods Enzymol 1994 233:229-240. 85. Halliwell B. What nitrates tyrosine Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo FEBS Lett 1997 411:157-160. 86. Davies MJ Dean RT. Radical-Mediated Protein Oxidation. From Chemistry to Medicine. Oxford England: Oxford University Press 1997. 87. Wells-Knecht MC Huggins TG Dyer DG et al. Oxidized amino acids in lens proteins with age. Measurement of o-tyrosine and dityrosine in the aging human lens. J Biol Chem. 1993 268:12348-12352. 88. Lubec G Weninger M Anderson SR. Racemization and oxidation studies of hair protein in the Homo tirolensis. FASEB J 1994 8:1166-1169. 89. Levine RL Garland D Oliver CN Amici A Climent I Lenz AG. Ahn BW Shaltiel S Stadtman ER. Determination of carbonyl content in oxidatively modi• fied protein. Methods Enzymol 1990 186:464-487. 90. Amici A Levine RL Tsai L Stadtman ER. Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal• catalyzed oxidation reactions. J Biol Chem 1989 264:3341-3346. 91. Lyras L Shaw PJ Evans PJ Halliwell B. Oxidative damage and motor neurone disease. Difficulties in the measurement of protein carbonyls in human brain tissue. Free Rad Res 1996 24:397-406. 92. Mayhan WG. Superoxide dismutase partially restores impaired dilatation of the basilar artery during diabetes mellitus. Brain Res 1997 760:204-209. 93. Cosentino F Hishikawa K Katusic ZS Li.ischer TF. High glucose increases ni-

slide 83:

52 Halliwell tric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 1997 96:25-28. 94. Horie K Miyata T Maeda K Miyata S Sugiyama S Sakai H van Ypsersele de Strihou C Monnier YM Witztum JL Kurokawa K. Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in dia• betic renal glomerular lesions. Implications for glycoxidative stress in the patho• genesis of diabetic nephropathy. J Clin Invest 1997 100:2995-3004. 95. Cominacini L Young MMR Capriati A Garbin U Fratta Pasini A Campagnola M Davoli A Rigoni A Contessi GB Lo Cascio Y. Troglitazone increases the resistance of low density lipoprotein to oxidation in healthy volunteers. Diabeto• logia 1997 40: 1211-1218. 96. Viana M Herrera E Bonet B. Teratogenic effects of diabetes mellitus in the rat. Prevention by vitamin E. Diabetologia 1996 39: l 041-1046. 97. Hagay ZJ Weiss Y Zusman I Peled-Kamar M Reece EA Eriksson UJ Groner Y. Prevention of diabetes-associated embryopathy by overexpression of the free radical scavenger copper zinc superoxide dismutase in transgenic mouse em• bryos. Am J Obstet Gynecol 1995 173:1036-1041.

slide 84:

4 Plasma Lipid Hydroperoxide and Vitamin E Profiles in Patients with Diabetes Mellitus To Kill Diabetes Forever Click Here Jaffar Nourooz-Zadeh University College London London England Patients with non-insulin-dependent diabetes mellitus NIDDM are at in• creased risk of developing vascular and other complications. This excess risk is only partially explained by the traditional risk factors including smoking hypertension and dyslipidemia 1-3. Therefore oxidative stress has been proposed as a possible explanation for the accelerated complications in NIDDM 4- 7. A major hypothesis is that low-density lipoprotein LDL modification by oxidation or glycosylation contributes to tissue damage through cytotoxic reactions with endothelial cells or through further reactions to generate "modified" LDL that is selectively accumulated by "scavenger" receptors 8. Despite the biochemical importance of oxidative stress its measurement in vivo has been difficult 9. Common approaches to assess oxidative stress in biological fluids are measurement of lipid peroxidation products oxidatively modified DNA or protein damage and measurement of the depletion of anti• oxidants. Enhanced lipid peroxidation in diabetics has been reported using thiobar• bituric acid reactive substances TBARS as an assay I0-14. The simple TBARS assay in fact measures many substances in addition to products of lipid peroxidation and is affected by the lipid content of the sample 15. Therefore

slide 85:

53

slide 86:

s 54 Nourooz-Zadeh 0.18 0.15 E c 0 0.12 0 0.09 c ll ..Q 0 0.06 J ..Q t: 0-TPP E3 +TPP 0.03 0.00 Plasma +5-HPETE +PC-OOH Figure 1 Detection of authentic ROOHs in plasma. PC-OOH phospholipid hydro• peroxides 5-HPETE 5-hydroperoxyeicotetraenoic acid. it is unclear to what extent plasma lipoprotein peroxidation assessed by this method accounts for biological changes associated with oxidative stress. We have used the ferrous oxidation with xylenol orange FOX assay coupled with triphenylphosphine TPP to determine plasma lipid hydroper• oxide ROOH levels in health and disease 16 17. TPP is used to reduce ROOHs. This maneuver is necessary to generate a proper control for each individual plasma sample because plasma contains interfering components mainly ferric ions that are detected by xylenol orange Fig. l. Other advan• tages of the FOX2 assay over existing techniques are kinetics of the reaction are independent of the chemical structure of the ROOHs and no extraction step is normally needed because the use of the 90 methanol-25 mM H2S04 denatures proteins sufficiently allow access of the ferrous ions to available ROOHs. The coefficient of variation for individual plasma samples using this method is typically less than 5 whereas that for the interassay coefficient of variation is 10 16-19. I. DIURNAL VARIATION OF PLASMA ROOHs No information is available on the effect of diurnal variation on plasma lipid peroxidation products. Using the FOX assay we examined this issue in two subjects one woman one man aged 30 and 36 respectively under fasting

slide 87:

• -• Subject 2 18 19 20 21 22 23 24 48 52 19 ROOH and Vitamin E in Diabetes 55 10 0-0 Subject 1 2 Sampling time hours Figure 2 Diurnal change in plasma ROOHs. From Ref. 18. and nonfasting conditions I 7. As shown in Figure 2 little fluctuation around the mean ROOH value was associated with either the fasted or fed state. These data suggest that dietary input of ROOHs appears to be small by comparison with metabolic production of ROOH. II. PLASMA ROOH LEVELS IN HEALTHY AND DIABETIC SUBJECTS Mean levels of ROOH of 9.04 :::: 4.3 and 9.4 :::: 3.3 µmol/L were detected in freshly prepared plasma from type II diabetics from two different studies n 22 and 87 respectively 17 18. The corresponding levels for healthy volunteers from three different studies were 3.02 ::: 1.85 3.76 :::: 2.48 and 4.1 :::: 2.2 µmol/L n 23 21 and 41 respectively 16-18. Data spread for plasma ROOHs and ratio of ROOHs/cholesterolstandardized a-tocopherol in NIDDM and control subjects are shown in Figure 3. Clinical characteristics of NIDDM and control subjects are summarized in Table I. Similar plasma ROOH concentrations have also been reported by other investigators using the FOX2 assay 20-22. These data together provide the evidence for the reliability of the FOX2 assay for the measurement of plasma ROOHs.

slide 88:

0000 0 s .c - c: II Ill co " - t 20 0 0 15 0 00 0 000 0000 0 0000· E :I. 10 0 0 00000 00 0000 0 0 0:: 00000 00000 000 0 00 000 5 000 000 0 O----i...........1.- 12 00 000 0 ... II c. 0 . .B 9 ti "C 8 0 II 0 N 0 0 " " C 6 o. Ill co 0 ... .s 0 0 .c c o ::: 3 0 0 0 0 0:: a L 0 0 Control NIDDM Figure 3 Data spread for ROOHs top and ROOHs/cholesterol-standardized a• tocopherol bottom in control and type II diabetic subjects. 56

slide 89:

Diabetes duration yr 21-69 17-86 12.0 :t 8.3 0.0-44 Total cholesterol mmol/L 5.0 :t 1.1 1.4-6.9 6.0 :t 1.3 3.3-9.9 0.002 Triglycerides mmol/L 0.9 :t 0.5 0.3-2.5 2.8 :t 1.8 0.6-3.5 0.0005 Fasting glucose mmol/L 4.9 :t 0.4 4.2-5.8 12.1 :t 5.1 1.9-28.9 0.0005 HbAlc 5-8 11.0 :t 2.4 5.9-17.8 ROOH µM 4.1 :t 2.2 0.4-10.3 9.4 :t 3.3 2.7-16.8 0.0005 u-Tocopherol µmol/L 23.8 :t 8.3 19.6 :t 7.5 0.05 I 0.6-4 7 .0 8.6-44.3 cx.-Tocopherol/cholesterol µmol/L/ 5.1 :t 2.3 3.3 :t 1.0 0.0005 l.9-13.0 1.5-6.2 ROOH/cx.-tocopherol/cholesterol 0.9 :t 0.6 0.1-2.7 3.2 :t 1.6 0.7-8.3 0.0005 Data in brackets are ranges. Data in parentheses are normal ranges. Source: Ref. 18. ROOH and Vitamin E in Diabetes 57 Table 1 Clinical Characteristics of Healthy and NIDDM Individuals Variables Healthy NlDDM Numbers 41 87 Sex F/M 24/17 40/47 Age yr 38.2 :t 12.3 58.4 :t 14.7 p 0.0005 mmol/L Ill. PLASMA ROOH IN DIABETICS WITH AND WITHOUT COMPLICATIONS The association between diabetic complications and plasma lipid peroxidation as measured by nonspecific techniques e.g. thiobarbituric acid- or ultraviolet• absorbing diene conjugates has been examined by a number of investigators but has yielded contradictory results 10-12. We have shown that the elevated level of plasma ROOHs in diabetic subjects was not influenced by diabetic complications Table 2. These data suggest that oxidative stress is an early stage in the disease pathology and not simply a consequence of the complica• tions.

slide 90:

58 Nourooz-Zadeh Table 2 Clinical Characterization for NIDDM Subjects With and Without Complications Variables No complications Complications p Numbers Age yr 38 53.3 ± 13.7 17-82 49 62.3 ::: 14.1 31-86 0.005 Total cholesterol mmol/L 5.6 ::: 1.2 6.4 ::: I.I 0.05 Triglycerides mmol/L 3.3-9.9 2.5 ± 1.5 4.2-9.9 3.1 ::: 1.9 NS 0.7-6.2 0.5-9.5 Fasting glucose mmol/L 11.4 ::: 5.4 12.7 ::: 5.1 NS HbAlc 1.9-28.9 10.9 ::: 2.5 2.3-28.5 I I.I ::: 2.3 NS 5.9-16.8 6.9-17.8 ROOH µmol/L 9.5 ::: 3.3 9.4 ::: 3.4 NS 2.7-15.5 2.7-16.8 o.-Tocopherol µ11101/L 18.6 ::: 5.6 20.4 ::: 8.7 NS o-Toccpherol/cholesterol 9.2-30.9 3.4 ± 0.9 8.6-44.3 3.2 ::: 1.0 NS µmol/L/mmol/L 1.9-5.0 1.5-6.2 ROOH/a-tocopherol/cholesterol 3.0 ::: 1.4 3.3 ±: 1.6 NS 0.6-6.1 0.9-8.3 Data in brackets are ranges. NS not significant. Source: Ref. 18. IV. PLASMA VITAMIN E STATUS IN HEALTHY AND DIABETIC SUBJECTS Data are conflicting on the rx-tocopherolstatus in diabetic subjects. Some stud• ies report no changes others a decrease and still others an increase 23-25. One problem with the previous studies was a failure to standardize n-tocoph• erol for lipid concentration which would produce misleading results in hyper• lipidemic patients. We have found that absolute plasma n-tocopherol levels in diabetic subjects were slightly but significantly lower than those of the control subjects Table 1 . Plasma o-tocopherol levels between the two groups differed markedly when o-tocopherol levels were expressed per unit of choles• terol. No difference was found in absolute plasma or cholesterol-standardized o:-tocopherol in the diabetic patients with and without complications Table 2. These findings have recently been confirmed by Borcea et al. 22. Further

slide 91:

ROOH and Vitamin E in Diabetes 59 studies are needed to address the question whether the low plasma n-toccpb• erol in the diabetic patients is related to increased oxidative stress or chronic low dietary intake of vitamin E. V. RELATIONSHIP BETWEEN ROOH AND GLYCEMIC CONTROL Two independent studies from this laboratory have shown no correlation be• tween plasma ROOHs and HbAle 17 18. On the other hand there was a scatter association between ROOH and fasting blood glucose r 0.2 p 0.05 in the diabetic subjects but not in the control group 17 18. ROOH/ cholesterol-standardized o-tocopherol ratio also showed a weak association with fasting blood glucose in the diabetic subjects but not in the control group r 0.23 p 0.05 18. VI. INFLUENCE OF INSULIN THERAPY ON PLASMA ROOHs Little information is available on the effect of glycemic control on plasma markers of oxidative stress. Berg et al. 26 compared the effect of continuous intensified insulin treatment CIIT and conventional insulin treatment CIT on plasma lipid peroxides as measured by the FOX assay. Plasma ROOHs in patients receiving CIIT fell by 31 as compared with baseline over a period of24 months. HbAlc fell by 15 during the same period Fig. 4. By contrast no difference was seen in patients receiving CIT over the same period. Faure et al. 27 also examined the effect of CIIT on plasma lipid peroxides using the TBA assay. They too reported a marked reduction in TBARs after CUT as compared with the baseline level 2.42 :::+::: 0.25 vs. 3.03 ± 0.27 umol/L: n 16 over a period of 7 years 27. These observations provide further sup• port for the hypothesis of a beneficial effect of insulin therapy on lipid peroxi• dation brought about by decreasing circulating HbA I c levels VII. EFFECT OF ANTIOXIDANT TREATMENT ON PLASMA ROOHs To the best of our knowledge there is one study addressing the effect of antioxidant therapy on plasma markers of oxidative stress in diabetic patients.

slide 92:

-------· t i . - e 6 --. 60 Nourooz-Zadeh 12 10 ee tJ . ... 8 ... .Q 4 --CIIT -+--CIT :c I 2 0 l 0 12 24 I r 5 ------------------------ ------ --- i 4 2: ::c 0 3 a:: 2 0 " I l I 0 12 24 months Figure 4 Change in plasma ROOHs and HbAlc during continuous intensified insu• lin therapy CIIT and conventional insulin therapy CIT. Borcea et al. 22 studied the effect of the antioxidant cc-lipoic acid on plasma ROOHs in diabetic patients n 33 receiving n-lipoic acid for 12 weeks 600 mg/day. Diabetics treated with n-lipoic acid had markedly lower levels of plasma ROOHs than the control group. A trend toward higher n-tocopherol concentration was seen in the n-Iipoic acid-treated subjects as compared with control subjects. n-Lipoic acid exists naturally in physiological systems as a cofactor for enzymatically catalyzed acyl transfer reactions 28. o-Lipoic acid and its intracellularly reduced form dehydrolipoate have been shown to scav-

slide 93:

ROOH and Vitamin E in Diabetes 61 enge a variety of reactive species such as Ho·ROO"HOC and peroxynitrite to regenerate both cc-tocopherol and ascorbate and to raise intracellular glu• tathione levels. Thus these data provide the first direct evidence for the hy• pothesis that treatment with the antioxidant o-lipoic acid reduces accumulation of ROOHs in the circulation. VIII. CONCLUSION Diabetes mellitus has been proposed to be associated with a high risk of athero• sclerosis and kidney and nerve damage. Preliminary work from this laboratory has shown that plasma from individuals with diabetes mellitus contains ele• vated levels of ROOHs. The diabetic subjects also had lower levels of plasma n-tocopherol as compared with control subjects which was unrelated to dys• lipidernia. Another important point from our data is that ROOH levels were similar in diabetics with and without complications suggesting that oxidative stress occurs at an early stage in the disease pathology. It predates the compli• cations not simply a consequence of the complications. In addition we have shown that insulin therapy and antioxidant therapy have a beneficial effect on oxidative stress. ACKNOWLEDGMENT Supported by the British Heart Foundation. REFERENCES I. Pyorala K Laakso M Uusitupa M. Diabetes and atherosclerosis: an epidemio• logical view. Diabetes 1987 3:463-524. 2. Kanell WB Hjorland M Castelli WP. Role of diabetes in cardiac disease: con• clusion from population studies. Am J Cardiol 1974 34:29-34. 3. Uusittupa M Niskanen LK Siitonen 0 Voutilainen E Pyorala K. Five-years incidence of atherosclerotic vascular disease in relation to general risk factors insulin level and abnormalities in lipoprotein composition in NIDDM and non• diabetic subjects. Circulation 1990 82:27-36. 4. Oberley LW. Free radicals and diabetes. Free Rad Biol Med 1988 5: 113-124. 5. Baynes JW. Perspectives in diabetes: role of oxidative stress in development of complications in diabetes. Diabetes 1991 40:405-412.

slide 94:

62 Nourooz-Zadeh 6. Wolff SP. Diabetes and free radicals. Br Med Bull 1993 49:642-652. 7. Giugliano D Ceriello A Paolisso G. Diabetes mellitus hypertension and cardio• vascular disease: which role for oxidative stress Metabolism 1995 44:363-368. 8. Steinberg D Parthasarathy S Carew TE Khoo JC Witztum JL. Beyond choles• terol: modification of LDL that increase atherogenicity. N Engl J Med 1989 320:915-924. 9. Halliwell B. Oxidative stress nutrition and health: experimental strategies for optimization of nutritional antioxidants intakes in humans. Free Rad Res Comm 1996 25:57-74. 10. Velazques E Winocour PH Kesteven P Alberti KGMM Laker MF. Relation of lipid peroxides to macrovascular disease in type 2 diabetes. Diabetic Med 1991: 8:752-758. 11. MacRury SM Gordon D Wilson R Bradley H Gemmell CG Paterson JR Rumley AG MacCuish AC. A comparison of different methods of assessing free radical activity in type 2 diabetes and peripheral vascular disease. Diabetic Med 1993 10:331-335. 12. Griesmacher A Kinder-Hauser M Andert S Schreiner W Toma C Knoebl P Pietschmann P Prager R. Enhanced serum levels of TBARS in diabetes mellitus. Am J Med 1995 98:469-475. 13. Niskanen LK Salonen JT Nyssonen K Uusitupa MIJ. Plasma lipid peroxidation and hyperglycaemia: a connection through hyperinsulinaemia. Diabetic Med 1995 12:802-808. 14. Sundaram RK. Bhaskar A Vijayalingam S Viswanatthan M Mohan R Shanmu• gasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes with and without complications. Clin Sci 1996 90:255-260. 15. Chirico S Smith C Marchant C Mitchinson MJ Halliwell B. Lipid peroxidation in hyperlipidaemic patients. A study of plasma using an HPLC-based thiobar• bituric acid test. Free Rad Res Commun 1993 19:51-57. 16. Nourooz-Zadeh J. Tajaddini-Sarmadi J Wolff SP. Measurement of plasma hy• droperoxide concentrations by the ferrous-oxidation in xylenol FOX assay in conjunction with triphenylphosphine. Anal Biochem 1994 220:403-409. 17. Nourooz-Zadeh J Tajaddini-Sarmadi J McCarthy S Betteridge DJ Wolff SP. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: I 054-1058. 18. Nourooz-Zadeh J Rahimi A Tajaddini-Sarmadi J Tritschler H Rosen P Halli• well B Betteridge DJ. Relationship between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997 40:647-653. 19. Sodergren E Nourooz-Zadeh J Berglund L Vessby B. Re-evaluation of the ferrous oxidation in xylenol orange assay for the measurement of lipid hydroper• oxides. J Biochem Biophys Methods 1998 37: 137-146. 20. Santini SA Marra G Giardina B. Cotroneo P Mordente A Martorana GE Manto A Ghirlanda G. Detective plasma antioxidant defenses and enhanced sus• ceptibility to lipid peroxidation in uncomplicated IDDM. Diabetes 1997 46: 1853-1858.

slide 95:

ROOH and Vitamin E in Diabetes 63 21. Nuttall SL Martin U Sinclair AJ Kendall. Glutathione: in sickness and in health. Lancet 1998 351 :645-646. 22. Borcea V Nourooz-Zadeh J Wolff SP Zumbach M Hofmann M Urich H Wahl P Ziegler R Tritschler HJ Nawroth PP. Oxidative stress and antioxidant depletion in patients with diabetes mellitus. Diabetes Care submitted. 23. Thompson KH Godin DY. Micronutrients and antioxidants in the progression of diabetes. Nutr Res 1995 15:1377-1410. 24. Tsai EC Hirsch IB Brunzell JD Chait A. Reduced plasma peroxyl radical trap• ping capacity and increased susceptibility of LDL to oxidation in poorly con• trolled IDDM. Diabetes 1994 43:IOI0-1014. 25. Asayama K Uchida N Nakane T Hayashibe H Dobashi K Amemiya S Kato K Nakazawa S. Antioxidants in the serum of children with IDDM. Free Rad Biol Med 1993 15:597-602. 26. Berg TJ Nourooz-Zadeh J. Wolff SP Tritschler HJ Bangstad HJ Hanssen KE. Hydroperoxides in plasma are reduced by intensified insulin treatment: a ran• domised controlled study of IDDM patients with microalbuminuria. Diabetes Care in press. 27. Faure P Corticelli P Richard MJ. Arnaud J Coudray C Halimi S Favier A Roussel AM. Lipid peroxidation and trace element status in diabetic ketonic pa• tients: Influence of insulin therapy. Clin Chem 1993 39:789-793. 28. Packer L. Witt EH Tritschler HJ. Alpha-lipoic acid as a biological antioxidant. Free Rad Biol Med 1995 19:227-250.

slide 96:

This Page Intentionally Left Blank

slide 97:

5 Concentrations of Antioxidative Vitamins in Plasma and Low-Density Lipoproteinof Diabetic Patients Click Here For Best Diabetes Treatment Wolfgang Leonhardt Technical University Dresden Germany Several recent studies indicate that oxidative stress is increased in diabetic patients. Oxidative stress is a major component in the development of late complications in diabetic patients 1-4. It is mainly based on hyperglyce• mia. During the cellular metabolization of glucose superoxide anions can be formed that shift the pro/antioxidative balance in blood 56. Intracellular activation of the polyol pathway produces an imbalance in the ratio of NADH/ NAD+. Elevated blood glucose concentrations also cause increased glycation of lipoproteins. Because of these factors reactive oxygen species and lipo• peroxides are formed in the blood of diabetic patients 7-12 and their lipo• proteins are more prone to in vitro oxidation 13 14. Glucose can act pro• oxidatively on low-density lipoprotein LDL in vitro 161516. Impaired protection of lipid membranes against damage by free radicals is important in insulin-dependent diabetes mellitus IDDM because islet cell destruction by leukocytes may be mediated through the generation of toxic oxygen radi• cals 17. There are many defenses to protect the organism from free radical pro• cesses 18. Antioxidant enzymes such as superoxide dismutase catalase and glutathione peroxidase are preventive antioxidants because they eliminate

slide 98:

species involved in the initiation of free radical chain reactions. Some small molecule antioxidants such as ascorbate the tocopherols ubiquinol urate 65

slide 99:

66 Leonhardt and glutathione are able to repair oxidizing radicals directly and therefore are chain-breaking antioxidants. I. VITAMIN E IN DIABETES Vitamin E RRR-a-tocopherol is the most important lipid-soluble antioxi• dant which protects lipoproteins and cell membrane lipids from oxidative damage. This ability is coupled to other antioxidant systems vitamin C gluta• thione lipoic acid that can recycle the vitamin E radical 19. In the absence of such systems vitamin E can behave as an oxidant 20. Dietary vitamin E is transported to the liver and secreted from the liver within very-low-density lipoproteins VLDL. It is distributed among VLDL and LDL during the trans• fer and metabolism of the lipoprotein lipids 21. Thermodynamic partitioning also permits some transfer into high-density lipoproteins 22. An important part of vitamin E is constituent of cell membranes where it protects the lipid moiety against peroxidation 23. About one-half of the total plasma vitamin E is a constituent of circulat• ing LDL. Interindividual variations in plasma vitamin E are closely related to those in LDL 2425. The concentration of vitamin E per LDL particle is rather low i.e. in the order of 5-9 molecules compared with 2200 molecules of cholesterol and 170 molecules of triglycerides 26. Nevertheless the level of vitamin E in LDL is an independent factor that influences susceptibility of LDL to oxidation. The lagtirne of in vitro LDL oxidation was found to be related to the level of vitamin E in LDL when diabetic patients were supple• mented with vitamin E 27 28. For persons with usual nutritional habits the corresponding relationship was observed in two studies 2930 but not in others 33132. There are several publications on vitamin E concentrations in the plasma of diabetic patients. The assumption that the oxidative stress in diabetes is due to deficient vitamin E in plasma could not be confirmed. In most stud• ies no statistically significant differences in the concentrations between dia• betic and control persons were observed. Two studies exhibited even signifi• cantly higher levels in diabetes Table 1 . The weighted mean values control subjects 26.4 ::: 1.9 urnol/L IDDM 24.4 ::: 1.6 µmol/L NIDDM 22.4 ::: 2.0 µmol/L are only marginally different for the plasma of control persons IDDM and non-IDDM NIDDM. Published data on vitamin E concentrations in LDL Table 2 show very similar mean values for diabetic persons and control subjects control subjects 7.5 mol/mol IDDM 7.0 mol/mol NIDDM 6.8 mol/mol. One study demon-

slide 100:

Control subjects M/F 28.6/27.9 210/240 IDDM M/F 23.6/26.2 60/63 41 Control subjects 23.8 :::: 1.3 41 NIDDM 19.5 :::: 0.8 87 42 Control subjects vs. NIDDM 33.3 :::: 3.3 20 Control subjects vs. IDDM 27.5 :::: 1.4 24 43 NIDDM 32.0 :::: 1.8 24 IDDM 25.2 :::: 1.4 28 Median. Ant ioxidant Vitamins in Diabetics 67 Table 1 Plasma Serum Vitamin E Concentrations in Diabetes Vitamin E Group umol/L 11 Sign. Ref. Control subjects 21.4 :::: 0.6 29 IDDM 23.7 :::: 0.8 27 33 Control subjects 24.9 :::: 1.4 20 IDDM 24.6 :::: 1.6 15 3 Control subjects 26.5 62 IDDM 22.7 77 34 NIDDM 21.4 81 Control subjects 24.8 :::: 2.3 180 NIDDM 21.1 :::: 1.5 164 35 Control subjects 23.9 :::: 1.4 20 IDDM 28.3 :::: 1.5 20 36 Control subjects 17.0 :::: 2.1 12 NIDDM 16.2 :::: 2.9 9 37 Control subjects vs. NIDDM 20.1 :::: 2.7 15 Control subjects vs. IDDM 18.0 :::: 1.9 10 30 NIDDM 22.6 :::: 6.5 53 IDDM 21.0 :::: 4.1 10 Control subjects 27.8 :::: 2.4 47 NIDDM 25.8 :::: 2.1 59 38 Control subjects 20.6 :::: 0.7 40 NIDDM 24.2 :::: 1.0 40 39 Control subjects 27.9 16.3-39.5 28 NIDDM 27.5 8.7-46.3 21 40

slide 101:

68 Leonhardt Table 2 LDL Vitamin E Concentrations in Diabetes Group Vitamin E mol/mol II Sign. Ref. Control subjects vs. NIDDM 7.5 :: 1.0 20 31 Control subjects vs. IDDM 7.1 :: 1.3 20 NIDDM 8.5 :: 3.5 20 IDDM 7.7 :: 2.1 20 Control subjects vs. NIDDM 8.1 1.20 15 30 Control subjects vs. lDDM 7.4 1.33 10 NIDDM 6.1 1.33 53 IDDM 5.7 1.32 10 Geometric mean and geometric standard deviation in parentheses. strated significantly lowered levels in NIDDM vitamin E in LDL was in• versely related to HbAlc and positively related to the lagtime of ex vivo oxi• dation of LDL. This means that vitamin E in LDL is a better marker of antioxidant deficiency in diabetes than vitamin E in plasma 30. II. PROBLEM WITH LIPID STANDARDIZATION OF VITAMIN E There are close correlations between the concentrations of vitamin E and lipids triglycerides cholesterol and phospholipids in plasma. They are due to the lipophilic properties of vitamin E and moreover they reflect that the antioxida• ti ve capacity of lipids is regulated in progression with the lipid mass. Epidemiological studies revealed that the mathematical relationship be• tween vitamin E and lipid concentrations 44 has the feature that it does not pass the origin. Rather it has a large ordinate section. This means that low lipid concentrations are associated with relatively more vitamin E and high lipid concentrations with relatively less vitamin E. Lipid standardization has the purpose to make vitamin E concentrations comparable irrespective of the corresponding lipid concentrations. The ratio of vitamin E to lipid concentra• tion does not fulfill this condition. It can be shown that this ratio is inversely related to the lipid concentrations. Therefore the widely used division of vi• tamin Eby cholesterol 33739414245 or cholesterol plus triglyceride 34 4045 concentrations is inadequate. By this procedure most persons with above-normal lipid concentrations appear vitamin deficient. An alternative is

slide 102:

Antioxidant Vitamins in Diabetics 69 a correction formula derived from the multiple regression of vitamin E on cholesterol and triglyceride concentrations. The constants in this formula should be derived from the population under study. For this purpose 15-20 complete data sets may be sufficient 44. Recently the formula was published with constants as follows vitamin E in µmol/L 46: Standardized vitamin E measured vitamin E -2.9 cholesterol -5.2 -1.5 triglycerides -1.3 The multiple regression on cholesterol and triglyceride concentrations was used for vitamin E standardization by several authors 4446-48. No statisti• cally significant difference of standardized values between diabetic patients and control persons was observed. Ill. VITAMIN A IN DIABETES Vitamin A trans-retinol is required for normal growth vision and resistance to radical-mediated processes during infections. The fat-soluble vitamin is taken up with animal food and partly formed from other carotenoids. After absorption vitamin A is transported in chylomicrons from the gut via the lymph duct and blood to the liver. Although the liver secretes a specific retinol• binding protein 3849 into the bloodstream plasma vitamin A correlates with plasma lipids almost as strongly as vitamin E does 50. The level of vitamin A in diabetes has found much attention in recent literature. Vitamin A deficiency may cause blindness. Poorly controlled diabe• tes mellitus is attributed to a decreased availability of retinol carrier protein and subsequently to depressed vitamin A levels in blood. The impaired vitamin A status may not be improved by vitamin A supplementation but by insulin administration 51. In persons with well-controlled NIDDM without insulin deficiency the metabolism of vitamin A appears not to be impaired. Interest• ingly persons with impaired glucose tolerance show increased 2.5 µmol/L vitamin A versus persons with normal glucose tolerance 2.1 µmol/L 46. Most studies demonstrated lowered levels in IDDM the difference being sig• nificant in six studies. Significantly lowered levels in NIDDM were found in two studies Table 3. The weighed mean values are similar for control subjects and NIDDM and lower for IDDM control subjects 1.95 :±: 0.23 µmol/L IDDM 1.52 + 0.46 µmol/L NIDDM 2.29 + 0.23 µmol/L.

slide 103:

70 Leonhard t Table 3 Plasma Serum Vitamin A Retinol Concentrations in Diabetes Vitamin A Group µmol/L /l Sign. Ref. Control subjects 2.32 :::: 0.36 52 Control subjects 1.86 :::: 0.35 53 IDDM Control subjects 1.63 :::: 0.33 1.82 :::: 0.45 IDDM Control subjects 1.45 :::: 0.36 1.95 :::: 0.11 20 54 IDDM 1.55 :::: 0.07 15 3 Control subjects 1.89 :::: 0.56 20 IDDM 1.71 :::: 0.99 20 36 Control subjects 2.14 :::: 0.10 47 NIDDM 2.23 :::: 0.10 59 38 Control subjects 2.3 :::: 0.18 180 NIDDM 2.2 :::: 0.11 164 35 lDDM 1.68 :::: 0.83 39 Control subjects 1.7 :::: 0.1 47 NIDDM 1.5 :::: 0.1 46 Control subjects 2.7 0.8-4.4 28 40 NIDDM 2.6 1.6-3.6 21 Control subjects M/F 1.86/ 1.54 210/240 IDDM M/F 1.46/ 1.28 60/63 41 Control subjects vs. NIDDM 2.23 :::: 0.18 20 Control subjects vs. IDDM 1.94 :::: 0.10 24 43 NIDDM 2.23 :::: 0.14 24 IDDM 1.30 ± 0.05 28 Control subjects 2.9 ± 0.6 35 IDDM 3.3 ± 1.0 JO 51 NIDDM 3.2 :±: 0.9 53 Median. IV. VITAMIN C IN DIABETES Vitamin C ascorbic acid is a powerful antioxidant and a cofactor in collagen biosynthesis which affects platelet activation prostaglandin biosynthesis and the polyol pathway. Vitamin C acts as an antioxidant both in vitro and in vivo and protects plasma lipids and lipid membranes. It has the power to spare and to increase plasma-reduced glutathione 56. The antioxidative ability of vitamin E can be continuously restored through its recycling by other antioxi• dants mainly vitamin C 1819.

slide 104:

Antioxidant Vitamins in Diabetics 71 Table 4 Plasma Vitamin C Concentrations in Diabetes Vitamin C Group urnol/L II Sign. Ref. Control subjects 82.9 ::: 30.9 22 NIDDM 55.6 ::: 20.0 21 NIDDM + Complic. 42.1 ::: 19.3 20 57 Control subjects 60.0 ::: 5.5 180 NIDDM 48.8 ::: 2.3 164 NlDDM + Complic. 29.9 - 41.2 163 35 Normal persons 61.3 ::: 9.7 20 Borderline FBG 47.7 ::: 7.4 IO NIDDM 28.4 ::: 5.7 30 58 Control subjects 71.6 37.5-IOS.6 28 NIDDM 57.9 51.7-64.2 21 40 ND PVD 47.1 7.4-86.9 21 NIDDM PVD 48.3 15.3-80.1 11 Control subjects A 28 yr 87.5 ::: 4.9 24 IDDM 63.6 ::: 6.0 28 43 Control A 63 yr 58.5 ::: 5.3 20 NIDDM 38.6 ::: 5.7 24 Cornplic. diabetic complications FBG fasting blood glucose PVD peripheral vascular disease. Abnormalities of vitamin C metabolism have been reported in experi• mentally induced diabetes and in diabetic patients. Hyperglycemia may be directly responsible for a vitamin C deficit. Exposure to glucose may inactivate antioxidant enzymes and impair the intracellular regeneration of vitamin C by removing reducing equivalents in the form of NADPH for the polyol path• way 59. Nearly all studies demonstrated significantly diminished levels of vita• min C in the plasma of NIDDM. The reduction was also observed in IDDM and in borderline fasting blood glucose and it was more pronounced when diabetic complications occurred in addition Table 4. The weighed mean values are very different for control subjects and NIDDM control subjects 65.0 :± 7.8 µmol/L NIDDM 46.8 :± 4.6 µmol/L. V. CONCLUSION The epidemiological data of this chapter refer to diabetic patients and control persons who are not supplemented with antioxidant vitamins and who do not

slide 105:

72 Leonhardt take antioxidative medications. There are many reports showing that increased dietary intake or supplementation with vitamins E A and C can largely in• crease the plasma levels of the antioxidant vitamins with positive conse• quences concerning metabolic control and late complications of diabetes. REFERENCES I. Hunt JV Smith CC Wolff SP. Autoxidative glycosylation and possible involve• ment of peroxides and free radicals in LDL modification by glucose. Diabetes 1990 39: 1420-1424. 2. Ceriello A Giugliano D Quatraro A Donzella C Dipalo G Lefebvre PJ. Vita• min E reduction of protein glycosylation in diabetes: new prospect for prevention of diabetic complications Diabetes Care 1991 14:68-72. 3. Tsai EC Hirsch 18 Brunzell JD Chait A. Reduced plasma peroxyl radical trap• ping capacity and increased susceptibility of LDL to oxidation in poorly con• trolled IDDM. Diabetes 1994 43:1010-1014. 4. Giugliano D Ceriello A Paolisso G. Diabetes mellitus hypertension and cardio• vascular disease: which role for oxidative stress Metabolism 1995 44:363-368. 5. Thomalley P. Monosaccharide auto-oxidation in health and disease. Environ Health Perspect 1985 64:297-307. 6. Rifici VA Schneider SH Khachadurian AK. Stimulation of low density lipopro• tein oxidation by insulin and insulin like growth factor I. Atherosclerosis 1994 I 07 :99-108. 7. Altomare F Vendemiale G et al. Increased lipid peroxidation in type 2 poorly controlled diabetic patients. Diabetes Metab 1992 18:264-271. 8. Chittar HS Nihalani KD Varthakavi PK Udipi SA. Lipid peroxide levels in diabetics with micro- and macroangiopathies. J Nutr Biochem 1994 5:442-445. 9. Evans RW Orchard TJ. Oxidized lipids in insulin-dependent diabetes mellitus: a sex-diabetes interaction Metabolism 1994 43: 1196-1200. I 0. Gallou G Ruelland A Campion L Maugendre D Lemoullec N Legras 8 Allan• nic H Cloarec L. Increase in thiobarbituric acid-reactive substances and vascular complications in type 2 diabetes mellitus. Diabetes Metab J 99420:258-264. 11. Haffner SM Agil A Mykkanen L Stem MP Jialal I. Plasma oxidizability in subjects with normal glucose tolerance impaired glucose tolerance and NIDDM. Diabetes Care 1995 18:646-653. 13. Babiy AV Gebicki JM Sullivan DR Willey K. Increased oxidizability of plasma lipoproteins in diabetic patients can be decreased by probucol therapy and is not due to glycation. Biochem Pharmacol 1992 43:995-1000. 14. Cestaro 8 Gandini R Viani P Maraffi F Cervato G Montalto C Gatti P Megali R. Fluorescence-determined kinetics of plasma high oxidizability in diabetic pa• tients. Biochem Mo Biol Int 1994 32:983-994. 15. Hicks M Delbridge L Yue DK Reeve TS. Catalysis of lipid peroxidation by

slide 106:

Antioxidant Vitamins in Diabetics 73 glucose and glycosylated collagen. Biochem Biophys Res Commun 1988 151: 649-655. 16. Kawamura M Heinecke JW Chait A. Pathophysiological concentrations of glu• cose promote oxidative modification of low density lipoprotein by a superoxide• dependent pathway. J Clin Invest 1994 94:771-778. 17. Oberley LW. Free radicals and diabetes. Free Rad Biol Med 1988 5:113-124. 18. Frei B Stocker R Ames BN. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci USA 1988 85:9748-9752. 19. Kagan VE Serbinova EA Forte T Scita G Packer L. Recycling of vitamin E in human low density lipoproteins. J Lipid Res 199233:385-397. 20. Stocker R Bowry VW Frei B. Ubiquinol-10 protects human low density lipopro• tein more efficiently against lipid peroxidation than does alpha-tocopherol Proc Natl Acad Sci USA 1991 88: 1646-1650. 21. Kostner GM Oettl K Jauhiainen M Ehnholm C Esterbauer H. Dieplinger H. Human plasma phospholipid transfer protein accelerates exchange/transfer of a• tocopherol between lipoproteins and cells. Biochem J 1995 305:659-667. 22. Behrens WA Thompson JN Madere R. Distribution of n-tocopherol in human plasma lipoproteins. Am J Clin Nutr 1982 35:691-696. 23. Buettner GR. The pecking order of free radicals and antioxidants: lipid peroxida• tion alpha-tocopheroland ascorbate Arch Biochem Biophys 1993 300:535-543. 24. Davies T Kelleher J Losowsky MS. Interrelation of serum lipoprotein and to• copherol levels. Clin Chim Acta 1969: 24:431-436. 25. Rubba P. Mancini M Fidanza F Leccia G Riemersma RA Gey KF. Plasma vitamin E apolipoprotein B and HDL-cholesterol in middle-aged men from southern Italy. Atherosclerosis 198977:25-29. 26. Esterbauer H Gebicky J Puhl H Ji.irgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992: 13: 341-390. 27. Reaven PD Herold DA. Barnett J Edelman S. Effects of vitamin Eon suscepti• bility of low-density lipoprotein and low-density lipoprotoin subfractions to oxi• dation and on protein glycation in NIDDM. Diabetes Care 1995 18:807-816. 28. Fuller CJ Chandalia M Garg A Grundy SM Jialal I. RRR-alpha-tocopheryl acetate supplementation at pharmacologic doses decreases low density lipopro• tein oxidative susceptibility but not protein glycation in patients with diabetes mellitus. Am J Clin Nutr 1996: 63:753- 759. 29. Frei B. Gaziano JM. Content of antioxidants preformed lipid hydroperoxides and cholesterol as predictors of the susceptibility of human LDL to metal ion• dependent and ion-independent oxidation. J Lipid Res 1993 34:2135-2145. 30. Leonhardt W Hanefeld M Muller G Hora C Meissner D Lattke P Paetzold A Jaross W Schroeder HE. Impact of concentrations of glycated hemoglobin alpha-tocopherol copper and manganese on oxidation of low density lipopro• reins in patients with type I diabetes type II diabetes and control subjects. Clin Chim Acta 1996 254:173-186. 31. Beaudeux JL Guillausseau PJ Peynet J Flourie F. Assayag M Tielmans D Warnet A Rousselet F. Enhanced susceptibility of low density lipoprotein to in

slide 107:

74 Leonhardt vitro oxidation in type I and type 2 diabetic patients. Clin Chim Acta 1995 239: 131-141. 32. Dimitriadis E. Griffin M Owens D Johnson A Collins P Tomkin GH. Oxida• tion of low density lipoprotcin in NIDDM: its relationship to fatty acid composi• tion. Diabetologia 1995 38: 1300-1306. 33. Asayarna K Uchida N Nakane T. Hayashibe H Dobashi K. Amemiya S Kato K Nakazawa S. Antioxidants in the serum of children with insulin-dependent diabetes mellitus. Free Rad Biol Med 1993 15:597-602. 34. Griesmacher A Kindhauser M Andert SE Schreiner W Toma C Knoebl P Pietschmann P Prager R Schnack C. Schernthaner G Mueller MM. Enhanced serum levels of thiobarbituric-acid-reactive substances in diabetes mellitus. Am J Med 1995 98:469-475. 35. Sundaram RK Bhaskar A Vijayalingam S Viswanthan M Mohan R Shanmu• gasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes mellitus with and without complications. Clin Sci 1996: 90:255-260. 36. Osterode W Holler C Ulberth F. Nutritional antioxidants red cell membrane fluidity and blood viscosity in type I insulin dependent diabetes mellitus. Dia• betic Med 1996 13: I 044-1050. 37. Galvan AQ Muscelli E Catalano C. Natali A Sanna G Masoni A Bernardini B Barsacchi R Ferrannini E. Insulin decreases circulating vitamin E levels in humans. Metabolism 1996 45:998-1003. 38. Basualdo CG Wein EE Basu TK. Vitamin A retinol status of first nation adults with non-insulin-dependent diabetes mellitus. J Am Coll Nutr 1997 16:39-45. 39. Ceriello A Bortolotti N Falleti E. Taboga C Tonutti L. Crescentini A Motz E Lizzio S Russo A Bartoli E. Total radical-trapping antioxidant parameter in NIDDM patients. Diabetes Care 1997 20:194-197. 40. Dyer RG. Stewart MW Mitcheson J George K Alberti MM Laker MF. 7- Ketocholesterol a specific indicator of lipoprotein oxidation and malondialde• hyde in non-insulin dependent diabetes and peripheral vascular disease. Clin Chim Acta 1997 260: 1-13. 41. Olmedilla B Granado F Gilmartinez E Blanco I Rojashidalgo E. Reference values for retinol tocopherol and main carotenoids in serum of control and insu• lin dependent diabetic Spanish subjects. Clin Chem 1997 43: I 066-1071. 42. Nourooz-Zadeh J Rahimi A. Tajaddinisarrnadi J Tritschler H Rosen P. Halli• well B Betteridge DJ. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997 40:647-653. 43. Maxwell SRJ Thomason H Sandler D Leguen C Baxter MA Thorpe GHG. Jones AF Barnett AH. Antioxidant status in patients with uncomplicated insulin• dependent and non-insulin-dependent diabetes mellitus. Eur J Clin Invest 1997 27:484-490. 44. Jordan P Brubacher D Moser U Stahelin HB Gey KF. Vitamin E and vitamin A concentrations in plasma adjusted for cholesterol and triglycerides by multiple regression. Clin Chem 1995 41 :924-927. 45. Thurnham DI Davies JA Crump BJ Situnayake RD Davis M. The use of differ-

slide 108:

Antioxidant Vitamins in Diabetics 75 ent lipids to express serum tocopherol: lipid ratios for the measurement of vita• min E status. Ann Clin Biochem 1986 23:514-520. 46. Tavridou A Unwin NG Laker MF White M Alberti KGMM. Serum concentra• tions of vitamins A and E in impaired glucose tolerance. Clin Chim Acta 1997 266: 129-140. 47. Salonen JT Nyyssonen K Tuomainen T-P Maenpaa TH Korpela H Kaplan GA Lynch J Heimrich SP Salonen R. Increased risk of non-insulin-dependent diabetes mellitus at low plasma vitamin E concentrations: a 4-year follow-up study in men. Br Med J 1995 311:1124-1127. 48. Nyyssonen K Porkkalasarataho E Kaikkonen J Salonen JT. Ascorbate and urate are the strongest determinants of plasma antioxidative capacity and serum lipid resistance to oxidation in Finnish men. Atherosclerosis 1997 130:223-233. 49. Krill D Oleary L Koehler AN Kramer MK Warty V Wagner MA Dorman JS. Association of retinol-binding protein in multiple-case families with insulin• dependent diabetes. Hum Biol 1997 69:89-96. 50. Gey KF Puska P Jordan P Moser UK. Inverse correlation between plasma vita• min E and mortality from ischemic heart disease in cross-cultural epidemiology. Am J Clin Nutr 1991 53:326S-334S. 51. Basu TK. Basualdo C. Vitamin A homeostasis and diabetes mellitus. Nutrition 1997 13:804-806. 52. Wako Y. Suzuki K Goto Y Kimura S. Vitamin A transport in plasma of diabetic patients. Tohoku J Exp Med 1986 49: 133. 53. Basu TK Tze WJ Leichter J. Serum vitamin A and retinol-binding protein in patients with insulin-dependent diabetes mellitus. Am J Clin Nutr 1989 50:329. 54. Martinoli L De Felice M Seghieri G et al. Plasma retinol and alpha-tocopherol concentrations in insulin-dependent diabetes mellitus: the relationship to micro• vascular complications. Int J Vitam Nutr Res 1993 63:87. 55. Leonhardt W Hanefeld M Lattke P Jarof W. Vitamin E-Mangel und Oxidier• barkeit der Low-Density-Lipoproteine bei Typ-l-und Typ-Il-Diabetes: EinfluJ3 der Qualitat der Stoffwechsclkontrolle. Diabetes 1997 6suppl 2:24-28. 56. Paolisso G D Amore A Balbi V Volpe C Galzerano D Giugliano D Sgambato S Varricchio M DOnofrio F. Plasma vitamin C affects glucose homeostasis in healthy subjects and in non-insulin-dependent diabetics. Am J Physiol 1994 266: E261-E268. 57. Sinclair AJ. Girling AJ Gray L Le Guen C Lunec J Barnett AH. Disturbed handling of ascorbic acid in diabetic patients with and without microangiopathy during high-dose ascorbate supplementation. Diabetologia 1997 34: 171-175. 58. Srinivasan KN Pugalcndi KV Sambandam G Rao MR Menon PV. Diabetes mellitus lipid peroxidation and antioxidant status in rural patients. Clin Chim Acta 1997 259: 183-186. 59. Maxwell SRJ Thomason H Sandler D Leguen C Baxter AM Thorpe GHG Jones AF Barnett AH. Poor glycaemic control is associated with reduced serum free radical scavenging antioxidant activity in non-insulin-dependent diabetes mellitus. Ann Clin Biochem 1997 34:638-644.

slide 109:

This Page Intentionally Left Blank

slide 110:

6 Oxidative Stress in Diabetes Want To Diabetes Free Life Click Here John W. Baynes and Suzanne R. Thorpe University of South Carolina Columbia South Carolina I. DEFINING OXIDATIVE STRESS The first step in addressing the role of oxidative stress OxS in diabetic com• plications is to define OxS. It is often defined as a shift in the pro-oxidant• antioxidant balance in the pro-oxidant direction. This definition of OxS is more descriptive than quantitative and chemical in nature. Philosophically it implies a null point a balance point at which there is no OxS-OxS occurs only when the balance is shifted toward the pro-oxidant direction. There is a conceptual flaw in this definition because it fails to recognize that OxS is a constant feature of biological systems. Peroxides superoxide hydroxyl radicals and other re• active oxygen species ROS the mediators of OxS are being formed continu• ously in the body and always exist at some steady-state concentration. The resulting oxidative damage to protein DNA and other biomolecules is a ubiq• uitous and universal consequence of life under aerobic conditions. OxS might be better defined as "a measure of the prevailing level of ROS in a biological system. This definition acknowledges the continuous presence of ROS in biological systems at some level determined by the rela• tive rates of their formation and consumption. It accepts OxS as a normal feature of cellular metabolism rather than a disturbance in an equilibrium• OxS waxes and wanes but never disappears from the biological scene. Like metabolites levels of ROS may differ at different stages in the feeding-fasting

slide 111:

and diurnal cycles among different subcellular compartments among differ• ent cell types in a cell at different stages in its growth and development and 77

slide 112:

78 Baynes and Thorpe even among cells of the same type but in different regions of a tissue. ROS are mediators of hormone action and growth factor and cytokine activity and variations in ROS concentrations and OxS in intracellular and extracellular environments appear to be a central feature of regulatory biology l . From a quantitative viewpoint OxS may be considered the sum of the products of the concentration and reactivity of numerous ROS in the cell. Should cells in which redox coenzyme systems are off-balance or in which reduced glutathione GSH is depleted also be considered oxidatively stressed This is not a trivial question because alterations in ascorbate or GSH homeo• stasis are often cited as evidence of OxS. However even a poor defense may be adequate in the absence of an oxidative challenge. Persons with glucose 6-phosphate dehydrogenase deficiency for example are asymptomatic until they are challenged by drugs or infection leading to hemolytic anemia. Thus a shift in the set point or concentration of redox coenzymes in a cell may predispose to oxidative stress but the perturbation per se does not necessarily indicate that the cell is oxidatively stressed. At this time it is not possible to quantify OxS but this may eventually be achievable. The total radical antioxidant potential TRAP of plasma can now be estimated for example as the sum of a variety of antioxidant concen• trations in plasma including ascorbate tocopherols uric acid and protein 2. Plasma TRAP is commonly expressed relative to that of a concentration of an antioxidant standard such as Trolox 3. It may eventually be possible to develop a standard such as "H202 equivalents" for assessing OxS in cells and tissues or actually to measure the concentration of specific oxyradicals by electron paramagnetic resonance spectroscopy 4. Because of the many components and factors affecting OxS Fig. I it is difficult if not impossible to assess the overall status of OxS in a biological system by measurement of the status of an individual or even several enzymes or antioxidant systems. Indeed the interpretation of these data is often gratu• itous. A low level of antioxidant enzyme is often interpreted as evidence of OxS but high levels of superoxide dismutase are associated with OxS in the lungs in response to hyperbaric oxygen. Similarly high plasma levels of uric acid are associated with inflammation in gout whereas high levels of ascorbic may be pro-oxidant in the presence of free or heme iron. In the absence of unambiguous assays or standards for measuring OxS measurement of the consequences of OxS has been used as a surrogate. One approach for assessing the status of OxS is to measure the rate of excretion of products of oxidation of DNA such as thymidine glycol or 8-oxodeoxy• guanosine 56. Another is to measure the level of activation or expression of protein kinases activator protein-I or nuclear factor kappa B growth factors transforming growth facror-B insulin-like growth factor- I and vascular en-

slide 113:

Oxidative Stress in Diabetes 79 Oxidative Stress a measure of the steady statelevel of reactive oxygen species Pro-Oxidant Hyperbaric oxygen Metals overload decompartmentalization Metabolic hyperglycemia glycation AGES polyol pathway activity Immunological inflammation Complement activation autoimmune disease phagocytosis NADPH oxidase myeloperoxidase Drugs Xenobiotics smoking alcohol Anti-Oxidant Antioxidant enzymes SOD CAT GPx Antioxidant vitamins ACE Other antioxidants bilirubin glutathione taurine ubiquinol urate Metal sequestration albumin transferrin ferritin hemopexin Dietary factors flavonoids micronutrients selenium Figure 1 Some factors determining the status of OxS in biological systems. dothelial growth factor or heme oxygenase all of which are involved in the response to oxidative stress 17 but also respond to other stresses such as reductive thermal heat/cold and osmotic shock. A third approach the focus of our research and of this article is the measurement of the extent of oxidative damage to long-lived proteins such as collagen which integrates the time• averaged ambient level of OxS. II. NATURE OF OXIDATIVE DAMAGE Oxidative damage to protein may be divided chemically into primary and sec• ondary damage Table I. Primary damage results from direct reaction of pro-

slide 114:

80 Baynes and Thorpe Table 1 Biomarkers of Oxidative Stress Precursors: Reactive Oxygen Species H202 02-· OOH OH HOC ONOOH metal:oxo complexes Primary Products of Oxidation of Protein Class Example Aromatic Sulfbydryl Amino acid hydroperoxides Protein carbonyls a-Tyrosine dityrosine chlorotyrosine nitrotyro- sine ditytrosine dihydroxyphenylalanine Protein disulfides methionine sulfoxide Valine leucine isoleucine Oxohistidine adipic semialdehyde Secondary Products of Oxidation of Protein Lipid Carbohydrate Mixed Precursor MDA-Lys Pentosidine CML CEL Products HNE-Lys His Cys Pyrroles Crosslines Vesperlysines GOLD MOLD Argpyrimidine teins with ROS. Products of these reactions include stable compounds isolable by acid hydrolysis of proteins such as a-tyrosine and methionine sulfoxide MetSO. Some products such as nitrotyrosine and chlorotyrosine provide insight into the source of the damage peroxynitrite and HOCl respectively. Other products such as protein carbonyls may be derived from several sources are unstable to acid hydrolysis and may be transient but important intermediates in the cell e.g. in marking proteins for turnover 8. Secondary oxidative damage results from reaction of proteins with prod• ucts of oxidation of small molecules including lipids carbohydrates and amino acids. The intermediates in this process are reactive carbonyl and dicar• bonyl compounds such as malondialdehyde a -unsaturated and hydroxy• aldehydes glyoxal and methylglyoxal MGO which react with nucleophilic groups on protein to form lipoxidation 9 and glycoxidation I 0 11 products Table 1. Lipoxidation products require oxidation peroxidation for their for• mation from lipids whereas glycoxidation products are a subclass of advanced glycation end products AGEs requiring autoxidation chemistry oxidation by molecular oxygen for their formation from reducing sugars or ascorbate. Some AGEs e.g. pyrraline and imidazolones formed by reaction of 3-deoxy• glucosone 3DG with lysine and arginine residues in protein do not require oxidation for their formation from reducing sugars. These AGEs are useful indicators of nonoxidative chemical modification of proteins.

slide 115:

Oxidative Stress in Diabetes 81 Some secondary oxidation products such as carboxymethyllysine CML carboxyethyllysine CEL pentosidine 12 13 and vesperlysines 14 are stable.to acid hydrolysis whereas others such as crosslines 15 and the malondialdehyde and 4-hydroxynonenal adducts to lysine MDA-lysine HNE-lysine 16 are labile. Some are characteristic of lipid peroxidation such as MDA-Lys and HNE adducts to Lys His and Cys whereas pentosi• dine vesperlysines and crosslines are derived exclusively from carbohydrates. CML and CEL are more general markers that may be formed after oxidation of carbohydrates lipids or amino acids 17 18. Some of these compounds such as CML CEL and the imidazolium salts glyoxal- and methylglyoxal• lysine dimer GOLD MOLD 19 are end products whereas others such as MDA and HNE adducts to lysine may progress to form crosslinks and pyrrole adducts in protein. Both primary and secondary oxidation products are measured as indica• tors of OxS. In the several cases in which stable end products are formed such as a-tyrosine MetSO CML CEL pentosidine GOLD and MOLD the products accumulate with age in long-lived proteins such as collagens and crystallines 12 13 17 1920. Because they are oxidation products these com• pounds are biomarkers of oxidative damage to protein and should provide insight into levels of oxidative stress. Primary oxidation products are formed directly by reaction of ROS with protein and their concentration in tissue proteins should provide a direct index of OxS. In contrast secondary oxidation products should be second-order products of OxS Fig. 2 that is their levels in tissues are determined by both the prevailing level of OxS and the ambient concentration of oxidizable substrates. Substrate lipidcarbohydrate aminoacid Protein Reactive _ Reactive oxygen --- ..· . --· carbonyl species species ......._ _ Modified protein Figure 2 Reaction pathway illustrating the role of both prevailing oxidative stress ROS concentration and ambient substrate concentration in formation of secondary oxidation products. According to this scheme the rate of production of reactive car• bonyl species intermediates in the formation of secondary oxidation products is first order in ROS first order in substrate. and second order overall. Increases in either ROS or substrate may increase the rate of formation of reactive carbonyl species and secondary oxidation products.

slide 116:

82 Baynes and Thorpe Ill. IS OxS INCREASED IN DIABETES OxS is a feature of all chronic diseases. Free Radicals in Biology and Medicine 21 one of the early monographs in this field lists a full page of diseases in which OxS is implicated as a pathogenic agent. OxS is a sign of cellular stress injury and apoptosis. At sites of overt pathology such as in the kidney in diabetes and in plaque formed in atherosclerosis Alzheimers disease and dialysis-related amyloidosis both primary and secondary biomarkers of OxS are detected together by immunohistochemicaltechniques 22-28. Although these observations indicate a broad spectrum of oxidative damage to tissue proteins in chronic disease it is unlikely that OxS is a primary pathogenic mechanism in most chronic diseases. This is most obvious in autoimmune diseases where an errant immunological response underlies the development of pathology. Even in the case of disease induced by environmental agents such as chronic pulmonary disease OxS associated with phagocytosis and complement activation is probably the major source of tissue damage. The question about the role of OxS in diabetes is therefore not whether OxS in increased but whether OxS is a primary pathogenic mechanism in diabetes. IV. WHAT DO CHEMICAL BIOMARKERS OF OxS TELL US ABOUT THE STATUS OF OxS IN DIABETES As summarized below and presented in greater detail elsewhere 11 there are several lines of evidence that OxS is not a primary pathogenic mechanism underlying diabetic complications. 1. Age-adjusted levels of the primary oxidation products o-Tyr and MetSO in skin collagen are not increased in diabetes Fig. 3 20. 2. Increases in secondary oxidation lipoxidation and glycoxidation products in plasma and tissue proteins and in urine can be explained by increases in substrate concentrationsalone without invoking an increase in oxidative stress 1113. 3. Increases in lipoxidation and glycoxidation products in plasma are more closely associated with the presence of vascular 2930 and renal 31 complications rather than diabetes itself suggesting that OxS is apparent only when advanced tissue damage has oc• curred. 4. Increased levels of 3DG a nonoxidative AGE precursor are ob-

slide 117:

B • 0 • Nondiabetic 0 Diabetic e 120 e Oxidative Stress in Diabetes 83 .::- GI :E 120 20 Gi s:. CL 0 :::: 80 0 e 15 o .§ 0 E . 0 40 GI :E 10.. . : .. 5 0 0 20 40 0 60 0 20 40 60 AGE years c 200 • 160 i :E 0 :: 0 e 80 §. 40 :E • i---..---...---.......................... 0 0 5 10 15 20 25 o-Tyr µmol/mol Phe Figure 3 Age-dependent increase in A MetSO and B o-tyrosine in human skin collagen from diabetic and nondiabetic subjects. Despite significant differences in the absolute concentrations of these biornarkers there is a strong correlation C between levels of these primary oxidation products in skin collagen. From Ref. 20.

slide 118:

84 Baynes and Thorpe served in diabetic serum 3233 suggesting a more generalized in• crease in carbonyl stress 11 in diabetes. V. CARBONYL STRESS IN DIABETES The increase in lipoxidation and glycoxidation products in diabetes is the di• rect result of an increase in carbonyl precursors however not all of these intermediates are derived from oxidative reactions. 3DG for example is formed nonoxidatively from Amadori compounds 34 or fructose-3-phos• phate 35 and is also increased in diabetes along with increases in both pyrra• line and 3DG-arginine imidazolone 36 adducts that are derived from 3DG by nonoxidative mechanisms. MGO adducts to protein including CEL and MOLD are also increased in diabetes 3738. Like 3DG MGO is formed by anaerobic mechanisms either enzymatically as an intermediate in amino acid catabolism or by l-elimination reactions of triose phosphates 3940. How• ever MGO may also be produced during the oxidative chemistry of both lipids and carbohydrates so that its precise origin in vivo is unknown. In any case the increase in 3DG and possibly MGO and their adducts to proteins suggests that limitations in the detoxification of reactive carbonyl compounds produced by both oxidative and nonoxidative mechanisms underlie the increased chemi• cal modification of proteins in diabetes Fig. 4. There are three major routes for detoxification of reactive carbonyl compounds: NAD+-dependent oxidation of aldehydes to carboxylic acids NADPH-dependent reduction of aldehydes to alcohols and rearrangement of ketoaldehydes to hydroxyacids. The first pathway is illustrated by the oxida• tion of HNE to hydroxynonenoic acid 4142 or of 3DG to 2-keto-3-deoxyglu• conic acid 43 the second by reduction of 3DG to 3-deoxyfructose 4445 and the third by the GSH-dependent glyoxalase pathway 40. Conjugation of reactive aldehydes to GSH thiohemiacetals by GSH S-transferases is also important in presenting substrates for oxidation by dehydrogenases 4246 for metabolism of MGO in the glyoxalase pathway 38 or for export of the GSH conjugates from cells 4247. There is widespread evidence that these pathways are compromised in diabetes as a result of shifts in redox coenzyme systems or that they are overwhelmed by an excess of carbonyl substrates. The shift in the redox potential of the NAO+ II NADH couple during pseudo• hypoxia 4849 may limit NAO+ -dependent oxidation reactions contributing to the increase in diacylglycerol concentration and activation of protein kinase C. Shifts in the NADP+ II NADPH system as a result of polyol pathway activity

slide 119:

l Oxidative Stress in Diabetes 85 Protein Primary ROS ----- ... oxidation products Substrates Lipid Carbohydrate Amino acids ::ROS Reactive Protein carbonyl intermediates Detoxification Oxidation NAD+ Reduction NADPH Rearrangement GSH Export GSH conjugates Secondary oxidation products nonoxidative AG Es Figure 4 Reaction scheme illustrating the role of impaired detoxification pathways in increased formation of lipoxidation and glycoxidation products and nonoxidative AGEs in tissues in diabetes. The increase in substrate concentrations and impairment of detoxification systems rather than increased oxidative stress is considered the most significant factor contributing to the increased chemical modification of proteins in diabetes. may limit the efficiency of NADPH-dependent reduction reactions 50 ex• plaining the increase in both 3DG 3031 and 3-deoxyfructose 51 in plasma of diabetic patients and decreases in GSH 52. Similarly the parallel in• creases in MGO n-lactate and S-lactoylglutathione 40 in diabetic blood suggests an overload on the glyoxalase pathway in diabetes resulting from a combination of increased substrate flux rate-limiting GSH production and excessive GSH-conjugate efflux from tissues. The combination of increased concentration of oxidizable substrates and decreased efficiency of detoxification pathways can cause an increase in the concentration of reactive carbonyl intermediates even in the absence of an increase in OxS. The resultant increase in secondary oxidation products lip• oxidation and glycoxidation products in tissues may appear at first glance to be the result of an increase in OxS. However the increase in secondary oxidation products lipoxidation and glycoxidation products 13 without a corresponding increase in primary oxidation products MetSO and a-tyrosine 20 suggests that this is not the case in diabetes. At least in the early stages of the disease before the appearance of overt complications the increase in

slide 120:

86 Baynes and Thorpe secondary oxidation products can be attributed to an increase in oxidizable substrates coupled with insufficient detoxification activity without invoking an increase in oxidative stress. VI. IS THERE A RATIONALE FOR ANTIOXIDANT THERAPY IN DIABETES ln principle all chronic disease should respond to antioxidant therapy. In fact however antioxidant therapy has had limited impact on the progress of chronic diseases such as chronic pulmonary disease or autoimmune diseases such as lupus erythematosus or rheumatoid arthritis. There is epidemiological evi• dence that antioxidants may limit atherogenesis but alternative therapies such as antihypertensive and lipid-lowering drugs are substantially more effective than antioxidant therapy. Antioxidant therapy in autoimmune diseases is also symptomatic therapy directed at limiting the damage rather than suppressing the pathogenic mechanism. Likewise antioxidant therapy to address the fulmi• nating OxS characteristic of advanced complications in diabetes may have limited effect in retarding the development of early diabetic complications. However "antioxidant" therapy with agents such as N-acetylcysteine and lipoic thioctic acid which yield an increase in cellular GSH may provide protection against tissue damage by enhancing detoxification systems for reac• tive carbonyl compounds. GSH is a bifunctional coenzyme-it supports both antioxidant and other detoxification activities in the cell. It is oxidized to GSSG when it functions as a coenzyme for antioxidant reactions e.g. in reduction of protein disulfides or in detoxification of hydrogen or organic per• oxides catalyzed by GSH peroxidase. However GSH is regenerated intact when it participates in the glyoxalase pathway or as a substrate for GSH S• transferases. In these instances it acts as a coenzyme not as an antioxidant. The therapeutic benefits of N-acetylcysteine and lipoic acid therapy may there• fore be attributable to their role in enhancing GSH-dependent detoxification rather than antioxidant pathways. At the same time other antioxidants such as ascorbate and vitamin E may spare GSH for use in detoxification functions providing some protective advantage to cells challenged by increase rates of reactive carbonyl formation in diabetes. The interplay between the antioxidant and carbonyl detoxification roles of GSH is critical for protecting the cell against both oxidative and carbonyl stress.

slide 121:

Oxidative Stress in Diabetes 87 VII. SUMMARY AND CONCLUSION In the foregoing discussion we have tried to define the nature of OxS and then using that definition to assess the status of OxS in diabetes. Based on analysis of various biomarkers of OxS in long-lived proteins we conclude that OxS is not overtly or systemically increased in diabetes except at later stages in the development of complications. Metabolic derangements in diabe• tes lead to an increase in concentration of oxidizable substrates and compro• mised detoxification pathways. The resulting increase in reactive carbonyls in tissues known as carbonyl stress leads directly to increased chemical modifi• cation of proteins in diabetes. Efforts directed at decreasing substrate concen• tration maintenance of euglycemia and normolipidemia bolstering detoxi• fication pathways GSH precursors or enhancers and trapping reactive carbonyl species AGE inhibitors carbonyl traps represent reasonable thera• peutic approaches for limiting the chemical modification and crosslinking of proteins in diabetes and inhibiting the development of diabetic complications. Antioxidant vitamins and drugs may spare coenzymes for detoxification path• ways during early stages of diabetes and may be useful as supportive therapy at later stages of the disease. ACKNOWLEDGMENT Supported by research grant DK-19971 from the National Institutes of Diabe• tes and Digestive and Kidney Diseases. REFERENCES I. Suzuki YJ Forman HJ Sevanian A. Oxidants as stimulators of signal transduc• tion. Free Rad Biol Med 1997 22:269-285. 2. Ghisclli A Serafini M Maiani G Azzini E Ferro-Luzzi A. A fluorescence-based method for measuring total plasma antioxidant capability. Free Rad Biol Med 1995 18:29-36. 3. Tubaro F Ghiselli A Rapuzzi P Maiorino M Ursini F. Analysis of plasma antioxidant capacity by competition kinetics. Free Rad Biol Med 1998 24: I 228- 1234. 4. Delmas-Beauvieux MC Peuchant E Thomas MJ Dugourg L Pinto AP Clerc M Gin H. The place of electron spin resonance methods in the detection of

slide 122:

88 Baynes and Thorpe oxidative stress in type 2 diabetes with poor glycemic control. Clin Biochem 1998 31:221-228. 5. Beckman KB Ames BN. Detection and quantification of oxidative adducts of mitochondrial DNA. Methods Enzymol 1996264:442-453. 6. Cadet J Berger M Douki T Ravanat JL. Oxidative damage to DNA: formation measurement and biological significance. Rev Physiol Biochem Pharmacol 1997 131:1-87. 7. Camhi SL Lee P Choi AM. The oxidative stress response. New Horizons 1995 3:170-182. 8. Stadtman ER. Covalent modification reactions are marking steps in protein turn• over. Biochemistry 1990 29:6323-6331. 9. Requena JR Fu MX Ahmed MU Jenkins AJ Lyons TJ Thorpe SR. Lipoxida• tion products as biomarkers of oxidative damage to proteins during lipid peroxi• dation reactions. Nephrol Dial Transplant I 996 11 suppl 1 :48-53. 10. Wells-Knecht KJ Brinkmann E Wells-Knecht MC Ahmed MU Zyzak DY Thorpe SR Baynes JW. New biomarkers of Maillard reaction damage to pro• teins. Nephrol Dial Transplant 1996 11 suppl 5:41-47. 11. Baynes JW Thorpe SR. The role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999 48: 1-9. I 2. Ahmed MU Brinkmann-Frye E Degenhardt TP Thorpe SR Baynes JW. N- car• boxyethyllysine a product of chemical modification of protein by methylglyoxal increases with age in human lens proteins. Biochem J 1997 324:565-570. 13. Dyer DG Dunn JA Thorpe SR Baillie KE Lyons TJ Mccance DR Baynes JW. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 1993 91 :2463-2469. 14. Nakamura K Nakazawa Y Ienaga K. Acid-stable fluorescent advanced glycation end products: vesperlysines A B and C are formed as crosslinked products in the Maillard reaction between lysine or proteins with glucose. Biochem Biophys Res Commun 1997 232:227-230. 15. Ienaga K Nakamura K Hochi T Nakazawa Y Fukunaga Y Kakita H Nakano K. Crosslines fluorophores in AGE-related cross-linked proteins. Contrib Nephrol I 995 112:42-51. 16. Requena JR Fu M-X Ahmed MU Jenkins AJ. Lyons TJ Baynes JW Thorpe SR. Quantitation of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low density lipoprotein. Biochem J 1997 322:317-325. 17. Fu M-X Requena JR Jenkins AJ Lyons TJ Baynes JW Thorpe SR. The ad• vanced glycation end-product N-carboxymethyllysine is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 1996 271 :9982- 9986. 18. Hazen SL Hsu FF dAvignon A Heinecke JW. Human neutrophils employ myeloperoxidase to convert alpha-amino acids to a battery of reactive aldehydes: a pathway for aldehyde generation at sites of inflammation. Biochemistry 1998 37:6864-6873.

slide 123:

Oxidative Stress in Diabetes 89 19. Brinkmann Frye E Degenhardt TP Thorpe SR Baynes JW. Role of the Maillard reaction in aging of tissue proteins: age-dependent increase in imidazolium cross• links in human lens protein. J Biol Chem 1998 273: 18714-18719. 20. Wells-Knecht MC Lyons TJ McCance DR Thorpe SR Baynes JW. Age• dependent accumulation of ort/10-tyrosine and methionine sulfoxide in human skin collagen is not increased in diabetes: evidence against a generalized increase in oxidative stress in diabetes. J Clin Invest 1997 I 00:839-846. 21. Halliwell B Gutteridge JMC. Free Radicals in Biology and Medicine. 2d ed. Oxford: Clarendon Press 1989. 22. Niwa T Katsuzaki T Miyazaki S Miyazaki T lshizaki Y Hayase F. Taternichi N. Takei Y. Immunohistochemical detection of imidazolone a novel advanced glycation end product in kidneys and aortas of diabetic patients. J Clin Invest 1997 99: 1272-1280. 23. Horie K Miyata T Maeda K. Miyata S Sugiyama S Sakai H van Ypersele C Monnier VM. Witztum JL Kurokawa K. Immunohistochemical colocalization of glycoxidation and lipid peroxidation products in diabetic renal disease: impli• cation of glycoxidative stress in the pathogenesis of diabetic nephropathy. J Clin Invest 1997 100:2995-3004. 24. Beckmann JS Yao ZY Anderson PG Chen J Accavitti MA Tarpey MM. White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe-Seyler 1994 375:81-86. 25. Hazen SL. Heinecke JW. 3-Chlorotyrosine a specific marker of myeloperoxi• dase-catalyzed oxidation is markedly elevated in low density lipoprotein isolated from human atherosclerotic plaque. J Clin Invest 1997 99:2075-2081. 26. Haberland ME Fong D Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science 1988: 241 :215- 218. 27. Kume S Takeya M Mori T Araki N Suzuki H Horiuchi S Kodama T Miyau• chi Y Takahashi K. lmmunohistochemical and ultrastructural detection of ad• vanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am J Pathol 1995 147:654-667. 28. Sayre LM Zelasko DA Harris PL Perry G. Salomon RG. Smith MA. 4-Hy• droxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimers disease. J Neurochem 1997 68:2092-2097. 29. Stringer MD Gorog PG Freeman A. Kakkar VY. Lipid peroxides and athero• sclerosis. Br Med J 1989 298:281-284. 30. Willems D. Dorchy H Dufrasne D. Serum antioxidant status and oxidized LDL in well-controlled young type I diabetic patients and without subclinical compli• cations. Atherosclerosis 1998: I 37suppl:S6 I -64. 31. Miyata T Fu MX Kurokawa K van Ypersele C Thorpe SR Baynes JW. Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: is the oxidative stress in uremia Kidney Int 1998 54: I 290- 1295. 32. Niwa T Takeda H Yoshizumi H Tatematsu A Ohara M. Timiyama S. Niimura

slide 124:

90 Baynes and Thorpe K. Presence of 3-deoxyglucosone a potent protein crosslinking intermediate of the Maillard reaction. Biochem Biophys Res Commun 1993 196:837-843. 33. Hamada Y Nakamura J Fujisawa H Yago H Nakashima E Koh N Hotta N. Effects of glycemic control on plasma 3-deoxyglucosone levels in NIDDM pa• tients. Diabetes Care 1997 20:1466-1469. 34. Hayashi T Namiki M. Role of sugar fragmentation in the Maillard reaction. In: Fujimaki M Namiki FM Kato H eds Amino-carbonyl Reactions in Food and Biological Systems. Amsterdam: Elsevier 1986:29-38. 35. Lal S Szwergold BS Taylor AH Randall WC Kappler F Wells-Knecht KJ Baynes JW Brown TR. Metabolism of fructose 3-phosphate in the diabetic rat lens. Arch Biochem Biophys 1995 318:191-199. 36. Niwa T Katsuzaki T Miyazaki S Miyazaki T Ishizaki Y Hayase F Tatemichi N Takei Y. lmmunohistochemical detection of imidazolone a novel advanced glycation end product in kidneys and aortas of diabetic patients. J Clin Invest 1997 99:1272-1280. 37. Degenhardt TP Thorpe SR Baynes JW. Chemical modification of proteins by methylglyoxal. Cell Mol Biol 1998 44:1139-1145. 38. Nagaraj RH Shipanova IN. Faust FM. Protein crosslinking by the Maillard reac• tion. Isolation characterization and in vivo detection of a lysine-lysine cross• link derived from methylglyoxal. J Biol Chem 1996 271: 19339-19345. 39. Richard JP. Kinetic parameters for elimination reaction catalyzed by triosephos• phate isomerase and an estimation of the reactions physiological significance. Biochemistry 1991 30:4581-4585. 40. Thornalley PJ. The glyoxalase system in health and disease. Mol Aspects Med 1993 14:287-371. 41. Siems W Grune T Beier B Zollner H Esterbauer H. The metabolism of 4- hydroxynonenal a lipid peroxidation product is dependent on tumor age in Ehr• lich mouse ascites cells. In: Emerit I Chance B eds. Free Radicals and Aging. Basel: Birkhauser 1992:124-135. 42. Srivastava S Chandra A Wang LF Seifert WE DaGue BB Ansari NH Srivas• tava SK Bhatnagar A. Metabolism of the lipid peroxidation product 4-hydroxy• trans-2-nonenal in isolated perfused rat heart. J Biol Chem 1998 273: 10893- 10900. 43. Fujii E Iwase H Ishii-Karakasa I Yajirna Y Hotta K. The presence of 2-keto- 3-deoxygluconic acid and oxoaldehyde dehydrogenase activity in human eryth• rocytes. Biochem Biophys Res Commun 1995 210:852-857. 44. Kato H van Chuyen N Shinoda T Sekiya F Hayase F. Metabolism of 3-deoxy• glucosone an intermediate compound in the Maillard reaction administered orally or intravenously to rats. Biochim Biophys Acta 1990 1035:71-76. 45. Kanazu T Shinoda M Nakayama T Deyashiki Y Hara A. Sawada H. Aldose reductase is a major protein associated with 3-deoxyglucosone reductase activity in rat pig and human livers. Biochem J 1991 279:903-906. 46. Srivastava S Chandra A Bhatnagar A Srivastava S Ansari NH. Lipid peroxida• tion product 4-hydroxynonenal and its conjugate with GSH are excellent sub-

slide 125:

Oxidative Stress in Diabetes 91 strates of bovine lens aldose reductase. Biochem Biophys Res Commun 1995 217:741-746. 47. Ishikawa T Esterbauer H Sics H. Role of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydroxy• nonenal in the heart. J Biol Chem 1986 261:1576-1581. 48. Williamson JR Chang K Frangos M Hasan KS Ido Y Kawamura T Nyen• gaard JR Yan den Enden M Kilo C Tilton RG. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993 42:801-813. 49. Jdo Y Kilo C Williamson JR. Cytosolic NADH/NAD free radicals and vascu• lar dysfunction in early diabetes mellitus. Diabetologia 1997 40:S 115-S 117. 50. Bravi MC Pietrangeli P Laurenti 0 Basili S Cassone-Faldetta M Ferri C De Mattia G. Polyol pathway activation and glutathione redox status in non-insulin• dependent diabetic patients. Metabolism 1997 46: 1194-1198. 51. Wells-Knecht KJ Lyons TJ. Mccance DR Thorpe SR Feather MS. Baynes JW. 3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes. Diabetes 1994 43: 1152-1156. 52. Samiec PS Drews-Betsch C Flagg EW Kurtz JC Sternberg P Jr. Reed RL Jones DP. Glutathione in human plasma: decline in association with aging. age• related macular degeneration and diabetes. Free Rad Biol Med 1998 24:699- 704.

slide 126:

This Page Intentionally Left Blank

slide 127:

7 AntioxidativeDefense in Diabetic Peripheral Nerve: Effects of DL-a• Lipoic Acid Aldose Reductase Inhibitor and Sorbitol Dehydrogenase Inhibitor Click Here If You Also Want To Be Free From Diabetes Irina G. Obrosova and Douglas A. Greene University of Michigan Medical Center Ann Arbor Michigan Hans-lochen Lang Hoechst Marion Roussel Frankfurt Germany Diabetes-induced oxidative stress in target tissues for diabetic complications including peripheral nerve results from at least three mechanisms Fig. 1 including glucose autooxidation formation of advanced glycation end prod• ucts and increased aldose reductase AR activity I. The contribution of oxidative stress to peripheral diabetic neuropathy has been well established 1-6. Diabetes-induced oxidative stress leads to decreased endoneurial blood flow with resulting endoneurial hypoxia 1-467. Increased formation of re• active oxygen species ROS impairs neurotrophic support 8 and causes re• dox imbalances 9 energy deficiency 9 and perhaps defects in ion-transport mechanisms which theoretically can be both mediated by and be independent of the corresponding changes in nerve blood flow. Although the role of oxidative stress in diabetes-induced nerve vascular

slide 128:

dysfunction and nerve conduction deficits is no longer a subject for debate there is still a number of questions that need to be addressed. It is still unclear 93

slide 129:

-- ... 94 Obrosova et al. HMfflii·H M·HiMI Figure 1 Mechanisms and pathogenetic consequences of oxidative stress in diabetic peripheral nerve. and no consensus has been reached so far what the relative contribution of oxidative stress-linked defects in endoneurial blood flow metabolism and neurotrophic support to nerve conduction deficits is. It remains to be estab• lished whether ROS-induced neurovascular dysfunction and resulting endo• neurial hypoxia mediate the diabetes-induced decrease in neurotrophic support as they at least partially mediate metabolic defects 10 considering that the levels of substance P a product of nerve growth factor NGF-influenced gene in primary afferents in the nerve are decreased under hypoxic conditions 11. Mechanisms leading from hyperglycemia to increased ROS formation require further studies as well. In particular the relative contribution of the three afore• mentioned ROS-generating mechanisms to diabetes-induced nerve free radical damage remains to be identified. Little information is available on the changes in antioxidative defense enzymes 1 and mechanisms of their downregulation in diabetic peripheral nerve and a possibility of modulation of their activity with antioxidants and other pharmacological interventions. In addition the role for AR in diabetes-induced changes in nerve antioxidant status needs further studies because reports 2 12 indicate an inconsistency between marked depletion of nerve total glutathione TG versus very minor increase in oxidized glutathione GSSG in diabetes and restoration of TG levels with

slide 130:

Antioxidative Defense in Diabetic Peripheral Nerve 95 AR inhibitor ARI treatment which points to the contribution of other AR• dependent mechanisms in addition to or instead of AR-mediated NADPH deficiency to nerve antioxidant deficit. Also it remains to be established whether there is any role for the second enzyme of the sorbitol pathway sorbi• tol dehydrogenase in diabetes-induced nerve oxidative injury. Some of the aforementioned questions were addressed in the present study which was designed to identify diabetes-related deficits of peripheral nerve antioxidative defense enzymes to evaluate a role of oxidative stress in impairment of protective mechanisms against superoxide hydrogen peroxide and semiquinone radical-induced oxidative injury by assessing a possibility of preventing downregulation of superoxide dismutase catalase and total quinone reductase by antioxidant DL-a-lipoic acid and to compare the ef• fects of ARI and sorbitol dehydrogenase inhibitor SDI on parameters of oxi• dative stress and antioxidative defense in diabetes and thus to identify the role for two enzymes of the sorbitol pathway in diabetes-induced nerve oxidative injury. I. MATERIALS AND METHODS Experiments were performed in accordance with regulations specified by the National Institutes of Health 1985 revised version of the principles of labora• tory animal care and University of Michigan Protocol for Animal Studies. A. Animals Banier-sustained cesarean-deliveredmale Wistar rats Charles River Wilming• ton MA body weight 250-300 g were fed a standard rat chow diet ICN Biomedicals Cleveland OH and had ad libitum access to water. Diabetes was induced by a single intraperitoneal injection of streptozotocin Upjohn Kalamazoo MI 55 mg/kg body weight IP in 0.2 mL of 10 mM citrate buffer pH 5.5 to animals that were fasted overnight. Blood samples for measure• ments of glucose were taken from the tail vein -48 h after streptozotocin injection and the day before they were killed. The rats with blood glucose 250 mg/dL were considered diabetics and the treatments were started -48 h after streptozotocin injection. Three experiments were performed. In experi• ment 1 the experimental groups included control 6-week diabetic and 6- week diabetic rats treated with nt-n-lipoic acid Sigma 100 mg/kg body weight/day IP 5 days a week. Experiment 2 was performed in control 3- week diabetic and 3-week diabetic rats treated with the SDI-157 Hoechst

slide 131:

96 Obrosova et al. Marion Roussel lOO mg/kg body weight/day in the drinking water. In exper• iment 3 the groups included control 6-week diabetic and 6-week diabetic rats treated with the ARI sorbinil Pfizer 65 mg/kg body weight/day in the diet for 2 weeks after 4 weeks of untreated diabetes. B. Reagents Unless otherwise stated all chemicals were of reagent-grade quality and were purchased from Sigma Chemical St. Louis MO. Methanol high-perfor• mance liquid chromatography grade perchloric acid hydrochloric acid and sodium hydroxide were purchased from Fisher Scientific Pittsburgh PA. Ethyl alcohol 200 proof dehydrated alcohol U.S.P. punctilious was pur• chased from Quantum Chemical Company Tiscola IL. C. Experimental Procedure Rats from each group were sedated with carbon dioxide and subsequently killed by cervical dislocation. Both nerves were rapidly dissected carefully blotted with fine filter paper to remove any accompanying blood and frozen in liquid nitrogen for subsequent biochemical analyses. In experiment 1 we measured levels of sorbitol pathway intermediates glucose sorbitol fructose and activities of antioxidative defense enzymes including superoxide dismu• tase SOD catalase glutathione reductase GSSGRed glutathione trans• ferase GSHTrans total quinone reductase TQR and DT-diaphorase. In experiment 2 and 3 in addition to the above-mentioned sorbitol pathway inter• mediates and antioxidative defense enzyme activities except DT-diaphorase measurements of total MDA and 4-hydroxyalkenal and reduced glutathione GSH levels and glutathione peroxidase GSH-Px activities were performed. D. Biochemical Measurements 1. Measurements of Sorbitol Pathway Intermediates Nerve segments -20 mg were weighed and homogenized in 0.8 mL 0.9 NaCL A lOO-µL volume of 0.3 M zinc sulfate followed by an equivalent of barium hydroxide was then added to homogenate for protein precipitation. The samples were centrifuged at 4000 X g for lO min Sorvall MC 12V and aliquots of the supernatant were taken for spectrofluorometric measure• ments of glucose sorbitol and fructose by enzymatic procedures using hexokinase/glucose 6-phosphate dehydrogenase 13 sorbitol dehydrogenase

slide 132:

Antioxidative Defense in Diabetic Peripheral Nerve 97 14 and fructose dehydrogenase 15. In brief the analytical mixture for glu• cose contained 0.9 mL of I mM MgCl2 0.5 mM dithiothreitol 300 µM ATP 5 µM NADP and 0.02 U/mL glucose 6-phosphate dehydrogenase in 25 mM Tris-HCI buffer pH 8.1 and deproteinized extract 0.1 mL for control and 0.02 plus 0.08 mL H20 for diabetic nerves. The reaction was initiated by addition of -0.14 U of hexokinase. Initial and final readings were taken at "A excitation 340 nm "A emission 460 nm slits 20 and 15 spectrofluorometer Perkin-Elmer LS-58 and were compared with the corresponding glucose standards 0.5-10 x I 0-9 M processed in the same nm. The analytical mix• ture for sorbitol contained 0.8 mL 0.5 mM NAD in 0.1 M glycine-NaOH buffer pH 9.5 and deproteinized extract 0.2 mL for control and 0.05 mL plus 0.15 mL H20 for diabetic nerves. The reaction was started by addition of -0.8 U of sorbitol dehydrogenase. Initial and final readings were taken at "A excitation 340 nm "A emission 460 nm slits 20 and 15. The analytical mix• ture for fructose contained 0.9 mL of rezasurin-containing 150 mM citrate buffer pH 4.5 I aliquot of rezasurin 5 mg-10 mL H20 was mixed with I 00 aliquots of citrate buffer and deproteinized extract 0.1 mL for control and 0.05 mL plus 0.05 mL H20 for diabetic nerves. The reaction was started by addition of -0.5 U of fructose dehydrogenase. Initial and final readings were taken at "A excitation 572 nm "A emission 585 slits 5 and 5. The differ• ences in initial and final readings for tested samples were compared with those with corresponding sets of standards for glucose sorbitol and fructose 0.1- 10 X 10-9 M processed in the same run. 2. Measurements of GSH Nerve segments -15-20 mg were weighed homogenized in I mL of ice• cold 6 HCl04 and centrifuged at 4000 X g for IO min. After centrifugation the samples were immediately neutralized with SM K2C03 to pH 6- 7 and were centrifuged again at 4000 X g for 5 min to precipitate insoluble KC104• A total of 0.1 mL of extract was mixed with 0.89 mL of 20 mM EDT A in 1.0 M Tris-HCI buffer pH 8.1 and the reaction was initiated by addition of 0.0 I mL of o-phthaldialdehyde 10 mg- I mL methanol. Initial and final read• ings were taken at "A excitation 345 nm "A emission 425 nm slits 5 and 5. The differences in initial and final readings were compared with those in corre• sponding GSH standards 1-10 X 10-9 M processed in the same run. 3. Measurements of Total MDA and 4-Hydroxyalkenal Measurements of total malondialdehyde MDA and 4-hydroxyalkenal levels were performed using kits from Oxis International Inc. LP0-586 assay. The

slide 133:

98 Obrosova et al. method is based on the reaction of a chromogenic reagent N-methyl-2-phenyl• indole with MDA and 4-hydroxyalkenals at 45 °C. The samples were prepared by homogenization of preweighed nerve segments -40 mg in I mL of 20 mM Tris buffer pH 7.4 containing 5 mM butylated hydroxytoluene a total of 200 µL of homogenate was used for measurements of total MDA and 4- hydroxyalkenal levels according to the procedure described in detail in the kit. The absorbance of chromogenic product was measured at 586 nm spectro• photometer Beckman DU 640 and was compared with the absorbance in cor• responding 4-hydroxyalkenal standards. 4. Measurements of Antioxidative Defense Enzyme Activities For measurements of antioxidative defense enzyme activities -30 mg of fro• zen sciatic nerves were homogenized in 2 mL of ice-cold 0.1 M sodium-phos• phate buffer pH 6.5 16. Homogenates were centrifuged at 20000 X g and supernatant fraction was used for assays of enzymatic activities and protein content. Protein levels were quantified with the Pierce BCA protein assay kits Rockford IL. SOD activity was measured by following spectrophotometri• cally at 480 nm the autooxidation of - -epinephrine at pH 10.4 17. The reaction mixture contained 0.8 mL 50 mM glycine buffer pH I 0.4 and 0.2 mL supernatant. The reaction was started by addition of 0.02 ml of - j-epi• nephrine due to poor solubility of --epinephrine in water the solution was prepared by suspending 40 mg of the compound in 2 mL water and then by adding two to three drops of 2 N HCI. SOD activity was expressed as nmol of - -epinephrine protected from oxidation after addition of the sample com• pared with the corresponding readings in the blank cuvette. The molar extinc• tion coefficient of 4.02 mM-1 crn " was used for calculations. Catalase activity was measured by following spectrophotometrically at 240 nm for 5 min the decrease in absorbance of hydrogen peroxide after addition of 0.1 ml of super• natant to 0.9 mL of H02-containing 50 mM phosphate buffer pH 6.8. The enzyme activity was calculated using 2.04 mM-1 crn " as molar extinction coefficient. GSHTrans activity toward 1-chloro-24-dinitrobenzene CDNB was measured according to Habig et al. 18. A total of 0.8 rnL of the reaction mixture contained 0.1 M sodium-phosphate buffer pH 6.5 I mM GSH I mM CDNB preliminary dissolved in ethanol and I mM EDTA. The reaction was slatted by addition of 0.2 mL supernatant and was monitored spectropho• tometrically at 340 nm for 5 min. Calculations were performed using extinc• tion coefficient of 9.6 mM-1 cm ". GSSGRed activity was measured spectro• photometrically at 340 nm by monitoring NADPH oxidation coupled to reduction of GSSG to GSH 0.8 mL of the reaction mixture contained 0.1 M

slide 134:

Antloxidative Defense in Diabetic Peripheral Nerve 99 potassium-phosphate buffer pH 7.0 2.5 mM GSSG and 125 µM NADPH. The reaction was started by addition of 0.2 mL of the sample and was moni• tored for 5 min. The calculations were performed by using a molar extinction coefficient of 6.22 mM-1 cm " Total quinone reductase and nr-diaphorase activities were measured with p-benzoquinone as a substrate and Twin-20 as an activator as described in detail by Ernster et al. 19. E. Statistical Analysis The results are expressed as means ::: SD. In experiment I differences among experimental groups were determined by ANOV A and the significance of differences between these groups assessed by the Student-Newman-Keuls multiple range test. Significance was defined at p 0.05. In experiments 2 and 3 analysis of the parameters was performed on natural logarithm trans• formed data with the SAS general linear models procedure. Overall differ• ences among experimental groups for each parameter were first assessed by the Van der Waerden test individual pairwise comparisons were evaluated by least-square means analysis only if the Van der Waerden test was significant at p 0.05 for a given parameter. A nonparametric Blom transformation of all data was performed before assessment of individual pairwise group differ• ences. Uncorrected p values based on two-tailed tests of significance are shown for the relevant comparisons. II. RESULTS The body weights were lower in diabetic rats compared with those in controls experiment 1 327.3 ::: 50.9 vs. 420.4 ::: 27.0 g p 0.01 experiment 2 313.6 ::: 25.7 vs. 372.8 ::: 20.8 g p 0.05 and experiment 3 300.1 ::: 44. l vs. 428.3 ::: 17 .9 g p 0.0 I. The initial body weights were similar in control and diabetic groups in all three experiments not shown. No statistically sig• nificant difference was found between body weights in diabetic rats treated with nt-u-Iipoic acid 305.5 ::: 32.0 g SDI 308.5 ::: 32.6 g or ARI 307.8 :::: 65.2 g and the corresponding untreated groups. Blood glucose levels were markedly increased in diabetic rats compared with those in controls experiment 1 340.0 :::: 60.3 vs. 66.8 ::: 9.3 mg/dL experiment 2 334. l ::: 62.3 vs. 71.4 ::: I 0.5 mg/dL and experiment 3 326.3 ::: 42.7 vs. 58.2 ::: 5.0 p 0.001 for all three comparisons. Blood glucose levels in diabetic rats were not affected by nt-u-Iipoic acid 322.6 ::: 43.5 mg/dl. SDI 348.5 ::: 87.0 mg/dl. or ARI 332.7 ::: 38.5 mg/dL.

slide 135:

100 Obrosova et al. Table 1 The Levels of Glucose Sorbitol and Fructose umol/g wet weight in the Sciatic Nerve of Control and Diabetic Rats Treated With/Without DL-a-Lipoic Acid LA 11 7-8 Control Diabetic Diabetic + LA Glucose Sorbitol 3.54 :±:: 0.69 0.260 :±:: 0.091 10.46 :±:: 2.32 1.34 :±:: 0.51 19.44 :±:: 3.79t 2.14 :±:: 0.88:j: Fructose 2.00 :±:: 0.42 7.47 :±:: 1.23 10.12 :±:: 1.20:j: Significantly different compared with those in controls p 0.0001 . t+ Significantly different compared with those in untreated diabetics p 0.01 and 0.001 respec• tively. Nerve glucose sorbitol and fructose levels in control and diabetic rats treated with/without nr.-u-Iipoic acid are presented in Table I. Glucose sorbi• tol and fructose levels in diabetic rats were increased 3.9- 19.6- and 6.4- fold respectively compared with those in controls. DL-a-Lipoic acid treat• ment further increased levels of glucose sorbitol and fructose 1.9- 1.6- and 1.4-fold respectively compared with those in untreated diabetics. Nerve glucose sorbitol and fructose levels in control and diabetic rats treated with/without SDI are presented in Table 2. Glucose sorbitol and fruc• tose levels in diabetic rats were increased 3.6- 11.1- and 5.5-fold respec• tively compared with those in controls. Glucose levels were indistinguishable in diabetic rats treated with/without SDI. Sorbitol levels in the SDI-treated diabetic rats were increased 4.4-fold compared with those in untreated diabet• ics whereas fructose levels were markedly reduced but not completely nor• malized. Table 2 The Levels of Glucose Sorbitol and Fructose µmol/g wet weight in the Sciatic Nerve of Control and 3-Week Diabetic Rats Treated With/Without SDI n 7-8 Control Diabetic Diabetic + SDI Glucose Sorbitol 3.24 :±:: 0.94 0.149 :±:: 0.034 11.78 :±:: 4.17 1.65 :±:: 0.36 11.32 :±:: 2.78t 7.34 :±:: 1.77:j: Fructose 1.22 :±:: 0.15 6.66 :±:: 2.10 1.73 :±:: 0.77:j: Significantly different compared with those in controls p 0.0001 . t:J: Significantly different compared with those in untreated diabetics p 0.0 I and 0.00 I respec• tively.

slide 136:

0 Antioxidative Defense in Diabetic Peripheral Nerve 101 Table 3 The Levels of Glucose Sorbitol and Fructose umol/g wet weight in the Sciatic Nerve of Control and 6-Week Diabetic Rats Treated With/Without ARI n 7-8 Control Diabetic Diabetic + ARI Glucose 3.58 :::: 0.44 13.43 :::: 2.44 13.54 :::: 1.78 t Sorbitol Fructose 0.128 :::: 0.045 1.29 :::: 0.40 1.36 :::: 0.37 6.54 :::: 0.19 0.062 :::: 0.042:j: 1.21 :::: 0.33:j: Significantly different compared with those in controls p 0.00I. t Significantly different compared with those in untreated diabetics p 0.01 and 0.001 respec• tively. Nerve glucose sorbitol and fructose levels in control and diabetic rats treated with/without ARI are presented in Table 3. Glucose sorbitol and fruc• tose levels in diabetic rats were increased 3.8- 10.6- and 5.1-fold over those in controls. Glucose levels in diabetic rats were not affected by the ARI treat• ment whereas sorbitol levels were decreased below those in nondiabetic con• trols and fructose levels were normalized. Nerve total MDA and 4-hydroxyalkenal levels in control and diabetic rats treated with/without ARI and SDI are presented in Fig. 2 A and B. Total MDA and 4-hydroxyalkenal levels were increased in both 3-week diabetic rats and 6-week diabetic rats vs. controls p 0.02 and 0.001 respectively. The increase in 6-week diabetic rats was completely corrected by the ARI treatment p 0.001 vs. untreated diabetics whereas the increase in 3-week A B s - : 1.2 1.2 ·a :: 0.8 :: 0.8 0.4 0.4 :I. 0 0 Control Diabetes Diabetes Control Diabetes Diabetes +ARI +SDI Figure 2 Total MDA and 4-hydroxyalkenal levels in sciatic nerve of control and diabetic rats treated with or without ARI A or SDI B mean :::: SD n 8-12.

slide 137:

12 - 102 Obrosova et al. .c .210.1 G : I 4i 0. : en 0.0 E ::t A B 0.24 0.18 0.12 0.06 O Control Diabetes Diabetes +SDI Figure 3 GSH levels in sciatic nerve of control and diabetic rats treated with or without ARI A or SDI B mean :: SD n 8-12. diabetic rats further progressed with the SDI treatment p 0.03 vs. untreated diabetics Nerve GSH levels in control and diabetic rats treated with/without ARI and SDI are presented in Fig. 3 A and B. GSH levels were decreased by 23.4 and 37 .6 in 3-week and 6-week diabetic rats vs. corresponding controls p 0.002 and 0.001 respectively. The decrease in 6-week diabetic rats was completely corrected by the ARI treatment p 0.001 vs. untreated diabetics whereas the decrease in 3-week diabetic rats further progressed with the SDI treatment p 0.02 vs. untreated diabetics. Antioxidative defense enzyme activities in control and diabetic rats treated with/without nt -n-lipoic acid are presented in Table 4. SOD catalase Table 4 Antioxidative Enzyme Activities in the Sciatic Nerve of Control and 6- Week Diabetic Rats Treated With/Without DL-a-Lipoic Acid LA nmol/mg protein per min n 5-8. Control Diabetic Diabetic + LA SOD 95.9 :::: 18.9 70.5 :: 9.8 136.8 :: 55.8t Catalase 109.7 :::: 28.6 77.4 :: 19.8 122.9 :: 3t.5t GSHTrans 39.6 :: 15.9 57.7 :: 12.9 56.3 :: 19.8 GSSGRed 9.0 :::: 2.6 10.5 :: 3.0 12.0 :: 3.6 TQRed 174.9 :: 29.7 131.2 :: 32.8 170.4 :: 44.2 t DT-diaphorase 119.7 :: 25.8 123.8 :: 30.8 119.7 :: 30.9 Significantly different compared with those in controls p 0.05. H Significantly different compared with those in untreated diabetics p 0.05 and 0.01.

slide 138:

Ant ioxidative Defense in Diabetic Peripheral Nerve Table 5 Antioxidative Enzyme Activities in the Sciatic Nerve of Control and 103 3-Week Diabetic Rats Treated With/Without SDI nmol/mg protein per min n 8-10 Control Diabetic Diabetic + SDI SOD Catalase GSHTrans GSSGRed GSH-Px TQRed 50.8 :±: 17.8 194.6 :±: 56.6 39.3 :±: 4.9 21.6 :±: 3.5 13.7 :±: 3.6 313.9 :±: 52.3 73.0 :±: 26.4 195.2 :±: 50.2 31.5 :±: 2.8:j: 18.3 :±: 2.6 9.8 :±: 4.6 236.8 :±: 22.51 1 93.0 :±: 31.8 300. I :±: 82.3 t 30.4 :±: 3.6§ 16.5 :±: 2.5:j: 17.1 :±: 8.6t 193.5 :±: 39.2:J:1 :J:§11 Significantly different compared with those in controls p 0.05 O.ol. 0.001. and 0.0001 respectively. tII Significantly different compared with those in untreated diabetics p 0.05 and 0.0 I respec• tively. and TQRed acnvities were decreased in 6-week diabetic rats vs. controls whereas GSHTrans GSSGRed and DT-diaphorase activities were indistin• guishable between the two groups. DL-a-Lipoic acid treatment prevented dia• betes-induced downregulation of SOD catalase and TQRed and did not affect GSHTrans GSSGRed and DT-diaphorase. Antioxidative defense enzyme activities in control and 3-week diabetic rats treated with/without SDI are presented in Table 5. SOD catalase GSH- Table 6 Antioxidative Enzyme Activities in the Sciatic Nerve of Control and 6-Week Diabetic Rats Treated With/Without ARI nmol/mg protein per min n 8-10 Control Diabetic Diabetic+ ARI SOD 89.7 :±: 22.5 64.7 :±: 8.5 76.9 :±: 1 l.6H Catalase 102.0 :±: 37.1 71.6 :±: 13.4 76.8 :±: 17.5 GSHTrans 33.8 :±: 4.5 44.5 :±: 10.8 36.2 :±: 4.4 GSSGRed 13.1 :±: 1.9 12.2 :±: 2.5 12. :±: 2.5 GSH-Px TQRed 11.2 :±: 2.2 197.8 :±: 64.4 13.0 :±: 4.8 134.6 :±: 38.2 11.3 :±: 1.7 160.4 :±: 25.3§11 t§ Significantly different compared with those in controls p 0.05 0.01 and 0.001 re• spectively. :J:11 Significantly different compared with those in untreated diabetics p 0.05 and 0.01. respec• tively.

slide 139:

104 Obrosova et al. Px and GSSGRed activities were similar in control and 3-week diabetic rats. SOD catalase and GSH-Px were increased in SDI-treated diabetic rats com• pared with untreated diabetic group whereas GSSGRed activity was slightly decreased. TQRed and GSHTrans were decreased in 3-week diabetic rats vs. controls and TQRed but not GSHTrans activity was further decreased by the SDI treatment. Antioxidative defense enzyme activities in control and 6-week diabetic rats treated with/without ARI are presented in Table 6. Similar to experiment 1 SOD catalase and TQRed activities were decreased in the diabetic group vs. controls whereas GSH-Px GSSGRed and GSHTrans remained unaf• fected. The ARI treatment partially corrected SOD and TQRed but not catalase activity. GSH-Px GSSGRed and GSHTrans activities were indistinguishable between the SDI-treated and untreated diabetic groups. Ill. DISCUSSION The findings of the present study are indicative of increased vulnerability of diabetic peripheral nerve to free radical-induced oxidative damage and are consistent with the studies of Low and colleagues 12021 demonstrating accumulation of conjugated dienes and lipid peroxide reduction of GSH levels and CuZn-SOD activity and changes of other markers of oxidative stress in nerve in the model of streptozotocin-induced diabetes. Comparison of Tables 5 and 6 suggests that antioxidative defense enzyme activities especially those of SOD catalase GSSGRed and TQRed are age-dependent consistent with observations of age dependence for other parameters related to oxidative stress 20. Also it is important to point out that diabetes-induced changes in most parameters in the present study revealed strong dependence on the duration of diabetes. For example depletion of GSH the major biological antioxidant progressed with the duration of diabetes being more advanced in 6-week than in 3-week diabetic model. SOD catalase and TQRed activities remained within the normal range in 3-week diabetic rats but were markedly reduced in 6-week diabetic rats whereas on the contrary GSHTrans deficit appeared to be transient and was present in the 3-week but not in the 6-week model of streptozotocin diabetes. Another important characteristic of diabetes-induced impairment of antioxidative defense is its selectivity manifested by deficits in SOD catalase and TQRed coexisting in 6-week diabetes with normal GSSGRed GSHTrans and diaphorase activities. Although the significance of these changes for diabetes-induced increased ROS production and antioxida• tive defense against certain free radical species still remains to be established

slide 140:

Antioxidative Defense in Diabetic Peripheral Nerve 105 the finding of the diabetes-induced deficit in TQRed together with normal DT• diaphorase activity DT-diaphorase represents a cytoplasmic component of TQRed 19 is probably indicative of increased attack of semiquinone free radicals 16 very strong reducing agents that can also rapidly react with molecular oxygen generating the superoxide anion radical 02 in mitochondria and is consistent with the concept that mitochondria are a primary target for oxidative damage 1. It is also interesting that only those enzymes that are affected by diabetes respond to antioxidant DL-a-Iipoic acid treatment. The latter observation is consistent with the report of Maitra et al. 22 that demonstrated restoration of buthionine sulfoximine-induced catalase and GSH-Px deficits with o-lipoic acid treatment and with studies in other models of oxidative stress 2324 suggesting a possibility of posttranslational down• regulation of some antioxidative defense enzymes by oxidative modification of their proteins. The concept of posttranslational regulation of antioxidative defense enzymes in diabetic peripheral nerve is supported by recent unpub• lished findings of Phillip Low s laboratory demonstrating the lack of any effect of streptozotocin-diabetes of different duration on CuZn-SOD Mn-SOD GSSGRed and GSH-Px mRNAs. The finding of a marked upregulation of SOD by o-lipoic acid in diabetic peripheral nerve in the present study is differ• ent from reports for other tissues in other models of oxidative stress 2225 demonstrating the lack of correcting effect of n-lipoic acid on SOD activity. Based on this apparent discrepancy it is possible to suggest the indirect modu• lation of SOD by n-Iipoic acid in diabetic peripheral nerve probably via NGF which is known to both upregulate and replace SOD in some types of oxidative injury 2627. This assumption is supported by the findings of decreased NGF levels in diabetic peripheral nerve and of partial prevention of diabetes-in• duced deficit in neurotrophic support with ot-o-Iipoic acid treatment 28. The elevated levels of total MDA and 4-hydroxyalkenals products of lipid peroxidation are consistent with the presence of peripheral nerve oxida• tive injury in both 3-week and 6-week streptozotocin-diabetic rat models. As MDA levels were reported 20 to be similar in control and diabetic rats re• gardless of duration of diabetes the increase in lipid aldehyde level in the present study probably reflects the accumulation of 4-hydroxy-23-transnon• enal HNE a major toxic product of lipid peroxidation 29. Interestingly and surprisingly the lipid aldehyde level is normalized by ARI despite the fact that AR has been reported to be involved in HNE metabolism 29. A possible explanation can be derived from a comparison of the results of the present study with the recent report of Srivastava et al. 30 for isolated perfused hearts from nondiabetic rats indicating that the major metabolic transformations of HNE involve conjugation with glutathione and oxidation to 4-hydroxy-2-non-

slide 141:

106 Obrosova et al. enoic acid and providing evidence that sorbinil does not affect formation of glutathione-HNE conjugates at the same time preventing their further AR• mediated reduction to glutathione-14-dihydroxy-2-nonene and stimulates HNE oxidation to 4-hydroxy-trans-2-nonenoic acid. Based on these findings and assuming that the pathways of HNE metabolism are similar in heart and peripheral nerve one would expect to find an accumulation of HNE in periph• eral nerve under diabetic conditions characterized by depletion of GSH 2 and in the present study and by a decrease in the free cytosolic NAD+ /NADH ratio 931 that may affect NAD-dependent oxidation of HNE to 4-hydroxy• trans-2-nonenoic acid by aldehyde dehydrogenase which is consistent with the results of the present study. Also taking into consideration that ARI treat• ment corrects both GSH levels 12 and in the present study and the redox state of free cytosolic NAO-couple 31 the metabolic basis for prevention of total MDA and 4-hydroxyalkenal accumulation by sorbinil treatment becomes understandable. The importance of GSH for neutralization of toxic products of lipid peroxidation is confirmed by the experiments with SDI which effectively by -90.8 inhibited increased flux through sorbitol dehydrogenase in the 3- week streptozotocin-diabetic rat model. GSH depletion in 3-week streptozo• tocin-diabetic rats was further exacerbated by the SDI treatment which is consistent with a further accumulation of HNE over the level in the untreated diabetic group. A more advanced GSH depletion in the SDI-treated diabetic rats compared with the corresponding untreated group is consistent with the findings of Geisen et al. 32 for diabetic lens. These findings implicate sorbitol accumulation-linked osmotic stress in the mechanisms underlying increased vulnerability of diabetic peripheral nerve to oxidative injury and are in accor• dance with other reports demonstrating the inconsistency between a substantial -40 depletion of GSH versus a very minor 2 or an absent 12 depletion of GSSG which calls into question the importance of glutathione redox cy• cling mechanism in diabetes-induced GSH depletion. In addition total gluta• thione depletion in the diabetic nerve and correction of both total and reduced glutathione levels by ARls 12 and in the present study points to the involve• ment of other AR mediated mechanisms in addition to or instead of NADPH deficiency to nerve GSH deficit. It is important to point out that involvement of the osmotic factor in GSH depletion in the diabetic nerve does not necessar• ily mean increased GSH leakage from the endoneurium although some stud• ies for the lens 33 implicated decreased amino acid uptake and increased GSH efflux under hypersmotic conditions in the GSH deficit. We suggest that osmotic stress can be involved in diabetes-induced energy deficiency

slide 142:

Antioxidative Defense in Diabetic Peripheral Nerve 107 9 10 and considering that ATP is required for both y-glutamyl cysteine syn• thetase and glutathione synthetase reactions contributes to diabetes-related GSH deficit through the impairment of cofactor supply of GSH biosynthesis. The assumption regarding the link between sorbitol accumulation-linked os• motic stress and energy deficiency in diabetic peripheral nerve is supported by observations in DL-a-lipoic acid-treated diabetic rats demonstrating an in• creased sorbitol accumulation and the lack of prevention of nerve energy defi• ciency despite correction of both mitochondrial and cytosolic redox state of NAD couple 934 and by the findings of exacerbated decrease of nerve phosphocreatine/creatine ratio a marker of free cytosolic ATP/ ADP ratio in diabetic rats treated with SDI CP-166572 Pfizer 200 mg/kg/day in the drinking water which had nerve sorbitol levels exceeding those in untreated diabetics -6.3-fold Obrosova 1996 unpublished observations. It has been suggested 35 that diabetes-induced increase in flux through sorbitol dehydrogenase causes increase in free cytosolic NADH/NAD+ ratio so called "pseudohypoxia" which in turn contributes to increased ROS formation due to activation of NADH oxidase a superoxide generating en• zyme studies of NADH oxidase in diabetic tissues reported contradictory re• sults 36-38. If this was true one would expect the SDI treatment to be beneficial on parameters of oxidative injury and antioxidative defense. How• ever the present study demonstrates that the SDI treatment resulted in an opposite effect because total MDA and 4-hydroxyalkenal accumulation GSH depletion and total quinone reductase deficit in the diabetic nerve were exacer• bated with the SDI treatment. Interestingly nerve SOD catalase and GSH• Px activities which were similar in control and 3-week streptozotocin-diabetic rats were increased in 3-week streptozotocin-diabetic rats treated with SDI a phenomenon probably indicative of a compensatory response to the increased attack of ROS of superoxide and hydrogen peroxide due to exacerbated GSH and total quinone reductase deficits. In conclusion the increased vulnerability of diabetic peripheral nerve to oxidative injury results from impairment of antioxidative defense mechanisms manifested by decreased GSH levels and SOD catalase and total quinone reductase activities. Downregulation of these enzymes under diabetic condi• tions is caused by ROS probably posttranslational regulation and is pre• vented by antioxidant DL-a-lipoicacid treatment. AR is an important although not the only mechanism of diabetes-induced oxidative injury whereas sorbitol dehydrogenase has a protective role and its inhibition exacerbates oxidative damage. These findings implicate sorbitol accumulation-linked osmotic stress in nerve antioxidant deficit in diabetes.

slide 143:

108 Obrosova et al. REFERENCES I. Low PA Nickander KK Tritschler HJ. The roles of oxidative stress and antioxi• dant treatment in experimental diabetic neuropathy. Diabetes 1997 462S:38S- 42S. 2. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Dia• betes Care 1995 18: 1160-1 167. 3. Cameron NE Cotter MA Archibald V et al. Anti-oxidant and pro-oxidant ef• fects on nerve conduction velocity endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats. Diabetologia 1994 37:449- 459. 4. Cotter MA Love A Watt MJ et al. Effects of natural free radical scavengers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia 1995 38:1285-1294. 5. Karaso C Dewhurst M Stevens EJ Tomlinson DR. Effects of anti-oxidant treat• ment on sciatic nerve dysfunction in streptozotocin-diabetic rats comparison with essential fatty acids. Diabetologia 1995 38: 129-134. 6. Cameron NE Cotter MA H01TObin DH Tritschler HJ. Effects of alpha-lipoic acid on neuro-vascular function in diabetic rats: interaction with essential fatty acids. Diabetologia 1998 41 :390-399. 7. Love A Cotter MA Cameron NE. Nerve function and regeneration in diabetic and galactosemic rats: antioxidant and metal chelator effects. Eur J Pharmacol 1996 31 :433-439. 8. Hounsom L Horrobin DR Tritschler H et al. A lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neu• ropathy. Diabetologia 1998 41:839-843. 9. Greene DA Cao X Van Huysen C Obrosova I. Effects of DL-a-lipoic acid on diabetic nerve function bioenergetics and antioxidative defense abstr. Diabetes 1998 47suppl I: A 136. IO. Obrosova I VanHeyningen D Cao X et al. Metabolic compensation for diabe• tes-induced endoneurial hypoxia abstr. J Periph Nerv Syst 1997 2:290. l I. Smith WJ Diemel LT Leach RM Tomlinson DR. Central hypoxaemia in rats provokes neurological defects similar to those seen in experimental diabetes mel• litus: evidence for a partial role of endoneurial hypoxia in diabetic neuropathy. Neuroscience 1991 45:255-259. 12. Hohman TC Banis D Basso M et al. Resistance to increased oxidative stress is decreased in experimental diabetic neuropathy abstr. J Periph Nerv Syst 1997 2:272. 13. Lowry OH Passonneau JV. A Flexible System of Enzymatic Analysis. Orlando: Academic Press 1972. 14. Bergmeyer HU. Methods of Enzymatic Analysis. Weinheim: Verlag Chemie 1974.

slide 144:

Antioxidative Defense in Diabetic Peripheral Nerve 109 15. Holmes EW. Coupled enzymatic assay for the determination of sucrose. Anal Biochem 1997 244:103-109. 16. Romero FJ Monsalve E Hermenegildo C et al. Oxygen toxicity in the nervous tissue: comparison of the antioxidant defense of rat brain and sciatic nerve. Neu• rochem Res 1991 16: 157-161. 17. Misra HP Fridovich I. The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem l 972 247:3170-3175. 18. Habig WH Pabst MJ Jacoby WB. Glutathione S-transferases. The first enzy• matic step in mercapturic acid formation. J Biol Chem 1974 249:7130-7139. 19. Ernster L Danielson L Ljunggren M. OT diaphorase. I. Purification from the soluble fraction of rat liver cytoplasm and properties. Biochim Biophys Acta 1962 58:171-188. 20. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes 1991 40:873-877. 21. Nickander KK Schmelzer JD Rohwer DA Low PA. Effect of alpha-tocopherol deficiency on indices of oxidative stress in normal and diabetic peripheral nerve. J Neurol Sci 1994 126:6-14. 22. Maitra I Serbinova E Trischler H Packer L. Alpha-lipoic acid prevents buthio• nine sulfo-ximine-induced cataract formation in newborn rats. Free Rad Biol Med 1995 18:823-829. 23. Sumathi R Jayanthi S Kalpanadevi V Varalakshmi P. Effect of DL alpha-lipoic acid on tissue lipid peroxidation and antioxidant systems in normal and glycollate treated rats. Pharm Res 1993 27:309-318. 24. Sandhya P Varalakshmi P. Effect of lipoic acid administration on gentamicin• induced lipid peroxidation in rats. J Appl Toxicol 1997 17:405-408. 25. Seaton TA Jenner P Marsden CD. Mitochondrial respiratory enzyme function and superoxide dismutase activity following brain glutathione depletion in the rat. Biochem Pharmacol 1996 52:1657-1663. 26. Nistico G Ciriolo MR Fisk.inK et al. NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats. Free Rad Biol Med 1992 12: 177-181. 27. Santos FX Escudero M Perez L et al. Comparison of the effects of nerve growth factor and superoxide dismutase on vascular extravasation in experimental bums. Bums 1995 21:445-448. 28. Garrett NE Malcangio M Dewhurst M Tomlinson DR. n-Lipoic acid corrects neuropeptide deficits in diabetic rats via induction of trophic support. Neurosci Lett 1997 222:191-194. 29. Spycher SE Tabataba-Vakili S ODonnell VB et al. Aldose reductase induc• tion: a novel response to oxidative stress of smooth muscle cells. FASEB 1997 11:181-188. 30. Srivastava S Chandra A Wang LF et al. Metabolism of the lipid peroxidation product 4-hydroxy-trans-2-nonenal in isolated perfused rat heart. J Biol Chem 1998 273:10893-10900.

slide 145:

110 Obrosova et al. 31. Obrosova I Marvel J Faller A Williamson JR. Reductive stress is a very early metabolic imbalance in sciatic nerve in diabetic and galactose-fed rats abstr. Diabetologia 1995 38suppl I :AS. 32. Geisen K Utz R Grotsch H Lang J Nimmesgern H. Sorbitol-accumulating pyrimidine derivatives. Arzneim Forsch Drug Res 1994 44: I 032-1043. 33. Lou MF Dickerson JE Jr Garadi R York BM Jr. Glutathione depletion in the lens of galactosemic and diabetic rats. Exp Eye Res 1988 46:517-530. 34. Low PA Yao JK Kishi Y et al. Peripheral nerve energy metabolism in experi• mental diabetic neuropathy. Neurosci Res Comm 1997 21:49-56. 35. Williamson JR Chang K Frangos M et al. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993 42:801-813. 36. Kuo TH Moore KH Giacomelli F Wiener J. Defective oxidative metabolism of heart mitochondria from genetically diabetic mice. Diabetes 1983 32:781- 787. 37. Askar MA Baquer NZ. Changes in the activity of NADH oxidase in rat tissues during experimental diabetes. Biochem Mol Biol Int 1994 34:909-914. 38. Ellis EA Grant MB Murray Ff et al. Increased NADH oxidase activity in the retina of the BBZ/Wor diabetic rat. Free Rad Biol Med 1998 24: 111-120.

slide 146:

8 Pathways of Glucose-Mediated Oxidative Stress in Diabetic Neuropathy To Stop Diabetes In Few Days Click Here Douglas A. Greene Irina G. Obrosova Martin J. Stevens and Eva L. Feldman University of Michigan Medical Center Ann Arbor Michigan Diabetic distal symmetric sensorimotor polyneuropathy DPN the most com• mon peripheral neuropathy in developed countries l-3 affects up to 60- 70 of diabetic patients 4 and is the leading cause of foot amputation 5. The typical slowing of nerve conduction and the advancing distal symmetrical sensorimotor deficits are thought to reflect an underlying slowly progressive distal axonopathy of the dying-back type primarily affecting sensory nerve fibers 6. Improved blood glucose control substantially reduces the risk of developing DPN in insulin-dependent type 1 diabetes 78 thereby strongly implicating hyperglycemia as a causative factor. Animal and in vitro experiments have implicated a variety of enzymatic and nonenzymatic metabolic mechanisms in the initiation of glucose-induced neurotoxicity. These "metabolic initiators" include nonenzymatic glycation of proteins with subsequent chemical rearrangements yielding complex pro• tein adducts known as advanced glycation end products AGEs 9 LO auto• oxidation of glucose 11 increased aldose reductase AR activity leading to sorbitol and fructose accumulation NADP-redox imbalances and alterations

slide 147:

in signal transduction 12-14 and activation of protein kinase C PKC per• haps due to increased de novo synthesis of diacylglycerol DAG from glucose 111

slide 148:

112 Greene et al. and inhibition of DAG kinase 15-18. These metabolic initiators are compart• mentalized within the rich anatomical complexity and cellular heterogeneity of the peripheral nervous system PNS and its supporting vasculature and connective tissue elements. This compartmentalization channels and shapes the physiological response to metabolic initiators into the specific nerve fiber damage and loss underlying DPN. The intervening physiological mediators include interruption of nerve blood flow NBF 19-22 mitochondrial dys• function 1923 impaired neurotropic support 24 osmolyte derangements 14 and induction of neuronal and/or Schwann cell apoptosis 25. Combina• tions and permutations of metabolic initiators cellular and subcellular com• partmentalization and physiological mediators give rise to the current spec• trum of pathogenetic hypotheses for DPN Table 1. The intellectual challenge to basic and clinical scientists exploring the pathogenesis of DPN is the identification and characterization of the important initiators compartments and mediators common to various pathogenetic hypotheses. These common elements may serve as a linchpins around which to array and perhaps unify otherwise competing pathogenetic mechanisms. Glucose-induced generation of reactive oxygen species ROS may subserve this purpose Fig. 1 . Autooxidation of glucose catalyzed by trace amounts of free transition metals such as iron and copper 26 generate ROS in vitro 27 and metal chelating agents to preserve normal nerve conduction velocity Table 1 Putative Metabolic Initiators and Physiological Mediators of Glucose Toxicity in Experimental Diabetic Neuropathy Metabolic initiators Tissue compartments Physiological mediators Nonenzymatic gly• cation Sorbitol pathway Glucose autooxidation Protein kinase C acti• vation Endoneurial microcircu• lation Perineurial/epineurial vessels Dorsal root/anterior horn neurons Myelinated/ unmyelinated axons Schwarm cells Perineurial cells Distal motor/sensory projections Interruption of nerve blood flow Mitochondrial dysfunc• tion Reduced neurotrophic support Osmolyte derangements

slide 149:

Oxidative Stress in Diabetic Neuropathy 113 Figure 1 Generation of ROS from elevated levels of glucose may occur by multiple putative metabolic pathways: nonenzymatic glucose autooxidation and nonenzymatic formation of AGEs yellow and activation of the enzymes of the sorbitol pathway by mass action red. Sorbitol pathway activation produces osmolyte and NADPH deple• tion that diminish antiooxidative defense and produce osmotic stress. Osmotic stress may trigger cellular stress-response mechanisms such as PKC that have been linked to endothelial dysfunction vasoconstriction and ischemia. Ischemia and osmotic stress impair mitochondrial function and integrity which can limit mitochondrial contribution to oxidative defense. lschemia/reperfusion and impaired oxidative defense further magnify ROS accumulation vascular function and mitochondrial integrity. Neuro• trophic support that upregulates oxidative defense mechanisms may be impaired by ROS further compromising oxidative defense. and NBF in diabetic rats 28 transition metal handling may be impaired in experimental diabetes 29 Fig. 1 yellow. Furthermore ROS may intercon• nect autooxidation and AGE formation: ROS accelerate AGE formation and AGEs in turn supply ROS "autooxidative glycosylation" 30 Fig. 1 yel• low. AGEs generate ROS through a series of complex biochemical and mo• lecular pathways 31-33. Binding of AGEs to their cell surface receptor RAGE is associated with activation and nuclear translocation of the tran• scription factor NF-KB 34 possibly contributing to endothelial dysfunction 3536 impaired NBF and ischemia Fig. 1 black. Activation of the AR pathway has also been linked to ROS generation Fig. 1 red. Reduction of glucose to sorbitol by AR oxidizes NADPH di• rectly impairing antioxidative defense 37. Sorbitol accumulation produces osmotic stress which may promote oxidative stress through depletion of gluta-

slide 150:

114 Greene et al. thione and other putative antioxidants such as taurine 38. The novel hypothe• sis that AR pathway activation produces mitochondrial dysfunction through osmotic stress is discussed in this volume. Mitochondrial dysfunction could impair antioxidative defense by diminishing ATP for the de novo synthesis of glutathione 39. AR pathway activation may also contribute to the activa• tion of PKC reported in some 1516 but not all tissues prone to diabetic complications total PKC activity is reduced rather than increased by diabetes in rat sciatic nerve 4041 but selective activation of specific isofonns in some tissue components has been described in diabetic kidney 42 and has not been excluded in diabetic PNS. Increased AR pathway activity could promote de novo DAG synthesis by diverting dihydroxyacetone phosphate toward formation of n-glycerophospbate or PKC activation through osmotic stimulation of the JNK-kinase cascade. PKC activation would further exacer• bate reciprocal osmolyte depletion promoted by sorbitol accumulation by in• hibiting the transport activity of the Na+ -myoinositol 43 and the Na+-taurine 44 cotransporters. If activation of endoneurial or perineurial vascular PKC promoted vasoconstriction and nerve ischemia then this would further exacer• bate mitochondrial dysfunction through oxygen deprivation ischemia. Mito• chondrial dysfunction impaired antioxidative defense and ischemia would all contribute further to the generation of ROS which would further exacerbate vasoconstriction Fig. I 19. ROS may interact with diminished neurotropic support impaired energy metabolism and ischemia in experimental DPN neurotrophism. Oxidative stress induced by diabetes 45 would be particularly injurious to the PNS which is particularly vulnerable to oxidative stress 46. Impaired neurotrophic support in diabetes 47 may be mediated by ROS 48. ROS contribute to inschemia-reperfusion injury 49. ROS-induced apoptosis may share similar cell death pathways with neurotrophic withdrawal 5051 and neurotrophins may protect against ROS damage by inducing antioxidative defense mecha• nisms 52-54. Recent data suggest that antioxidant therapy may ameliorate some aspects of reduced neurotrophic support in experimental DPN 55. Thus oxidative stress and ROS link all of the potential intiators encom• pass most of the cellular compartments in the PNS and relate to virtually all physiological mediators implicated in the progressive nerve fiber dysfunction damage and loss in DPN Table 1. In each of these pathogenetic elements generation of ROS may initiate a feed-forward cycle because oxidative stress itself impairs antioxidative defense mechanisms Fig. 1 double-headed black arrow 56 resulting in a "viscous" cycle of metabolic damage. The role of ROS and oxidative stress in DPN has only recently begun to emerge and its

slide 151:

Oxidative Stress in Diabetic Neuropathy 115 potential as a therapeutic target for DPN holds great but as yet unfulfilled promise. REFERENCES l. The DCCT Research Group. Factors in the development of diabetic neuropathy: baseline analysis of the neuropathy in the feasibility phase of the Diabetes Con• trol and Complication Trial DCCT. Diabetes 1988 37:476-481. 2. Pirart J. Diabetes mellitus and its degenerative complications: a prospective study of 4400 patients observed between 1947 and 1973. Diabetes Care 1978 I: I 68- 188. 3. Young MJ Boulton AJM Macleod AF Williams ORR Sonksen PH. A multi• centre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic populations. Diabetologia 1993 36: 150-154. 4. Division of Diabetes Translation Center for Disease Control American Diabetes Association National Center for Health Statistics The Bureau of the Census. Diabetes in America. 2nd ed. NIH Publication 95-1468. Bethesda MD: National Institutes of Diabetes and Digestive and Kidney Diseases 1995. 5. Brand PW. The diabetic foot. In: Ellenberg M Rifkin H eds. Diabetes Mellitus. New York: Medical Examination Publishing Co. Inc. 1982:829-849. 6. Thomas PK Brown MJ. Diabetic polyneuropathy. In: Dyck PJ Asbury AK Winegrad Al Porte D eds. Diabetic Neuropathy Philadelphia: W.B. Saunders 1987:56-65. 7. The DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993 329:977-986. 8. The DCCT Research Group. The effect of intensive diabetes therapy on the de• velopment and progression of neuropathy. Ann Intern Med 1995 122:561-568. 9. Schmidt AM Hasu M Popov D Zhang JH Chen J Yan SD Brett J Cao R Kuwahara K Costache G Simionescu N Simionescu M Stern D. Receptor for advanced glycation end products AGEs has a central role in vessel wall inter• actions and gene activation in response to circulating AGE proteins. Proc Natl Acad Sci USA. 1994 91:8807-8811. 10. Varma SD Devamanoharan PS Ali AH. Formation of advanced glycation end AGE products in diabetes: prevention by pyruvate and u-kcroglutarate. Mol Cell Biochem 1997 171:23-28. 11. Love A Cotter MA Cameron NE. Nerve function and regeneration in diabetic and galactosemic rats: antioxidant and metal chelator effects. Eur J Pharmacol 1996 31:433-439. 12. Greene DA Sima AA Stevens MJ Feldman EL Killen PD Henry DN Thomas

slide 152:

116 Greene et al. T Dananberg J Lattimer SA. Aldose reductase inhibitors: an approach to the treatment of diabetic nerve damage. Diabetes Metab Rev 1993 9:189-217. 13. Stevens MJ Dananberg J Feldman EL Lattimer SA Kamijo M Thomas TP Shindo H Sima AA Greene DA. The linked roles of nitric oxide aldose reduc• tase and Na" K+-ATPase in the slowing of nerve conduction in the streptozo• tocin diabetic rat. J Clin Invest 1994 94:853-859. 14. Cameron NE Cotter MA Basso M Hohman TC. Comparison of the effects of inhibitors of aldose reductase and sorbitol dehydrogenase on neurovascular function nerve conduction and tissue polyol pathway metabolites in streptozo• tocin-diabetic rats. Diabetologia 1997 40:271-281. 15. Inoguchi T Battan R Handler E Sportsman JR Heath W King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 1992 89: 11059-11063. 16. Bursell SE Takagi C Clermont AC Takagi H Mori F Ishii H King GL. Spe• cific retinal diacylglycerol and protein kinase C beta isoform modulation mimics abnormal retinal hemodynamics in diabetic rat. Invest Ophthalmol Vis Sci 1997 38:2711-2720. 17. Ishii H Koya D King GL. Protein kinase C activation and its role in the develop• ment of vascular complication in diabetes mellitus. 1 Mo Med 1998 78:21-31. 18. Cameron NE Cotter MA Lai K Hohman TC. Effects of protein kinase C inhibi• tion on nerve function blood flow and Na K+ -ATP-ase defects in diabetic rats abst. Diabetes 1997 46suppl I :3 IA. 19. Low PA Nickander KK. Tritschler HJ. The roles of oxidative stress and antioxi• dant treatment in experimental diabetic neuropathy. Diabetes 1997 462S:38S- 42S. 20. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Dia• betes Care 1995 18:1160-1167. 21. Cameron NE Cotter MA Archibald V Dines KC Maxfield EK. Anti-oxidant and pro-oxidant effects on nerve conduction velocity endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats. Diabetologia 1994 37:449-459. 22. Cotter MA Love A Watt MJ Cameron NE Dines KC. Effects of natural free radical scavengers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia 1995 38: 1285-1294. 23. Obrosova I VanHeyningen D Cao X Stevens M Greene D. Metabolic compen• sation for diabetes-induced endoneurial hypoxia. J Periph Nerv Syst 1997 2: 290. 24. Fernyhough P Diemel LT Tomlinson DR. Target tissue production and axonal transport of neurotrophin-3 are reduced in streptozotocin-diabetic rats. Diabeto• logia 1998 41:300-306.

slide 153:

Oxidative Stress in Diabetic Neuropathy 117 25. Ekstrom PA. Neurones and glial cells of the mouse sciatic nerve undergo apoptosis after injury in vivo and in vitro. Neuroreport 1995 6: 1029-1032. 26. Wolf SP. Transition metals and oxidative stress in the complications of diabetes. In: Gries FA Wessels K eds. The Role of Anti-oxidants in Diabetes Mellitus. Frankfurt am Main: pmi Verlagsgrupe 1993:82-101. 27. Jiang Z-Y Woollard ACS Wolf SP. Hydrogen peroxide production during ex• perimental protein glycation. FEBS Lett 1990 268:69- 71. 28. Cameron NE Cotter MA. Neurovascular dysfunction in diabetic rats. Potential contribution of autoxidation and free radicals examined using transition metal chelating agents. J Clin Invest 1995 96: 1159-1163. 29. Cutler P. Deferoxamine therapy in high-ferritin diabetes. Diabetes 1989 38: 1207-1210. 30. Baynes JW. Role of oxidative stress in the development of complications of diabetes. Diabetes 1991 40:405-412. 31. Van SD Schmidt AM Anderson GM Zhang J Brett J Zou YS Pinsky D Stem D. Enhanced cellular oxidant stress by the interaction of advanced glycation endproducts with their receptors/binding proteins. J Biol Chem 1994 269:9889- 9897. 32. Mullarkey CJ Edelstein D Brownlee M. Free radical generation by early glyca• tion products: a mechanism for accelerated atherogenesis in diabetes. Biochem Biophys Res Commun 1990 173:932-939. 33. Giardino I Fard AK Hatchell DL Brownlee M. Aminoguanidine inhibits reac• tive oxygen species formation lipid peroxidation and oxidant-induced apoptosis. Diabetes 1998 47: 1114-1120. 34. Bierhaus A Illmer T Kasper M Luther T Quehenberger P Tritschler H Wahl P Ziegler R Muller M Nawroth PP. Advanced glycation end product AGE• mediated induction of tissue factor in cultured endothelial cells is dependent on RAGE. Circulation in press. 35. Amore A Cirina P Mitola S Peruzzi L Gianoglio B Rabbone I Sacchetti C Cerutti F Grillo C Coppo R. Nonenzymatically glycated albumin Amadori ad• ducts enhances nitric oxide synthase activity and gene expression in endothelial cells. Kidney Int 1997 51 :27-35. 36. Pieper GM Riaz-ul-Haq. Activation of nuclear factor-kappaB in cultured endo• thelial cells by increased glucose concentration: prevention by calphostin C. J Cardiovasc Pharmacol 1997 30:528-532. 37. Hohman TC Banis D Basso M Cotter MA Cameron NE. Resistance to in• ceased oxidative stress is decreased in experimental diabetic neuropathy. J Periph Nerv Syst 1997 2:272. 38. Aruoma 01 Halliwell B Hoey BM Butler J. The antioxidant action of taurine hypotaurine and their metabolic precursors. Biochem J 1988 256:251-255. 39. Hothersall JS Taylaur CE McLean P. Antioxidant status in an in vitro model for hyperglycemic lens Cataract formation: effect of aldose reductase inhibitor statil. Biochem Med Metab Biol 1988 40: 109-117.

slide 154:

118 Greene et al. 40. Kim J Rushovich EH Thomas TP Ueda T Agranoff BW. Greene DA. Dimin• ished specific activity of cytosolic protein kinase C in sciatic nerve of strepto• zocin-induced diabetic rats and its correction by dietary myo-inositol. Diabetes 1991 40:1545-1554. 41. Kowluru RA Jirousek M Stramm L Farid N Engerman RL Kern TS. Abnor• malities of retinal metabolism in diabetes or experimental galactosemia. V. Rela• tionship between protein kinase C and ATPases. Diabetes 1998 47:464-469. 42. Koya D Lee IK Ishii H Kanoh H. King GL. Prevention of glomerular dysfunc• tion in diabetic rats by treatment with d-alpha-tocopherol.J Am Soc Nephro 1997 8:426-435. 43. Karihaloo A Kato K Greene DA Thomas TP. Protein kinase and Ca" modula• tion of myo-inositol transport in cultured retinal pigment epithelial cells. Am J Physiol 1997 273:C671-C678. 44. Brandsch M Miyamoto Y Ganapathy V. Leibach FH. Regulation of taurine transport in human colon carcinoma cell lines HT-29 and Caco-2 by protein kinase C. Am J Physiol 1993 264:G939-G946. 45. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes 1991 40: 873-877. 46. Romero FJ Monsalve E Hermenegildo C Puertas FJ Higueras V Nies E Seg• ura-Aguilar J Roma J. Oxygen toxicity in the nervous tissue: Comparison of the antioxidant defense of rat brain and sciatic nerve. Neurochem Res 1991 16: 157-161. 47. Fernyhough P Diemel LT Brewster WJ Tomlinson DR. Altered neurotrophin mRNA levels in peripheral nerve and skeletal muscle of experimentally diabetic rats. J Neurochem 1995 64:1231-1237. 48. Hounsom L Horrobin DR Tritschlcr H Corder R Tomlinson DR. A lipoic acid• gamma linolenic acid conjugate is effective against multiple indices of experi• mental diabetic neuropathy. Diabetologia in press. 49. Chan PH Epstein CJ Li Y Huang TT Carlson E Kinouchi H Yang G Kamii H Mikawa S Kondo T. Transgenic mice and knockout mutants in the study of oxidative stress in brain injury. J Neurotrauma 1995 12:815-824. 50. Pm·k DS Morris EJ Stefanis L Troy CM Shelanski ML Geller HM Greene LA. Multiple pathways of neuronal death induced by DNA-damaging agents NGF deprivation and oxidative stress. J Neurosci 1998 18:830-840. 51. Luo Y Umegaki H Wang X Abe R Roth GS. Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem 1998 273:3756-3764. 52. Jackson GR Apffel L Werrbach-PerezK Perez-Polo JR. Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hy• drogen peroxide resistance. J Neurosci Res 1990 25:360-368. 53. Jackson GR Werrbach-Perez K Perez-Polo JR. Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. II. A conditioning lesion para• digm. J Neurosci Res 1990 25:369-374. 54. Nistico G Ciriolo MR Fiskin K Iannone M de Martino A Rotilio G. NGF

slide 155:

Oxidative Stress in Diabetic Neuropathy 119 restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats. Free Rad Biol Med 1992 12:177-181. 55. Garrett NE Malcangio M Dewhurst M Tomlinson DR. Alpha-lipoic acid cor• rects neuropeptide deficits in diabetic rats via induction of trophic support. Neu• rosci Lett 1997 222:191-194. 56. Magwere T Naik YS Hasler JA. Primaquine alters antioxidant enzyme profiles in rat liver and kidney. Free Rad Res 1997 27: 173-179.

slide 156:

This Page Intentionally Left Blank

slide 157:

9 Experimental Diabetic Neuropathy: Oxidative Stress and Antioxidant Therapy To Cure Diabetes Naturally Click Here Hans J. Tritschler ASTA Medica AWD CmbH Frankfurt Germany James D. Schmelzer Yutaka Kishi Yoshiyuki Mitsui Masaaki Nagamatsu Kim K. Nickander Paula J. Zollman and Phillip A. Low Mayo Foundation Rochester Minnesota There is ample evidence of oxidative stress in both experimental EDN and human diabetic neuropathy. Most studies have been done on plasma with limited study on neural tissues. We briefly review and update our studies. I. MECHANISMS OF OXIDATIVE STRESS A. Endoneurial lschemia/Hypoxia There is a perfusion deficit of approximately 50 that affects peripheral nerve endoneurium I and the parent cell bodies in relevant dorsal root and sympa• thetic ganglia 2. The onset of ischemia occurs within the first week 3 and is due to a reduction in nutritive rather than arteriovenous flow. There is atten• dant hypoxia seen in both experimental 4 and human diabetes 5.

slide 158:

121

slide 159:

122 Tritschleret al. B. Hyperglycemic Glycation and Autooxidation Glucose by a process of autooxidation in the presence of decompartmenta• lized trace transitional metals can cause lipid peroxidation 6. We have evalu• ated the role of hyperglycemia in lipid peroxidation in vitro using an in vitro lipid peroxidation model with an ascorbate-iron-EDTA system. The addition of 20 mM glucose to the incubation medium increased lipid peroxidation four• fold confirming rapid and marked glucose-mediated autooxidative lipid per• oxidation 7. Glucose autooxidation results in the production of protein reactive ketoaldehydes hydrogen peroxide highly reactive oxidants and the fragmentation of proteins free radical mechanisms. Glycation and oxidation are simultaneous and inextricably linked 8. II. ANTIOXIDANT ENZYMES Free radical defenses of peripheral nerve are reduced relative to brain and liver especially involving glutathione GSH-containing enzymes 9. Cuprozinc superoxide dismutase SOD is reduced in sciatic nerve of experimental dia• betic neuropathy and this reduction is improved by insulin treatment 10. Glutathione peroxidase GSH-Px is reported to be further reduced in experi• mental diabetic neuropathy in alloxan diabetic mice 7-21 days after induction of diabetes and enzyme activity inversely regresses with glucose level 11 . We recently evaluated the gene expression of the antioxidant enzymes GSH• Px SOD cuprozinc czSOD and manganese mnSOD separately and cata• lase CAT in L4-L6 dorsal root ganglia DRG and superior cervical ganglion SCG of rats that had been diabetic for 3 and 12 months Kishi et al. unpub• lished data. cDNA fragments for rat GSH-Px czSOD mnSOD CAT and cyclophilin was obtained by reverse transcriptase polymerese chain reaction of rat DRG RNA using specific primers for each probe and evaluated by North• ern blot analysis. We also evaluated GSH-Px activity in sciatic nerve DRG and superior cervical ganglion of these animals. GSH-Px CAT czSOD and mnSOD were not reduced in EDN at either 3 or 12 months. CAT mRNA was significantly increased in EDN more than 12 months. GSH-Px enzyme activity was normal in sciatic nerve. We conclude that gene expression is not reduced in peripheral nerve tissues in EON. Changes in enzyme activity may be due to posttranslational modifications.

slide 160:

Y-- ·· I I Ii if /x I .WI 5 Experimental Diabetic Neuropathy 123 Ill. ANTIOXIDANT THERAPY WITH a-LIPOIC ACID IN EDN Hyperglycemia causes lipid peroxidation of brain and sciatic nerve in vitro. We evaluated the effectiveness of the R + - S--enantiomers and racemate of cc-lipoic acid in reducing thiobarbituric acid reactive substances generation in rat brain and sciatic nerve. Studies were also done in an incubation medium containing 20 mM glucose which increased lipid peroxidation up to fourfold. A dose-dependent and statistically significant reduction in lipid peroxidation was seen in both tissues with similar potencies for both enantiomers 7. We also evaluated if lipoic acid will reduce oxidative stress in diabetic peripheral nerve and improve neuropathy in vivo using the model of strepto• zotocin diabetic neuropathy. End points were nerve blood flow NBF electro- 16 P 0.05 Nerve 12 I J blood flow mU100 g/min 8 lj A i/ _ 0 4 Con Con50 STZ STZ20 STZ50 STZ100 P 0.05 25 20 \ Nerve vascular 15 resistance mm Hg/mU100 g/min 10 i - iW. I eg.1 Con Con50 STZ STZ20 STZ50 STZ100 CA-1667C.Olt01A Figure 1 NBF and nerve vascular resistance of control Con 11 8 streptozotocin• diabetic neuropathy STZ 11 5 and animals given lipoic acid supplements at doses of 20 mg/kg STZ20 11 6 50 mg/kg Con50 STZ50 11 5 and 100 mg/kg STZIOO 11 8. Lipoic acid supplementation results in normal flow and nerve vascular resistance. Significance of difference STZ-supplemented vs. STZ. Bars SE. From Ref. 12.

slide 161:

a. .§ 6 I I 124 Tritschleret al. 9 1 c 8 Q 2 i----· 1 ---i 0 7 - r a --:::i 0 E c Cf I I I I . 5 - ·5 :: 8 .1 9 4 ::: "-::· ·:::_ ·v1-f Figure 2 Sciatic nerve GSH concentrations in controls Con and on restricted calo• ric intake ConR st.reptozotocindiabetic STZ u-tocopherol-depleted - and sup• plemented with lipoic acid at 20 50 and 100 mg/kg. Lipoic acid supplementation resulted in a dose-dependent prevention of GSH depletion. Significance of difference vs. control p 0.05 p 0.001 vs. STZ: a p 0.05 r p 0.001. From Low et al. Diabetes 1997 46 suppl 2: S38-S42. physiology and indices of oxidative stress in peripheral nerve at I month after onset of diabetes and in age-matched control rats 12. Lipoic acid in doses of 20 50 and 100 mg/kg was administered intraperitoneally five times per week after onset of diabetes. NBF in EDN was reduced by 50 lipoic acid did not affect NBF of normal nerves but improved that of EDN in a dose• dependent fashion. After 1 month of treatment lipoic acid-supplemented rats 100 mg/kg had normal NBF Fig. 1. The most sensitive and reliable indica• tor of oxidative stress was a reduction in GSH which was significantly reduced in EDN it was improved in a dose-dependent manner in lipoic acid-supple• mented rats Fig. 2. The conduction velocity of digital nerve was reduced in EDN and was significantly improved by lipoic acid. IV. ANTIOXIDANT THERAPY WITH a-LIPOIC ACID IN ISCHEMIA-REPERFUSION INJURY Reperfusion after peripheral nerve ischemia results in reduced reperfusion and a breakdown of the blood-nerve barrier 13 . After l h of ischemia the perme• ability-surface area product PA is unaltered but becomes significantly greater with reperfusion. After 3 h of ischemia PA is increased and becomes further increased with reperfusion 13. Reperfusion results in a significant increase

slide 162:

Experimental Diabetic Neuropathy 125 in endoneurial lipid hydroperoxides 14. Because o-lipoic acid is a powerful lipophilic antioxidant we evaluated its efficacy in protecting peripheral nerve from reperfusion injury using our established model of ischemia-reperfusion injury. We used male Sprague-Dawley rats 300 :::: 5 g. Surgical ligation of the supplying arteries to the sciatic-tibial nerve of the right hindlimb was per• formed for predetermined periods of ischemia either 3 or 5 h followed by the release of the ligatures. Lipoic acid 100 mg/kg/day was given by intra• peritoneal injection daily for 3 days pre- and postsurgery. The same dose of saline was given intraperitoneally to the control rats. A behavioral score of clinical neurological deficits and electrophysiology of motor and sensory nerves was analyzed at 1 week after the surgery. After the electrophysiological examination the sciatic-tibial nerve was fixed in situ and embedded in epon. One-micron sections with toluidine blue staining were evaluated for ischemic fiber degeneration IFD and edema using previously described methodology 15. Distal sensory conduction amplitude of sensory action potential and sensory conduction velocity of digital nerve was significantly improved in 3-h ischemia treated with lipoic acid p 0.05. Lipoic acid also improved IFD and edema. The changes after a longer duration of ischernia 5 h were fewer. These results suggest that the therapeutic window of o-lipoic acid might be relatively narrow but still has some protective effect on peripheral nerve against mild ischemia and reperfusioninsults especiallyon distal sensory nerves. V. NEUROPATHOLOGY Neuropathological alterations in sciatic nerve have been modest in EDN. How• ever with more recent focus on nerve root marked alterations in the spinal roots have been reported in long-standing streptozotocin-diabetic rats 16. We recently undertook a study addressing the status of vascular perfusion and neuropathology of DRG 17. Vascular perfusion and neuropathologic evaluation of the lumbar spinal roots and DRG were studied in long-standing duration of 12-18 months streptozotocin-induced diabetic rats and age- and sex-matched control rats. We also undertook nerve conduction studies includ• ing F wave recordings. We have undertaken both perfusion-fixed and immersion-fixed ganglia. Light microscopically changes of the myelin sheath in the dorsal and ventral roots and vacuolated cells in the DRG were the major findings being signifi• cantly higher in diabetic rats than in control rats. The effects of the diabetic state on myelin splitting was greater in the dorsal than ventral roots. Electron

slide 163:

126 Tritschler et al. microscopic studies revealed the consecutive changes of myelin from mild separation to severe ballooning of myelin with relative axonal sparing Fig. 3. DRG cells showed vacuoles of all sizes with cristae-like residua suggestive of mitochondria. These findings suggest that diabetes mellitus has a dual ef• fect: It accelerates the normal age-related degenerative changes in the spinal roots and DRG and it also has a selective effect on the sensory neuron. Nerve conduction studies showed markedly reduced conduction veloci• ties in the distal nerve segments and prolonged F wave latency and proximal conduction time despite the shorter conduction pathway in diabetic rats. We suggest that the combination of hyperglycemia and ischemia results in oxida• tive stress and a predominantly sensory neuropathy. Figure 3 Electron micrographs of representative ventral root fibers showing a pro• gression of rnyelinopathy in experimental diabetes. A Myelin decompaction. B A rim of intact myelin surrounds degenerating myelin with early myelin balls and a de• nuted atrophic axon. C An atrophic axon is surrounded by myelin showing residual rims separated by myelin degeneration and assuming a prominent honeycombed ap• pearance. D A completely demyelinated axon is seen. From Ref. 17.

slide 164:

Experimental Diabetic Neuropathy 127 VI. GLUCOSE UPTAKE AND ENERGY METABOLISM n-Lipoic acid has a number of actions in addition to its antioxidant properties. These include its effect on glucose uptake. We therefore evaluated glucose uptake nerve energy metabolism and the polyol pathway in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid at various doses 0 10 25 50 and I 00 mg/kg. Duration of diabetes was 1 month and n-lipoic acid was administered intraperitoneally 5 times during the final week of the experiment. Nerve glucose uptake was reduced to 60 37 and 30 of control values in the sciatic nerve L5 DRG and superior cervical ganglion respectively in EDN. n-Lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose but reversed the deficit in EDN with a threshold between IO and 25 mg/kg. Endoneurial glucose fructose sorbitol and myo-inositol were measured in sciatic nerve and L5 DRG. ATP creatine phosphate and lactate were mea• sured in sciatic nerve and superior cervical ganglion. o-Lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In contrast it significantly increased glucose fructose and sorbitol but paradoxically increased rather than reduced endoneurial myo-inositol. n-Lipoic acid prevented the reduction in superior cervical ganglion creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by o-lipoic acid a change associated with an improvement in peripheral nerve function possibly by improving energy metabolism in ischemic nerve and by increasing endoneurial myo-inositol. VII. CONCLUSION Oxidative stress occurs in EDN due to ischemic and autooxidative lipid peroxi• dation with resultant neuropathy. Antioxidant therapy with lipoic acid will improve perfusion electrophysiology and indices of oxidative stress. The drug has additional effects improving glucose uptake and energy metabolism. REFERENCES I. Low PA Lagerlund TD McManis PG. Nerve blood flow and oxygen delivery in normal diabetic and ischemic neuropathy. Int Rev Neurobiol 1989 31 :355- 438.

slide 165:

128 Tritschler et al. 2. Brook WH. Postural hypotension and the anti-gravity suit. Aust Fam Physician 1994 23:1948 1948-1949. 3. Cameron NE Cotter MA Low PA. Nerve blood flow in early experimental dia• betes in rats: relation to conduction deficits. Am J Physiol 1991 261:El-E8. 4. Tuck RR Schmelzer JD Low PA. Endoneurial blood flow and oxygen tension in the sciatic nerves of rats with experimental diabetic neuropathy. Brain 1984 107:935-950. 5. Newrick PG Wilson AJ Jakubowski J Boulton AJ Ward JD. Sura nerve oxy• gen tension in diabetes. Br Med J 1986 293: 1053-1054. 6. Wolff SP Jiang ZY Hunt JV. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Rad Biol Med 1991 10:339-352. 7. Nickander KK McPhee BR Low PA Tritschler H-J. n-Lipcic acid: antioxidant potency against lipid peroxidation of neural tissue in vitro and implications for diabetic neuropathy. Free Rad Biol Med 1996 21:631-639. 8. Hunt JV Wolff SP. Oxidative glycation and free radical production: a causal mechanism of diabetic complications. Free Rad Res Commun 1991 I:115-123. 9. Romero FJ Monsalve E Hermenegildo C Puertas FJ Higueras V Nies E Seg• ura-Aguilar J Roma J. Oxygen toxicity in the nervous tissue: comparison of the antioxidant defense of rat brain and sciatic nerve. Neurochem Res 1991 16: 157- 161. 10. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes 1991 40:873-877. 11. Hermenegildo C Raya A Roma J Romero FJ. Decreased glutathione peroxidase activity in sciatic nerve of alloxan-induced diabetic mice and its correlation with blood glucose levels. Neurochem Res 1993 18:893-896. 12. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Dia• betes Care 1995 18:1160-1167. 13. Schmelzer JD Zochodne DW Low PA. Ischemic and reperfusion injury of rat peripheral nerve. Proc Natl Acad Sci USA 1989 86: 1639-1642. 14. Nagamatsu M Schmelzer JD Zollman PJ Smithson IL Nickander KK Low PA. Ischemic reperfusion causes lipid peroxidation and fiber degeneration. Mus• cle Nerve 1996 19:37-47. 15. Kihara M Zollman PJ Schmelzer JD Low PA. The influence of dose of micro• spheres on nerve blood flow electrophysiology and fiber degeneration of rat peripheral nerve. Muscle Nerve 1993 16:1383-1389. 16. Tamura E Parry GJ. Severe radicular pathology in rats with longstanding diabe• tes. J Neurol Sci 1994 127:29-35. 17. Sasaki H Schmelzer JD Zollman PJ Low PA. Neuropathology and blood flow of nerve spinal roots and dorsal root ganglia in longstanding diabetic rats. Acta Neuropathol 1997 93: 118-128.

slide 166:

10 Antioxidantsin the Treatment of Diabetic Polyneuropathy: Synergy with Essential Fatty Acids To Get Best Natural Diabetes Treatment Click Here Norman E. Cameronand Mary A. Cotter University of Aberdeen Aberdeen Scotland I. INTRODUCTION A. Neuropathy and Nerve Blood Flow Neuropathy is a common complication of diabetes mellitus. Studies in patients and animal models have shown that endoneurial hypoxia caused by impaired nerve blood flow is a major factor in the etiology of diabetic neuropathy 1- 4. Changes in vascular function particularly of the endothelium occur early after diabetes induction in experimental models and in some preparations this may even be partially mimicked by acute exposure to hyperglycemia 56. In streptozotocin-induced diabetic rats sciatic nerve blood flow is reduced by approximately 50 within a week of diabetes induction 78 and this pre• cedes changes in nerve conduction velocity NCV. Large diameter sensory and motor fibers are particularly susceptible to endoneurial hypoxia in experi• mental diabetes 9 10. Several treatment strategies have been used to prevent or correct the blood flow deficit in diabetic rats and when achieved this results in improve•

slide 167:

ments of nerve function measures such as sensory and motor NCV and the increased resistance to hypoxic conduction failure 3. Powerful evidence for 129

slide 168:

130 Cameron and Cotter a direct link between impaired blood flow and nerve dysfunction in diabetes comes from studies using peripheral vasodilators. These do not change the hyperglycemic state or consequent alterations in nerve metabolism such as increased polyol pathway activity however they can completely correct re• duced NCV and attenuate the development of resistance to hypoxic conduction failure 11-15. Vasodilator treatment has also been used to improve nerve function in diabetic patients 16. Other approaches such as chronic electrical nerve stimulation 17 and drugs that correct metabolic changes in diabetes including L-carnitine analogues 11-6 essential fatty acids aldose reductase in• hibitors ARls protein kinase C PKC inhibitors antiadvanced glycation agents and antioxidants ameliorate NCV defects via their effects on nerve blood flow 3 18. For several of these agents the vascular endothelium nitric oxide NO system appears to be a primary target because their effects on NCV and blood flow are abolished by NO synthase inhibitor cotreatment whereas many of their other direct biochemical effects on nerve remain un• changed for example ARI-mediated suppression of polyol pathway metabo• lite levels 19-22. Several studies have identified a diminished vasa nervorum NO system in experimental diabetes 2324. Thus impaired nerve perfusion lies at the heart of the etiology of diabetic neuropathy. The relationship be• tween sciatic motor NCV and nutritive capillary endoneurial blood flow is shown in Figure I for groups of diabetic rats pooled from a large number of experiments in which various doses of these drugs were used reviewed in Refs. 3 and 4. This includes antioxidants which are the main subject of this review. It is clear that the results of these diverse treatments all fit the same relationship: NCY increases with increasing perfusion and reaches asymptote at blood flow levels within the normal range. B. Sources of Reactive Oxygen Species in Diabetes NO and Vasorelaxation Reactive oxygen species ROS are increased by diabetes. NO is an important vascular target for ROS superoxide neutralizes NO 25 and the peroxynitrite formed is a source of hydroxyl radicals that can cause endothelial damage 26. Glucose-induced oxidative stress therefore diminishes vessel endothe• lium-dependent relaxation 27 which contributes to impaired vasa nervorum function 34. There are several sources of ROS in diabetes including those derived from altered metabolism such as autoxidation of glucose and its me• tabolites the advanced glycation/glycoxidation process altered prostanoid production inefficient mitochondrial function and upregulation of the vascu• lar NADPH oxidase system 28-30. ROS are also produced as a result of

slide 169:

I I Antioxidants in the Treatment of Diabetic Polyneuropathy 131 66 .---------- I I 62 54 50 o 5 10 15 20 25 Figure 1 Relationship between sciatic nutritive endoneurial blood flow and motor conduction velocity in groups of streptozotocin-diabetic rats 11 6-16 given different drug treatments in our laboratory. Diabetes duration was 1-3 months and treatment was preventive or corrective. Groups treated with various vasodilators groups treated with essential fatty acids miscellaneous metabolically active compounds such as L-carnitinederivatives aminoguanidine sorbitol dehydrogenase inhibitors PKC in• hibitors and myo-inositol 0 groups treated with different antioxidants e and data from aldose reductase inhibitor studies ". The solid curve is the best-fitting Boltz• mann sigmoid curve r2 0.95 for df 97. The dashed rectangle denotes the non• diabetic range :::: 1 SD n 40. All treatment effects appear to follow a similar relationship conduction velocity is low at low flow rates and reaches an asymptote that approximates the nondiabetic level as perfusion increases. the blood flow problems they cause during episodes of ischemia-reperfusion by the xanthine oxidase mechanism 31. Another potential source that may be relevant during infection and inflammatory disease is the macrophage respi• ratory burst. The degree of oxidative stress seen in diabetic patients is inversely proportional to the degree of metabolic regulation 32 and very tight meta• bolic control is necessary to slow the development of the major diabetic com• plications including neuropathy 33. Because strict glycemic control is diffi• cult to achieve and carries with it the risk of hypoglycemic episodes there is

slide 170:

132 Cameron and Cotter a strong case for supplementary treatment with antioxidants to further reduce ROS activity. II. OXIDATIVE STRESS AND ANTIOXIDANT TREATMENT EFFECTS ON NEUROVASCULAR FUNCTION IN EXPERIMENTAL DIABETES Antioxidant protection mechanisms are compromised in nerves of diabetic rats lipid peroxidation is increased and the levels of superoxide dismutase and reduced glutathione GSH are decreased although glutathione peroxidase and reductase remain unchanged 34-37. Long-term exposure to elevated ROS coupled with diminished endogenous antioxidant protection could lead to cumulative neurodegenerative changes involving axonopathy and demye• lination and damage to dorsal root ganglion cell bodies and their mitochondria has been observed 3738. However in the short term ROS effects on vasa nervorum are more important being responsible for the earliest defects in nerve function in diabetic rats. A. Antioxidant Treatment VascularEndothelium and Nerve Function Defective endothelium-dependent relaxation has been found in diabetic ani• mals and in type I and type 2 patients 39-47 and is an important target for antioxidant treatment. An example is shown in Figure 2 where the lipophilic ROS scavenger o-tocopherol. protected rat aorta against a diabetic deficit in NO-mediated endothelium-dependent relaxation to acetylcholine 39. A simi• lar protective effect of ROS scavengers has been noted for vasa nervorum blood flow and NCV. The magnitude of effects possible by this approach is illustrated in Figure 3 where a high dose of the probucol analogue BM 150639 completely corrected motor NCV and blood flow deficits in diabetic rats. These effects were attenuated by cotreatment with a NO synthase inhibi• tor emphasizing the importance of the vasa nervorum NO system 19. The effectiveness of a variety of scavengers has been assessed over the last 5 years including lipophilic drugs like butylated hydroxytoluene n-tocopherol vita• min E P-carotene and probucol and the hydrophilic scavengers N-acetyl• L-cysteine and ascorbic acid vitamin C 48-54. n-Lipcic acid is the subject of considerable current interest and is both lipid and water soluble 55. In experiments on diabetic rats n-Iipoic acid has been shown to improve nerve antioxidant protection by increasing GSH con-

slide 171:

Antioxidants in the Treatment of Diabetic Polyneuropathy 133 100 75 e..... c: 0 E 50 nl Q a: 25 Om--i.:L--.-.--.-.- -9 -8 -7 -6 -5 -4 Concentration log M Figure 2 Effects of diabetes and antioxidant treatment with u-tocopherol on endo• thelium-dependent relaxation of phenylephrine-precontracted aortas to acetylcholine in vitro. Groups 11 14-17 nondiabetic control 0 8-week streptozotocin-diabetic e a-tocopherol I g I kg/day treated from diabetes induction•. From Ref. 39. tent and to correct blood flow and motor and sensory NCV deficits 365657. R and S enantiomers of o-lipoic acid had similar efficacy on impaired nerve function and perfusion 57 and for the inhibition of lipid peroxidation of neural tissues in vitro 56. u-Lipoic acid was found to be approximately IO times more potent than u-tocopherol in correcting motor NCV deficits in dia• betic rats 5057. Clinical trials of symptomatic and cardiac autonomic neu• ropathy revealed beneficial effects of cc-Iipoic acid treatment 5859. In rats the diabetes-induced decrease in sciatic nutritive blood How was accompanied by a reduction in mean endoneurial oxygen tension which was prevented by probucol treatment 49. In nondiabetic rats prooxidant treat• ment with the antimalarial drug primaquine mimicked the reductions in blood flow endoneurial oxygen tension and NCV found in experimental diabetes while having no effect on plasma glucose levels. These effects were blocked by probucol which stresses the importance of ROS to neurovascular dysfunc• tion. Furthermore both diabetes and primaquine treatment caused an increase in plasma angiotensin-converting enzyme activity a marker of endothelial damage which was attenuated by probucol treatment 49. Effects on plasma

slide 172:

------ - ----- 40 ---- 134 A 25 Cameron and Cotter 20 OJ 0 0 15 .E i::: f i I I 10 0 u::: i:iii:ii 5 ::::::. 0 c 1D 20 DA DAN B 65 60 U .s 55 · z u - 0 50 oi HH\/ iiiiiiiiii ••• 45 :::::::::: ::. ----- c 1D 20 DA DAN Figure 3 Reversal of sciatic nutritive endoneurial blood flow A and motor conduc• tion velocity B deficits in diabetic rats by treatment with the probucol analogue BM 150639. Groups 11 9-10 C nondiabetic control 10 2D I-month and 2-month streptozotocin-diabetic DA I-month untreated diabetes followed by I-month BM 150639 400 mg/kg/day treatment DAN as for DA but cotreated with the nitric oxide synthase inhibitor Na-nitro-L-arginine 10 mg/kg/day during lhe second month. Data are mean ::: SEM. The diabetic deficits in blood flow and motor conduction velocity were well developed after I month. BM 150639 treatment was highly effective in reversing these defects blood flow being approximately 33 supranormal and conduc• tion velocity in the nondiabetic range. The effects of BM 150639 on blood flow and conduction velocity were almost completely blocked by -nitro-L-arginine which suggests that blood flow modulates conduction velocity and lhat antioxidant treatment corrects a diabetic deficit in the vasa nervorum nitric oxide mechanism. See Ref. 24 for further details.

slide 173:

Antioxidants in the Treatment of Diabetic Polyneuropathy 135 angiotensin-converting enzyme activity suggest increased activation of the va• soconstrictor renin-angiotensin system in diabetes and studies with angioten• sin-converting enzyme inhibitors and angiotensin AT I receptor antagonists have shown that this is important for vasa nervorum these inhibitors correct blood flow endoneurial hypoxia and NCY defects in diabetic rats 60. In• creased local angiotensin II also causes upregulation of endothelial NADH oxidase which may exacerbate dysfunction by increasing ROS production 30. Furthermore oxidative stress also stimulates endothelial endothelin-l synthesis and this interacts with the angiotensin II system to increase vasa nervorum vasoconstriction 61 . Thus ROS cause a self-reinforcing cycle that compromises vasodilation by NO and increases local vasoconstrictor mecha• nisms. This cycle may be interrupted by antioxidant treatment. B. Antioxidant Dose Considerations The normal dietary intake of natural antioxidants could in theory influence NCY and blood flow in diabetic rats. However high doses that far exceed normal availability are required in practice for example 0.62 g/kg/day of a• tocopherol was necessary to give -50 protection of sciatic motor NCY in diabetic rats 50. For ascorbic acid 150 mg/kg/day gave an optimal level of protection that was relatively modest -35. At high doses 500 mg/kg/ day protection was less probably because ascorbic acid is susceptible to autoxidation acting as a prooxidant 5062. Under physiological conditions ascorbic acid may aid the recycling of c-tocopberol from its tocopheroxyl radical form 63 however with the pharmacological doses used in diabetic rats there was no evidence of a synergy between ascorbic acid and n-toco• pherol cotreatment on NCY. Instead their effects were simply additive as if they were acting independently in lipid and aqueous phases 50. c. Studies Using Transition Metal Chelators As an alternative to scavenging ROS it may be therapeutically preferable to prevent their formation by autoxidation the Fenton reaction and the advanced glycation process all of which are catalyzed by free transition metal ions. This can be accomplished using transition metal chelators. Low doses of defer• oxamine relatively specific for iron and trientine relatively specific for cop• per completely corrected sciatic nerve blood flow and motor and sensory NCV deficits in diabetic rats 64. In vessels such as aorta chronic trientine and deferoxamine treatment prevented the development of defective NO-me• diated endothelium-dependent relaxation 6566. For diabetic rat vasa nerv-

slide 174:

00 0 0 0 0 -8 -7 -6 -5 -4 136 Cameron and Cotter 1 x E cf. Q g l:J t3 ::: "O c: 8 4 15 Concentration Jog M Figure 4 Cumulative dose-response curves for changes in sciatic vasa nervorum vascular conductance after suffusion of saline containing increasing concentrations of norepinephrine. Blood flow in epi- and perineurial vessels was monitored by laser• Doppler flowmetry and the results are expressed as a percentage of the maximum vascular conductance. Groups n 8-12: nondiabetic control 0 8-week duration diabetic control e 8-week diabetic rats treated with deferoxamine 8 mg/kg/day for the last 2 weeks •. Data are mean ± SEM. Diabetes caused a leftward shift of the dose-response curve indicating greatly enhanced sensitivity to vasoconstriction by norepinephrine. In nondiabetic rats cosuffusion with the nitric oxide synthase inhibitor JVG-nitro-L-arginine 100 µM caused a similar leftward shift 0. Treatment of diabetic rats with deferoxamine completely restored norepinephrine sensitivity the interpreta• tion being that it corrected a vasa nervorum NO deficit. See Refs. 52 and 67 for further details. orum the NO deficit markedly increases reactivity to norepinephrine Fig. 4 and this was corrected by deferoxamine treatment 2467. The relatively high potency of o-lipcic acid compared with other natural scavengers such as cx.• tocopherol could be due to the additional property of transition metal chelation 55. Thus transition metal-catalyzed ROS production makes an important contribution to nerve and vascular dysfunction in experimental diabetes. D. Nerve Growth Regeneration and Small Fiber Function In addition to effects on NCV and blood flow antioxidant treatment improves other aspects of nerve function including growth and regenerative responses

slide 175:

50 Antioxidants in the Treatment of Diabetic Polyneuropathy 137 and the performance of small fibers which do not normally contribute to NCV measurements. Thus the hydrophilic scavenger N-acetyl-L-cysteine allowed normal nerve maturation in young diabetic rats preventing a reduction in mean nerve fiber size caused by impaired growth. N-acetyl-L-cysteine improved the regenerative response to nerve trauma which is blunted by diabetes Fig. 5 inhibited an increase in plasma tumor necrosis factor activity and prevented red cell lipid peroxidation 5152. The lipophilic scavengers o-tocopherol and butylated hydroxytoluene and the metal chelator trientine also prevent blunted nerve growth and regeneration in young diabetic rats 5354. Recently im• proved regeneration remyelination and muscle reinnervation have been noted for o-Iipoic acid treatment Flint H Cotter MA and Cameron NE unpublished observations 1998. Interestingly butylated hydroxytoluene and trientine also prevented nerve regeneration and growth deficits in the galactosemic rat model of enhanced polyol pathway and PKC activity 54. Vasodilator treatment had similar effects on these nerve growth parameters in diabetic rats 60 there• fore it is likely that antioxidant-mediated improvements in perfusion and their consequences for the supply of energy and nutrients to nerve fibers and cell 20-t-..--.----.-.--.- 8 9 10 11 12 13 14 15 Postlesion time days Figure 5 Effects of 4 weeks of diabetes and N-acetyl-L-cysteinetreatment on sciatic nerve myelinated fiber regeneration distance 9 and 14 days after a freeze lesion. Groups n 7-10: nondiabetic control 0 diabetic control e diabetic rats treated with 250 mg/kg/day N-acetyl-L-cysteine from diabetes induction •. Data are mean :: SEM. At both time points there was a significant regeneration deficit p 0.0 I with untreated diabetes which was completely prevented by N-acetyl-L-cysteine treatment. See Ref. 52 for further details.

slide 176:

138 Cameron and Cotter bodies are primarily responsible for their growth and regeneration-promoting actions. The effects of antioxidant treatment on small fiber function has not been examined in as much detail as large fibers. However in the isolated corpus cavernosum preparation from diabetic rats a deficit in vasorelaxation to ni• trergic nerve fiber stimulation was completely prevented by chronic n-lipoic acid treatment and partially prevented by trientine 68. E. Polyol Pathway Oxidative Stress and Neurovascular Dysfunction Polyol pathway activity also contributes to oxidative stress therefore ARis have an indirect antioxidant action. The reductions in nerve GSH content found in experimental diabetes and galactosemia were rapidly corrected by ARI treatment 69 which also prevented the elevation of nerve malondialde• hyde a marker of lipid peroxidation in diabetic rats 70. ARis correct nerve blood flow and NCY defects in diabetic rats 7172. They improve defective NO-mediated endothelium-dependent relaxation in vessels from diabetic ani• mals and similar dysfunction resulting from acute hyperglycemic exposure of vessels from nondiabetic animals 36. The first half of the polyol pathway catalyzed by aldose reductase is much more important than the second half catalyzed by sorbitol dehydrogenase because sorbitol dehydrogenase inhibi• tors did not correct blood flow or NCV in diabetic rats 73 and did not rectify the nerve GSH deficit Hohman TC personal communication 1997. Aldose reductase requires NADPH as a cofactor and NADPH is also used by glutathione reductase for maintaining GSH concentrations. Therefore competition for NADPH in diabetes probably contributes to diminished GSH levels. A further potential polyol pathway action is to increase the formation of advanced glycation end products AGEs. ARI treatment reduces tissue AGE accumulation perhaps by inhibiting the synthesis of fructose or by de• creasing elevated flux through the pentose phosphate pathway processes that produce sugars that are considerably more potent glycating agents than glucose 74. Alternatively the ARI-induced increases in tissue GSH and antioxidant capacity may be sufficient to oppose AGE formation by glycoxidation 75. AGE reactions are an important source of ROS therefore their reduction would decrease oxidative stress. Aminoguanidine although not directly an antioxidant irreversibly binds to reactive carbonyl intermediates thus blocking AGE formation. Aminoguanidine treatment of diabetic rats has simi• lar functional effects to ARis and antioxidants in preventing and correcting NCV nerve blood flow and NO-mediated endothelium-dependent vasorelax-

slide 177:

Antioxidants in the Treatment of Diabetic Polyneuropathy 139 Increased sugars Elevated free radical production Transition metal catalyzed reactions Advanced Autoxidation glycation \ NERVE DYSFUNCTION J Blood flow J Conduction velocity t Damage J Regeneration GLUCOSE GALACTOSE aldose reductase Sorbitol Galactitol I I Glutathione redox.._ cycle GS0SG Increased po/yo/ pathway flux -----+ Impaired antioxidant protection Figure 6 Relation of oxidative stress and nerve dysfunction to elevated polyol path• way activity and transition metal-catalyzed reactions in diabetes or galactosemia. The increased autoxidation and advanced glycation reactions produce ROS and flux through the first half of the polyol pathway consumes NADPH. This impairs the gluta• thione redox cycle so that endogenous antioxidant protection is reduced. ation deficits 344076. The putative interrelations between the polyol path• way advanced glycation and autoxidation processes in the production of oxi• dative stress under hyperglycemic conditions are summarized in Figure 6. F. AntioxidantsN F-KB and PKC One of the cellular events stimulated by oxidative stress-related biochemical changes in hyperglycemia either directly or via AGE receptors oxidized low• density lipoprotein or cytokine receptors is the activation of NF-KBan effect that may be prevented by antioxidant treatment with o-lipoic acid 77. This transcription factor is responsible for changes in gene expression that have important effects on vascular function relevant to diabetes including elevated endothelin-1 synthesis and upregulation of intercellular and vascular cell adhe• sion molecules. It has also been linked to increased NADH oxidase activity 3078. PKC is another cell-signaling mechanism activated by diabetes particu• larly in vascular tissue 79 although not in the nerve itself 80. In the retina

slide 178:

140 Cameron and Cotter of diabetic rats there is an early reduction in blood flow paralleling that for nerve. In both retina and nerve flow was restored by PKC inhibitor treatment 1881 and nerve NCV deficits were corrected. Diabetes activates PKC via increased de novo synthesis of diacylglycerol from glucose however even in the absence of hyperglycemia PKC is also stimulated by oxidative stress 82. Antioxidants such as n-tocopherol inhibit PKC both directly and via stimulation of diacylglycerol kinase which breaks down diacylglycerol 7982. When activated PKC can modulate several important vascular sys• tems. For example it is involved in cell signaling mechanisms for endothelin-1 action and can also stimulate NF-KB which increases endothelin-1 gene expression in endothelial cells 83. Phosphorylation by PKC controls endothelial constitutive NO synthase reducing its activity 84. Furthermore phosphorylation of vascular smooth muscle contractile proteins promotes vasoconstriction 85. Thus PKC is at the heart of altered vascular responses in experimental diabetes and forms a major component of the dysfunctional mechanisms targeted by antioxidant treatment. G. Therapeutic Implications From this brief literature review it is clear that antioxidant strategies based on the use of ROS scavengers and transition metal chelators can be very effective against experimental models of diabetic neuropathy and vasculopathy. The drawback with the scavenger approach is that very large doses of drug are required one-two orders of magnitude greater than necessary if using transi• tion metal chelators. However it is possible that with lower blood glucose concentrations than normally found in the experimental models more physio• logical doses for example of n-tocopherol could be effective as an adjunct to tight metabolic control in patients. The use of agents with both scavenger and chelator properties such as n-lipoic acid or combined therapy with drugs that improve endogenous antioxidant protection mechanisms such as ARis or drugs that target key cell signaling events such as PKC inhibitors could provide exciting future strategic approaches to the therapy of diabetic compli• cations including neuropathy. Ill. INTERACTIONS BETWEEN ANTIOXIDANTS AND ESSENTIAL FATTY ACIDS Most of the preceding discussion has dealt with effects of ROS on the NO system and cell-signaling pathways. Another major ROS target is the polyun-

slide 179:

Antioxidants in the Treatment of Diabetic Polyneuropathy 141 saturated fatty acids including the essential fatty acids necessary for eicosa• noid production and normal membrane structure and fluidity. ROS subject these important molecules to self-propagating destructive chain reactions initi• ated by lipid peroxidation. A. Essential Fatty Acids Diabetes and Neurovascular Dysfunction The most common dietary essential fatty acids are n-6 linoleic acid and n-3 a.-linolenic acid. They are metabolized by an alternating series of desaturation and elongation steps to produce n-6 arachidonic acid and n-3 eicosapentaenoic acid which are important precursors of prostanoids leukotrienes and other mediators. In addition to being subjected to destruction by the elevated ROS in diabetes metabolism of these essential fatty acids is rate limited by the desaturation steps that are inhibited by diabetes. Depressed hepatic d-6 desatu• ration results in lower plasma levels of the n-6 metabolites y-linolenic acid GLA and arachidonic acid. This reduces synthesis of the vasodilator prosta• cyclin PGl2 by vasa nervorum 86. The desaturation deficit may be by• passed by GLA or arachidonic acid treatments which improve vasa nervorum prostacyclin production nerve blood flow and NCV 3487. In contrast to the n-6 essential fatty acids n-3 metabolites such as eicosapentaenoic and docosahexaenoic acids have relatively little effect on nerve function in experi• mental diabetes 88. B. Multiple Endothelial Dysfunction in Diabetes and the Potential for Synergistic Therapies There are multiple defects of vasa nervorum endothelium and possibly smooth muscle in diabetes that cause reduced nerve blood flow and function. The NO deficit is compounded by diminished PGI2 synthesis and increases in endo• thelin-I and angiotensin II. Recently it was shown that relaxation mediated by endothelium-derived hyperpolarizing factor EDHF is also affected by diabetes. In the rat mesenteric vascular bed EDHF was 76 reduced after 8 weeks of diabetes. In common with the NO defect the EDHF deficit was attenuated by antioxidant treatment with u-lipoic acid 89. Vasodilator prostanoid synthesis is also deleteriously affected by oxidative stress high levels of lipid peroxides inhibit cyclooxygenase 90 and u-tocopherol treat• ment corrected the lowering of the PGii/thromboxane A2 ratio found in dia• betic rats 91.

slide 180:

142 Cameron and Cotter Normally these different mechanisms provide an integrated local sys• tem for nerve blood flow control however by disrupting several mechanisms simultaneously diabetes disintegrates control and markedly shifts the balance toward vasoconstriction. These individual mechanisms do not exist in isolation but are mutually interactive 92 therefore changes in one will affect the others. In experiments on nondiabetic rats designed to mimic some of the vasa nervorum changes in diabetes and assess the consequences for nerve function chronic treatment with a low dose of a cyclooxygenase inhibitor to reduce PGl2 synthesis or a NO synthase inhibitor caused modest NCY reductions. However with combined treatment there was a fivefold amplification of drug effects on NCV compared with that expected for simple summation 93. This demonstrates a marked synergism between blockade of the prostanoid and the NO systems suggesting that they normally act in a mutually compensatory manner to limit nerve perfusion changes. The converse effect joint treatment of diabetes with drugs that target the NO and prostanoid mechanisms as sche• matized in Figure 7 could potentially offer a marked therapeutic advan• tage. C. Synergy Between Antioxidant and n-6 Essential Fatty Acid Treatments Low doses of an ARI or evening primrose oil which contains GLA had modest effects on NCV and blood flow in diabetic rats however in combina• tion improvements matching those obtained by an eightfold increase in the dose of either drug alone were noted 20. A similar magnitude of synergistic interaction was seen for joint treatment with low-dose GLA and the lipophilic scavenger BM 150639 94. The notion of antioxidant-n-6 essential fatty acid combination therapy has led to the synthesis of hybrid drugs such as ascorbyl- 6-GLA. Although ascorbic acid alone is not particularly effective against NCY deficits in diabetic rats its combination with GLA increases lipid solubility and therefore the ability to enter cell membranes. It also places the ascorbate moiety in a good physical position to protect the GLA component from ROS damage. Compared with GLA alone ascorbyl-6-GLA was 4.4-fold more effi• cacious in correcting NCV and nerve blood flow deficits in diabetic rats 95. Recent attention has focused on GLA-a-lipoic acid combinations be• cause both drugs have similar dose-response relationships for correcting NCY deficits and because of the greater effective antioxidant power of n-lipoic acid than ascorbate in experimental diabetic neuropathy. Effects on motor NCV and nerve blood flow are shown in Figure 8. Low doses of GLA or o-lipoic

slide 181:

Im ide des • Antioxidants in the Treatment of Diabetic Polyneuropathy 143 DIABETES Superox Antioxidants INTERACTION paired aturation gamma• linolenic acid NITRIC OXIDE 4 Jlr PROSTACYCLIN VASODILATION / NERVE BLOOD FLOW NERVE FUNCTION Figure7 Schematic for the synergistic interaction between NO and prostacyclin pathways. Increased oxidative stress in diabetes and the generation of superoxide neu• tralizes endothelial NO production which impairs vasa nervorum vasodilation. Diabe• tes also reduces hepatic 11-6 essential fatty acid metabolism particularly the rate-lim• iting desaturation steps in the conversion of dietary linoleic acid to y-linolenic acid. In turn this reduces vasa nervorum prostacyclin production and vasodilation Normally at the level of the endothelial cell. and possibly vascular smooth muscle there is a mutual facilitatory interaction between NO and prostacyclin systems. This may give a therapeutic advantage for the use of joint treatment with antioxidant which corrects the NO defect and y-linolenic acid which bypasses the desaturation block to boost prostacyclin production. acid alone had modest effects on NCV and no statistically significant effects on blood flow. However when the drugs were combined NCV and blood flow were in the nondiabetic range showing a greater than additive effect 57. Synergy was found for GLA-a.-lipoic acid mixtures in ratios between I : 3 and 3: I the greatest amplification of drug action being for ratios near I : I or with GLA slightly in excess. Similar interactive effects were noted for the novel orally active drug SOC0150 which contains equimolar amounts of GLA and u-lipoic acid Fig. 9. The dose-response curve for correction of sciatic motor NCV showed that SOCO150 had an ED50 of 9.3 mg/kg/day giv• ing 3.5 mg/kg/day a.-lipoic acid and 4.5 mg/kg/day GLA. This compares very favorably with the ED50 of o-lipoic acid alone 38 mg/kg/day. In con• trast to SOCO 150 n-lipoic acid-containing compounds in which the GLA was substituted by the n-3 essential fatty acids docosahexaenoic acid 57 or eico-

slide 182:

.s 50 144 Cameron and Cotter 65 A 60 0 55 . ·"5 0 ai 45 20 B en : 15 0 0 c D G L LG . i E ::: 10 I ::: 0 u::: 5 c D G L LG Figure 8 Effects of low-dose y-linolenic acid and u-lipoic acid treatment alone and in combination on A sciatic motor conduction velocity and B sciatic endoneurial blood flow in streptozotocin-diabetic rats. Groups 11 8-12: C nondiabetic control D 8-week diabetic control G or L 8-week diabetic treated for the final 2 weeks with y-linolenic acid 20 mg/kg/day as the monoester or «-Iipoic acid 20 mg/kg/day LG 8-week diabetic given combined y-linolenic acid and u-lipoic acid treatment for the final 2 weeks. Data are mean ± SEM. The horizontal lines show the predicted conduction velocity and blood flow values for additive drug effects. The LG group greatly exceeded this level for both measures indicating a marked synergistic drug interaction. See Ref. 57 for further details.

slide 183:

---- s+: / •. Antioxidants in the Treatment of Diabetic Polyneuropathy 145 SOC0150 65 1-zzz-octadeca-69 12-trienoic acid-3-DL-12-dithiolane- 3-pentanoic acid-propyl diester nondiabetic ranll _ 60 CJ 5 :::: ·c3 0 Q 55 -------- ....._. / / I I I I I I I I I I I I Ji I / _ - - - - - - - - diabetic range 0 0.5 1.0 1.5 2.0 2.5 o-Lipolc acid dose log mg kg·1 Figure 9 Dose-response relationship for the correction of sciatic motor conduction velocity by the novel drug SOCO 150 containing equimolar amounts of y-linolenic acid and a-lipoic acid. The chemical name and structure of SOC0150 are shown at the top of the figure. Diabetes duration was 8 weeks and oral treatment was given for the final 2 weeks. The graph shows data points:::: SEM 11 7-10 for SOCOISOe plotted in terms of a-lipoic acid content and the best-fitting sigmoid dose-response relationship solid curve for comparison with the dose-response relationship for a• lipoic acid dashed curve. The SOC0150 curve is displaced approximately I log unit to the left of that for rx-lipoic acid indicating an approximately IO-fold increase in efficacy. Also shown are data points for similar drugs in which the y-linolenic acid moiety was replaced with the 11-3 components eicosapentaenoic acid 11 6 or docosahexaenoic acid 0 11 8. Conduction velocity was not significantly different from that predicted from their o-Iipoic acid content alone showing that synergistic interactions with antioxidants are specific to n-6 essential fatty acids. From Ref. 57 and Cameron NE Cotter MA unpublished observations 1998.

slide 184:

146 Cameron and Cotter sapentaenoic acid Cameron NE Cotter MA unpublished observations 1998 were no more effective than their a-lipoic acid component alone Fig. 9. This suggests that synergistic actions are relatively specific to the n-6 series. IV. SUMMARY AND CONCLUSIONS Studies on antioxidant treatment have shown that ROS makes a marked contri• bution to the etiology of nerve dysfunction in experimental diabetes. Effects on vasa nervorum predominate in the short term ROS cause dysfunction of vascular endothelium which at the very least reduces NO-mediated vasodila• tion and increases local vasoconstrictor production and reactivity. The effects of oxidative stress are crucial complex and far reaching causing basic changes in cell signaling such as PKC and NF-KB that affect a plethora of systems involved in the maintenance of vascular control and integrity. ROS effects also impinge on prostanoid and EDHF systems further exacerbating a diabetic deficit of substrate availability in the former. The result is reduced nerve perfusion causing endoneurial hypoxia which in turn is responsible for NCV and other functional deficits. Autooxidation of glucose and its metabo• lites and other transition metal-catalyzed reactions such as advanced glycation are important sources of ROS. Polyol pathway activity contributes to oxidative stress by compromising the glutathione redox cycle. Antioxidant treatment strategies in combination with good metabolic control offer a potential way forward in the prevention or control of diabetic neuropathy and other vascular complications. The powerful synergistic interactions between ROS-NO and n-6 essential fatty acid-prostanoid mechanisms on nerve perfusion offer a potential therapeutic advantage for the use of antioxidant-GLA mixtures and novel compounds. REFERENCES 1. Low PA Lagerlund TD McManis PG. Nerve blood flow and oxygen delivery in normal diabetic and ischemic neuropathy. Int Rev Neurobiol 1989 31 :355- 438. 2. Tesfaye S Malik R Ward JD. Vascular factors in diabetic neuropathy. Diabeto• logia 1994 37:847-854. 3. Cameron NE Cotter MA. The relationship of vascular changes to metabolic fac• tors in diabetes mellitus and their role in the development of peripheral nerve complications. Diabetes Metab Rev 1994 10: 189-224.

slide 185:

Antioxidants in the Treatment of Diabetic Polyneuropathy 147 4. Cameron NE Cotter MA. Metabolic and vascular factors in the pathogenesis of diabetic neuropathy. Diabetes 1997 46suppl 2:S31-S37. 5. Mayhan WG Patel KP. Acute effects of glucose on reactivity of cerebral micro• circulation: role of activation of protein kinase C. Am J Physiol 1995 269: Hl297-Hl302. 6. Cohen RA. Dysfunction of vascular endothelium in diabetes mellitus. Circulation 1993 87suppl V:Y67-Y76. 7. Cameron NE Cotter MA Low PA. Nerve blood flow in early experimental dia• betes in rats: relation to conduction deficits. Am J Physiol 1991 261 :EI -ES. 8. Wright RA Nukada H. Vascular and metabolic factors in the pathogenesis of experimental diabetic neuropathy in mature rats. Brain 1994 117:1395-1407. 9. Cameron NE Cotter MA Harrison J. The effects of diabetes on motor nerve conduction velocity in different branches of the rat sciatic nerve. Exp Neurol 1986 92:757-761. I 0. Cameron NE Cotter MA Robertson S. The effect of aldose reductase inhibition on the pattern of nerve conduction deficits in diabetic rats. Q J Exp Physiol 1989 74:917-926. 11. Cameron NE Cotter MA Ferguson K Robertson S Radcliffe MA. Effects of chronic n-adrenergic receptor blockade on peripheral nerve conduction hypoxic resistance polyols Na+-K+ -ATPase activity and vascular supply in STZ-D rats. Diabetes 1991 40: 1652-1658. 12. Cotter MA Cameron NE. Correction of neurovascular deficits in diabetic rats by z agonist and a1 adrenoceptor antagonist treatment: interactions with the nitric oxide system. Eur J Pharmacol 1988 343:217-223. 13. Kihara M Schmelzer JD Low PA. Effect of cilostazol on experimental diabetic neuropathy in the rat. Diabetologia 1995 38:914-918. 14. Hatta N Koh N Sakakibara F Nakamura J Kakuta H Fukasawa H Sakamoto N. Effects of beraprost sodium and insulin on the electroretinogram nerve con• duction and nerve blood flow in rats with streptozotocin-induced diabetes. Diabe• tes 1996 45:361-366. 15. Cameron NE Cotter MA Robertson S. Angiotensin converting enzyme inhibi• tion prevents the development of muscle and nerve dysfunction and stimulates angiogenesis in streptozotocin-diabetic rats. Diabetologia 1992 35:12-18. 16. Reja A Tesfaye S Harris ND Ward JD. ls ACE inhibition with lisinopril helpful in diabetic neuropathy Diabet Med 1995 12:307-309. 17. Cameron NE Cotter MA Robertson S Maxfield EK. Nerve function in experi• mental diabetes in rats: effects of electrical stimulation. Am J Physiol 1993 264: El61-El66. 18. Cameron NE Cotter MA Jack A Hohman TC. Inhibition of protein kinase C corrects nerve conduction and blood flow deficits in diabetic rats abstr. Diabeto• logia 1997: 40suppl I :A3 I. 19. Cameron NE Cotter MA. Reversal of peripheral nerve conduction and perfusion deficits by the free radical scavenger BM 15.0639 in diabetic rats. Naunyn Schmiedeberg s Arch Pharmacol 1995 321 :685-690.

slide 186:

148 Cameron and Cotter 20. Cameron NE Cotter MA Hohman TC. Interactions between essential fatty acid prostanoid polyol pathway and nitric oxide mechanisms in the neurovascular deficit of diabetic rats. Diabetologia 1996 39: 172-182. 21. Cameron NE Cotter MA. Rapid reversal by aminoguanidine of the neurovascu• lar effects of diabetes in rats: modulation by nitric oxide synthase inhibition. Metabolism 1996 45:1147-1152. 22. Cameron NE Cotter MA. Neurovascular effects of L-carnitine treatment in dia• betic rats. Eur J Pharmacol 1997 319:239-244. 23. Kihara M Low PA. Impaired vasoreactivity to nitric oxide in experimental dia• betic neuropathy. Exp Neurol 1995 132: 180-185. 24. Maxfield EK Cameron NE Cotter MA. Effects of diabetes on reactivity of sci• atic vasa nervorum in rats. J Diabet Complications 1997 11:47-55. 25. Gryglewski RJ Palmer RMJ Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 1986 320: 454-456. 26. Beckman JS Beckman TW Chen J Marshall PA Freeman BA. Apparent hy• droxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA I 990 87: I 620- 1624. 27. Tesfamariam B Cohen RA. Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol 1992 263:H321-H326. 28. Baynes JW. Role of oxidative stress in the development of complications in diabetes. Diabetes 1991 40:405-412. 29. Wolff SP. Diabetes mellitus and free radicals. Br Med Bull 1993 49:642- 652. 30. Harrison DG. Endothelial function and oxidant stress. Clin Cardiol 1997 20suppl 2:11-17. 31. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985 312:159-163. 32. Wieruszwysocka B Wysocki H Byks H Zozulinska D Wykretowicz A Kazier• czak M. Metabolic control quality and free radical activity in diabetic patients. Diabetes Res Clin Pract 1995 27:193-197. 33. Diabetes Control and Complications Trial Research Group. The effect of inten• sive diabetes therapy on the development and progression of neuropathy. Ann Intern Med 1995 122:561-568. 34. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes I 991 40:873-877. 35. Nickander KK Schmelzer JD Rohwer D Low PA. Effects of n-toccphercl• deficiency on indices of oxidative stress in normal and diabetic peripheral nerve. J Neural Sci 1994 126:6-14. 36. Nagarnatsu M Nickander KK. Schmelzer JD Raya A Wittrock A Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care 1995 18: 1160-1167.

slide 187:

Antioxidants in the Treatment of Diabetic Polyneuropathy 149 37. Low PA Nickander KK Tritschler HJ. The roles of oxidative stress and antioxi• dant treatment in experimental diabetic neuropathy. Diabetes 1997 46suppl 2: S38-S42. 38. Sasaki H Schmelzer JD Zollman PJ Low PA. Neuropathology and blood flow of nerve spinal roots and dorsal root ganglia in longstanding diabetic rats. Acta Neuropathol 1997 93:118-128. 39. Keegan A Walbank H Cotter MA Cameron NE. Chronic vitamin E treatment prevents defective endothelium-dependent relaxation in diabetic rat aorta. Diabe• tologia 1995 38: 1475-1478. 40. Archibald V Cotter MA Keegan A Cameron NE. Contraction and relaxation of aortas from diabetic rats: effects of chronic anti-oxidant and aminoguanidine treatments. Naunyn Schmiedebergs Arch Pharmacol 1996 353:584-591. 41. Rosen P Ballhausen T Bloch W Addicks K. Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995 38:1157-1168. 42. Pieper GM Gross GJ. Oxygen free radicals abolish endothelium-dependent re• laxation in diabetic rat aorta. Am J Physiol 1988 255:H825-H833. 43. Kamata K Miyata N Kasuya Y. Impairment of endothelium-dependent relax• ation and changes in levels of cyclic GMP in aorta from streptozotocin-induced diabetic rats. Br J Pharmacol 1989 97 :614-618. 44. McVeigh GE Brennan GM Johnston GD McDermott BJ McGrath LT Henry WR Andrews JW Hayes JR. Impaired endothelium -dependent and independent vasodilation in patients with type 2 non-insulin-dependent diabetes mellitus. Diabetologia 1992 35:771- 776. 45. Johnstone MT Creager SJ Scales KM Cusco JA Lee BK Creager MA. Im• paired endothelium-dependent vasodilation in patients with insulin-dependent di• abetes mellitus. Circulation 1993 88:2510-2516. 46. Nitenberg A Valensi P Sachs R Dali M Aptecar E Attali JR. Impairment of coronary vascular reserve and ACh-induced coronary vasodilation in diabetic patients with angiographically normal coronary arteries and normal left ventricu• lar systolic function. Diabetes 1993 42:1017-1025. 47. Morris SJ Shore AC Tooke JE. Responses of the skin microcirculation to acetyl• choline and sodium nitroprusside in patients with NIDDM. Diabetologia 1995 38:1337-1344. 48. Cameron NE Cotter MA Maxfield EK. Antioxidant treatment prevents the de• velopment of peripheral nerve dysfunction in streptozotocin-diabetic rats. Diabe• tologia 1993 36:299-304. 49. Cameron NE Cotter MA Archibald V Dines KC Maxfield EK. Anti-oxidant and pro-oxidant effects on nerve conduction velocity endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats. Diabetologia 1994 37:449-459. 50. Cotter MA Love A Watt MJ Cameron NE Dines KC. Effects of natural free radical scavengers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia 1995 38:1285-1294.

slide 188:

150 Cameron and Cotter 51. Sagara M Satoh J Wada R Yagihashi S Takahashi K Fukuzawa M Muto G Muto Y Toyota T. Inhibition of development of peripheral neuropathy in streptozotocin-induced diabetic rats with N-acetylcysteine. Diabetologia 1996 39:263-269. 52. Love A Cotter MA Cameron NE. Effects of the sulphydryl donor N-acetyl-L• cysteine on nerve conduction perfusion maturation and regeneration following freeze-damage in diabetic rats. Eur J Clin Invest l 996 26:698-706. 53. Love A Cotter MA Cameron NE. Effects of u-tocopherol on nerve conduction velocity and regeneration following a freeze lesion in immature diabetic rats. Naunyn Schmeidebergs Arch Pharmacol 1997 355:126-130. 54. Love A Cotter MA Cameron NE. Nerve function and regeneration in diabetic and galactosaemic rats: antioxidant and metal chelator effects. Eur J Pharmacol 1996 314:33-39. 55. Packer L Tritschler HJ Wessel K. Neuroprotection by the metabolic antioxidant u-Iipoic acid. Free Rad Biol Med 1996 22:359-378. 56. Nickander KK McPhee BR Low PA Tritschler H. Alpha-lipoic acid: antioxi• dant potency against lipid peroxidation of neural tissues in vitro and implications for diabetic neuropathy. Free Rad Biol Med 1996 21:631-639. 57. Cameron NE Cotter MA Horrobin DH Tritschler HJ. Effects of u-Iipoic acid on neurovascular function in diabetic rats: interaction with essential fatty acids. Diabetologia 1998 41 :390-399. 58. Ziegler D Hanefeld M Ruhnau KJ MeiBner HP Lobisch M Schi.itte K Gries FA The ALADIN Study Group. Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidant a-lipoic acid. A 3-week multicentre randomized controlled trial ALADIN Study. Diabetologia 1995 38:1425-1433. 59. Ziegler D Schatz H Gries FA Ulrich H Reichel G. Effects of treatment with the antioxidant a-lipoic acid on cardiac autonomic neuropathy in NIDDM pa• tients. A 4-month randomized controlled multicenter trial DEKAN Study. Dia• betes Care 1997 20:369-373. 60. Maxfield EK Love A Cotter MA Cameron NE. Nerve function and regenera• tion in diabetic rats: effects of ZD-7155 an AT1 receptor antagonist. Am J Phys• iol 1995 269:E530-E537. 61. Cameron NE Cotter MA. Effects of a nonpeptide endothelin-I ET" antagonist on neurovascular function in diabetic rats: interaction with the renin-angiotensin system. J Pharmacol Exp Ther 1996 278: 1262-1268. 62. Hunt JV Bottoms MA Mitchison MJ. Ascorbic acid oxidation: a potential cause of the elevated severity of atherosclerosis in diabetes mellitus. FEBS Lett 1992 311:161-164. 63. Packer LE Slater TF Wilson RL. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 1979 278:737- 738. 64. Cameron NE Cotter MA. Neurovascular dysfunction in diabetic rats: potential contribution of autoxidation and free radicals examined using transition metal chelating agents. J Clin Invest 1995 96:1159-1163. 65. Pieper GM Siebeneich W. Diabetes-induced endothelial dysfunction is pre-

slide 189:

Antioxidants in the Treatment of Diabetic Polyneuropathy 151 vented by long-term treatment with the modified iron chelator hydroxyethyl starch conjugated-deferoxamine. J Cardiovasc Pharmacol 1997 30:734- 738 66. Keegan A Cotter MA Archibald V Cameron NE. Metal chelator and free radi• cal scavenger treatments prevent chronic aorta relaxation defects in diabetic rats abstr. Diabetologia 1996 39suppl l :A240. 67. Cameron NE Cotter MA Maxfield EK. Correction of impaired neurovascular function by desferrioxamine in anaesthetized diabetic rats abstr. J Physiol 1995 487:84P-85P. 68. Keegan A Cotter MA Cameron NE. Impaired endothelial and autonomic relax• ation of the corpus cavernosum in diabetic rats: effects of antioxidants abstr. Diabetic Med 1998 l 5suppl I :S7. 69. Hohman TC Banas D Basso M Cotter MA Cameron NE. Increased oxidative stress in experimental diabetic neuropathy abstr Diabetologia 1997 40suppl l:A549. 70. Lowitt S Malone JI Salem AF Korthals J Benford S. Acetyl-L-camitine cor• rects altered peripheral nerve function in experimental diabetes. Metabolism 1995 44:677-680. 71. Cameron NE Cotter MA Dines KC Maxfield EK Carey F Mirrlees DJ. Aldose reductase inhibition nerve perfusion oxygenation and function in streptozo• tocin-diabetic rats: dose-response considerations and independence from a myo• inositol mechanism. Diabetologia 1994 37:651-663. 72. Hotta N Kaskuta H Fukasawa H Koh N Sakakibara F Nakamura J Hamada Y Wakao T Hara T Mori K Naruse K Nakashima E lnukai S Sakamoto N. Effect of a potent new aldose reductase inhibitor 5-3-thienyltetrazol- l-yl• acetic acid TAT on diabetic neuropathy in rats. Diabetes Res Clin Pract 1995 27:107-117. 73. Cameron NE Cotter MA Basso M Hohman TC. Comparison of the effects of inhibitors of aldose reductase and sorbitol dehydrogenase on neurovascular function nerve conduction and tissue polyol pathway metabolites in streptozo• tocin-diabetic rats. Diabetologia 1997 40:271-28 l. 74. Hamada Y Araki N Horiuchi S Hotta N. Role of polyol pathway in nonenzy• matic glycation. Nephrol Dial Transplant 1996 11 :95-98. 75. Fu MX Wells-Knecht KJ Blackledge JA Lyons TJ Thorpe SR Baynes JW. Glycation glycoxidation and cross-linking of collagen by glucose: kinetics mechanisms and inhibition of late stages of the Maillard reaction. Diabetes 1994 43:676-683. 76. Cameron NE Cotter MA. Rapid reversal by aminoguanidine of the neurovascu• lar effects of diabetes in rats: modulation by nitric oxide synthase inhibition. Metabolism 1996 45: 1147-1152. 77. Bierhaus A Chevion S Chevion M Hofmann M Quehenberger P Illmer T Luther Y Berentshtein E Tritschler H Millier M Wahl P Ziegler R Nawroth PP. Advanced glycation end product-induced activation of NF-KB is sup• pressed by n-Iipoic acid in cultured endothelial cells. Diabetes 1997 46: 1481- 1490.

slide 190:

152 Cameron and Cotter 78. Collins T. Endothelial nuclear factor-KB and the initiation of the atherosclerotic lesion. Lab Invest 1993 68:499-508. 79. Kunisaki M Bursell S Umeda F Nawata H King GL. Normalization of diacyl• glycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes 1994 43:1372-1377. 80. Borghini l Ania-Lahuerta A Regazzi R Ferrari G Gjinovci A Wollheirn CB Pralong W-F. a I II B and E protein kinase C isoforms and compound activ• ity in the sciatic nerve of normal and diabetic rats. J Neurochern 1994 62:686- 696. 81. Ishii H Jirousek MR Koya D Takagi C Xia P Clermont A Bursell S-E Kern TS Ballas LM Heath WF Stramm LE Feener EP King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC inhibitor. Science 1996 272:728- 731. 82. Keaney JF Jr Guo Y Cunningham D Shwaery GT Xu A Vita JA. Vascular incorporation of alpha-tocopherol prevents endothelial dysfunction due to oxi• dized LDL by inhibiting protein kinase C stimulation. J Clin Invest 1996 98: 386-394. 83. Rubanyi GM PolokoffMA. Endothelins: molecular biology. biochemistry phar• macology physiology and pathophysiology. Pharmacol Rev 1994 46:325- 415. 84. Davda RK Chandler LJ Guzman NJ. Protein kinase C modulates receptor-inde• pendent activation of endothelial nitric oxide synthase. Eur J Pharmacol 1994 266:237-244. 85. Shimamoto Y Shimamoto H Kwan C Daniel EE. Differential effects of putative protein kinase C inhibitors on contraction of rat aortic smooth muscle. Am J Physiol 1993 264:HI 300-Hl 306. 86. Ward KK Low PA Schmelzer JD Zochodne DW. Prostacyclin and noradrena• line in peripheral nerve of chronic experimental diabetes in rats. Brain 1989 112:197-208. 87. Cotter MA Cameron NE. Effects of dietary supplementation with arachidonic acid rich oils on nerve conduction and blood flow in streptozotocin-diabetic rats. Prostaglandins Leukot Essent Fatty Acids 1997 56:337-343. 88. Dines KC Cotter MA Cameron NE. Contrasting effects of treatment with ro-3 and ro-6 essential fatty acids on peripheral nerve function and capillarization in streptozotocin-diabetic rats. Diabetologia 1993 36: 1132-1138. 89. Jack A Cotter MA Cameron NE. Impaired endothelium-derived hyperpolariz• ing factor in the mesenteric vasculature of diabetic rats: effects of antioxidant treatment abstr. Diabetic Med 1998 15suppl I :S3 l. 90. Moncada S Gryglewski RJ Bunting S Vane JR. A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoper• oxides the substance prostaglandin X which prevents platelet aggregation. Pros• taglandins 1976 12:715-737. 91. Karpen CW Pritchard KA Arnold JH Cornwell DG Pangonamala RV. Restora-

slide 191:

Antioxidants in the Treatment of Diabetic Polyneuropathy 153 lion of the prostacyclin/thromboxane A2 balance in the diabetic rat: influence of vitamin E. Diabetes 1982 31:947-951. 92. De Nucci G Gryglewski RJ Warner TD Vane JR. Receptor mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endo• thelial cells is coupled. Proc Natl Acad Sci USA 1988 85:2334-2338. 93. Cameron NE Cotter MA Dines KC Maxfield EK. Pharmacological manipula• tion of vascular endothelium in non-diabetic and streptozotocin-diabetic rats: ef• fects on nerve conduction hypoxic resistance and endoneurial capillarization. Diabetologia 1993 36:516-522. 94. Cameron NE Cotter MA. Interaction between oxidative stress and y-linolenic acid in the impaired neurovascular function of diabetic rats. Am J Physiol 1996 271 :E47l-E476. 95. Cameron NE Cotter MA. Comparison of the effects of ascorbyl y-linolenic acid and y-linolenic acid in the correction of neurovascular deficits in diabetic rats. Diabetologia 1996 39:1047-1054.

slide 192:

This Page Intentionally Left Blank

slide 193:

11 A Thioctic Acid-Gamma-Linolenic Acid Conjugate Protects NeurotrophicSupport in Experimental Diabetic Neuropathy To Get Rid Of Diabetes Permanently Click Here Luke Hounsom Queen Mary and Westfield College London England David R. Tomlinson University of Manchester Manchester England Defects of the peripheral nervous system are common in patients with diabetes mellitus and a large fraction of patients will develop a form of diabetic neu• ropathy within 25 years after diagnosis 1. Although a rigid classification of diabetic neuropathy is difficult at least three major syndromes are recognized: symmetrical distal polyneuropathy symmetrical proximal motor neuropathies and focal asymmetrical neuropathies 2-4. As in most polyneuropathies sen• sory motor and autonomic nerves are concomitantly involved although sen• sory dysfunction usually predominates. Recent attempts to rationalize the etiology of diabetic neuropathy have focused initially on the identification of the biochemical defects that follow directly from hyperglycemia in this case the polyol pathway protein glyca• tion and oxidative stress for recent reviews see Refs. 5 and 6. Second there is the issue of the importance of reduced nerve blood flow and possible consequent ischemic damage 7-9. Related to this is the proposition that

slide 194:

defective metabolism of essential fatty acids resulting in reduced levels of y• linolenic acid and subsequently arachidonic acid and vasoactive prostanoids also has a large part to play in the pathogenesis of diabetic neuropathy see 155

slide 195:

156 Hounsom and Tomlinson Ref. 10 for review. A chain of consequences comprising hyperglycemia ex• aggerated polyol pathway flux oxidative stress impaired endoneurial nitric oxide production reduced nerve blood flow endoneurial hypoxia and im• paired nerve conduction has been suggested 11-13. Impaired neurotrophic support from nerve growth factor NGF and neurotrophin 3 NT-3 14-17 is also important because a failure of neurotrophic factors to regulate neuronal phenotype might be expected to result in such a clinical picture as presents in symptomatic diabetic neuropathy. Recently the antioxidant thioctic u-Iipoic acid has been found to pro• tect against a broad range of defects in diabetic rats 18-20 including both those related to nerve blood flow and those related to neurotrophins. I. NEUROTROPHIC FACTORS Neurotrophic factors were discovered as agents with the capacity to stimulate neurite outgrowth in culture systems of embryonic sensory or sympathetic neurons. Some of these factors such as ciliary neurotrophic factor a member of the interleukin-6 cytokine family and basic fibroblast growth factor a member of the fibroblast growth factor family are quite distinct from NGF the archetypal neurotrophic factor. It has now been established that NGF be• longs to a family of neurotrophic factors known as the neurotrophins that now includes NT-3 brain-derived neurotrophic factor BDNF and neurotrophin- 4/5 see Ref. 21 for review. NGF undergoes retrograde transport in sensory and sympathetic neurons in adult rats 2223 and about 50 of adult lumbar sensory neurons can bind NGF with high affinity 24. BDNF and NT-3 also undergo retrograde trans• port from an injection site in the sciatic nerve to the dorsal root ganglia DRG and motor neurons of adult rats 25. The neurotrophins bind to two distinct sites on responsive neurons. Low• affinity binding is associated with the p75 neurotrophin receptor p75NTR and shows no selectivity between the neurotrophins. High-affinity binding is provided by a family of trk proto-oncogene receptors that have intrinsic tyro• sine kinase activity. trkA is specific for NGF 2627 trkB for BDNF and NT- 4/5 2829 and trkC for NT-3 30. However NT-3 is promiscuous and will also bind to trkB and trkA at increasing concentrations. There is a large body of evidence suggesting that NGF and the other neurotrophins are involved not just in the survival of neurons during embry• onic development but also in the regulation of neuronal phenotype in the adult. Deprivation of trophic support by nerve transection provokes a pattern of

slide 196:

Neurotrophic Supportin Experimental DiabeticNeuropathy 157 change in the nerve cell body that is prevented by the administration of exoge• nous neurotrophin. Direct evidence for modulation of adult phenotype was provided when it was demonstrated that NGF can regulate the expression of substance P SP and calcitonin gene-related peptide CGRP in primary cul• tures of adult rat DRG neurons 31. Additionally deprivation of NGF has been found to cause a proportional reduction in the expression of these neuro• peptides 3233. 11. NEUROTROPHINS IN DIABETIC NEUROPATHY Decreased capture and retrograde transport of iodinated NGF in the sciatic nerve was observed in diabetic rats many years ago 34. Reduced retrograde transport of iodinated NGF in ilea mesenteric nerves has also been demon• strated 35. These observations imply that even in the absence of any deficit in production of NGF in diabetes a deficit in the amount delivered to the cell body might be expected. In diabetic rats there are reduced levels of NGF in the submandibular gland superior cervical ganglion and sciatic nerve 36- 38. NGF levels have also been shown to be decreased in the serum of diabetic patients with symptomatic peripheral neuropathy 39. Work in our laboratory has shown that with increasing duration of diabe• tes progressive reductions in NGF mRNA appear in leg muscle and sciatic nerve followed by reductions in skin. There is a profound reduction in the retrograde transport of NGF in the sciatic nerve which can be reversed by intensive insulin treatment and dose-related increases in sciatic nerve NGF retrograde transport were seen with recombinant human NGF rhNGF treat• ment 33. Additionally it is clear that there are also deficits in the production of NGF target genes and deficits in expression of SP and CGRP are easily demonstrable in experimental diabetic neuropathy 32. It is quite apparent that there are deficits in NGF expiation in experimen• tal diabetes but this does not explain the earlier observation of reduced capture and transport of exogenous NGF. Recent work in our laboratory has revealed a marked decrease in the retrograde transport of the p75NTR which closely follows the changes seen with NGF transport in diabetes 40. No changes in transport of trkA were observed but it is not yet possible to suggest which are the precedent changes because NGF availability is known to affect the expression of the p75NTR 41. NT-3 mRNA levels are reduced in leg muscle from diabetic rats but an assessment of NT-3 neurotrophic support is difficult because gene targets of NT-3 have yet to be identified. Work in our laboratory showed that treat-

slide 197:

158 Hounsom and Tomlinson ment of diabetic rats with rhNT-3 for the last 4 weeks of a 12-week period of diabetes could completely normalize the reduced sensory nerve conduction velocity which is characteristic of diabetic rats 16. This implies that NT-3 may be even more instrumental than NGF in the development of important functional deficits in diabetic neuropathy. However although treatment of ani• mals with NGF or NT-3 prevents some of the deficits characteristic of experi• mental neuropathy 14 16 these agents do not influence reduced nerve blood flow or motor nerve conduction velocity 42 other classic hallmarks of exper• imental neuropathy. Ill. NEUROTROPHINS AND OXIDATIVE STRESS Shifts in cellular redox balance due to increased levels of free radicals may cause or result from neuronal injury 4344. Oxygen free radicals are gener• ated as a consequence of ischemia-reperfusion inflammation traumatic and oxidative injury and are associated with neuronal cell death 4546. Peripheral nerves including the sciatic nerve have inherent low antioxi• dant defenses compared with the central nervous system because total reduced glutathione GSH content and activities of GSH utilizing enzymes like gluta• thione peroxidase GSH-Px are about I 0-fold lower than they are in brain 47. NGF stimulates cellular resistance to oxidative stress in PC 12 cells 48. In particular NGF protects from oxidative injury induced by hydrogen perox• ide and 6-hydroxydopamine 48-50 both of which generate hydroxyl radi• cals. From these observations it might be expected that NGF regulates cellular oxidant-antioxidant equilibrium. NGF has been found to regulate the expression of the antioxidant en• zymes catalase Cat and GSH-Px. Application of NGF to PC 12 cells in culture results in an increase in the transcription of the mRNA for Cat and GSH-Px and in addition appears to stabilize the transcript for Cat 51 . This is associated with an increase in the activities of both enzymes 52. Furthermore newborn rat astrocytes in culture synthesise NGF in a dose-dependent fashion in response to superoxide anion as generated by xanthine/xanthine oxidase and to hydrogen peroxide 53. IV. OXIDATIVE STRESS IN DIABETES Diabetes is associated with increases in oxidative stress in humans and in experimental animal models. Chronic hyperglycemia per se results in autoxi-

slide 198:

Neurotrophic Support in Experimental Diabetic Neuropathy 159 dative glycation/oxidation and lipid peroxidation 54-56 and hyperglycemia alone will cause lipid peroxidation of peripheral nerve in vitro 57. Diabetic peripheral nerve has increased levels of conjugated dienes end products from peroxidation of polyunsaturated fatty acids 57 58 decreased levels of GSH 19. and reduced activity of copper/zinc-superoxide dismutase that is reversible with reinstatement of moderate glycemic control 58. Further support for the role of oxidative stress in the pathogenesis of diabetic neuropathy come from the effectiveness of antioxidant treatment in reversing some of the functional neurological deficits observed in experimen• tal animal models. Probucol a powerful free radical scavenger normalizes both decreases in endoneurial nerve blood flow and motor nerve conduction velocity 5960. Dietary treatment with l butylated hydroxytoluene or n-tocopherol also has similar effects 6162. Intravenous administration of GSH can also partially prevent motor nerve conduction velocity slowing in diabetic rats 63. Benefi• cial effects on conduction velocity have also been reported using the metal chelator deferoxamine 64. V. THIOCTIC ACID Thioctic acid TA was first isolated in 1951 65 and is now known by a variety of different names including n-Iipoic acid 12-dithiolane-3-pentanoic acid 12-dithiolane valenic acid and 68-thioctic acid. As lipoamide it func• tions as a cofactor in the multienzyme complexes that catalyze the oxidative decarboxylations of u-keto acids such as pyruvate a-ketoglutarate and branched chain n-keto acids 66. In addition TA is a powerful antioxidant 67 and a potent free radical scavenger in peripheral nerve 5768 that would appear to act by substituting for c-tocophercl 57. Interestingly endogenous TA is depleted in diabetes 6970. A study carried out by Kahler et al. 71 confirmed the association of long-term diabetic complications with increased lipid peroxidation and en• hanced lipid peroxidation. Treatment with TA decreased peroxidation rate improved thermal and vibration sensibilities and improved patellar and Achil• les tendon reflexes. Furthermore results from the u-Lipoic Acid in Diabetic Neuropathy study 72 revealed that TA at a dose of 600 or 1200 mg adminis• tered intravenously significantly reduced the incidence of burning sensation parasthesiae and numbness in patients with symptomatic diabetic neuropathy. TA has effects beyond that of a simple antioxidant. TA stimulates glu• cose utilization by tissues 7374 and may also act as a chelator of transition

slide 199:

160 Hounsom and Tomlinson metal ions which are thought to contribute to oxidative stress 75- 77. Fur• thermore TA also inhibits nonenzymatic glycation/glycosylation which is now thought to play a role in the pathogenesis of diabetic late complications see Ref. 78 for review. Unrelated to this TA is also able to induce neurite outgrowth 79 and promote nerve regeneration 80. TA can also stimulate NGF synthesis and secretion 81. VI. IN VIVO STUDIES WITH THIOCTYL·y-LINOLENIC ACID y-Linolenic acid GLA is an Q-6 essential fatty acid that is thought to be the active ingredient of evening primrose oil EPO. Previous studies using EPO as a dietary supplement have shown complete reversal of motor nerve conduc• tion deficits in diabetic rats 8283. EPO has also been found to increase prostacyclin release in diabetic sciatic nerve 84 increase nerve blood flow reduce subsequent nerve trunk ischemia 85 and reduce resistance to isch• emic conduction failure 8687. Given the known effects of TA in experimental diabetes 1920 and the ability of EPO to reverse some of the deficits seen in experimental diabetes the rationale for use of a TA-y-linolenic acid conjugate thioctyl y-linolenic acid GLAMT ATA are not difficult to realize. However in the search for compounds that are active against a broad spectrum of defects encountered in diabetes no such compound has fit that bill. Therefore in vivo studies with the conjugate were designed to reflect two classes of defect with motor and sensory nerve conduction velocities indicating the polyol-nerve blood flow pathway and NGF and neuropeptides as markers of neurotrophic support. In vivo studies evaluated the efficacy of dietary supplementation with 2.5 GLAMTA see Fig. 1 for structural representation 100 mg/kg TA IP five times per week and dietary supplementation with 1.5 butylated hydroxytoluene BHT in 8-week streptozotocin-induced diabetic rats. Untreated diabetic animals showed both motor and sensory nerve con• duction velocity deficits Fig. 2A and reduced levels of NGF neuropeptide Y NPY and SP Fig. 28 in their sciatic nerves. Treatment with BHT was without effect on any of these variables. Treatment with TA increased the NGF content of sciatic nerve and produced small arithmetic increases in both SP and NPY which did not attain significance. There was no effect on either sensory or motor nerve conduction velocity. Treatment with the GLAMT A conjugate increased sciatic nerve levels of both SP and NPY so that they were significantly higher than those measured in nerves from untreated diabetic rats p 0.05 although they remained significantly lower than those of controls

slide 200:

Neurotrophic Support in Experimental Diabetic Neuropathy 161 CH2-0-GLA I CH2-0-TA Figure 1 Structure of thioctyl-y-linolenic acid GLN\1\TA. The molecule is a 123- propanediol diester with one molecule of GLA and one molecule of TA joined to the propanediol backbone by ester linkages. p 0.05 and O.Ql respectively Fig. 28. GLA1v\TA also increased NGF levels Fig. 28 and both motor and sensuous nerve conduction velocities Fig. 2A so that these values were not significantly different from those of control rats. The potential dependence of the neuropeptide changes on NGF levels was examined by regression analyses. For the dependence of SP on NGF r was 0.257 p 0.005 but the levels of NPY were less closely related to those of NGF where r was 0.145 and the regression was barely significant p 0.05. NPY in the sciatic nerve may derive from mixed fiber populations• some must be in sympathetic postganglionic fibers 8889 but there may also be some in somatic sensory fibers. However the level of expression in somatic afferents is low unless they are damaged when it increases 90 after axo• tomy this increased expression may be reduced by either NT-3 or NGF 9192. Expression of NPY by the sympathetic phenotype is clearly stimu• lated by NGF 93 and NGF-responsive elements have been identified on the NPY promoter 94. Thus the findings reported here might be most easily explained by the proposition that the NPY deficit in sciatic nerves of diabetic rats is also derived from reduced NGF neurotrophic support. However our previous study showed that treatment of diabetic rats with NGF although normalizing the SP levels in sciatic nerve did not affect the NPY deficit 20. Thus there may be control of NPY expression in these fibers by another neuro• trophin and the NGF response elements become functional only when other influences are removed. This might explain the NGF effects on NPY in vitro 9395 and increases in NPY after axotomy in vivo 9096. Thus the evolu• tion of the decrease in NPY expression in our diabetic rats cannot be explained as yet though the deficit clearly responds to treatment with the GLAMTA conjugate and in our previous study also responded to TA 20.

slide 201:

- -- c: 162 A Control Diabetic Diabetic Hounsom and Tomlinson llI MNCV o SNCV B 160 Q 140 I NPY SP Q i:: D NGF i:: Q Q Q 120 i:: -:: E 100 100 s 5 0 80 80 5 E :::- 60 60 a.. a. - . Q o z 40 40 20 z c: rn . v c i ::: 0 0 / Control Diabetic Diabetic +GLAMTA Figure 2 A Bar chart showing nerve conduction velocities in control diabetic and 2.5 GLA/V\T A-treated diabetic rats. Control versus diabetic p 0.01 for both motor and sensory nerve conduction velocity 2.5 GLA MTA treated animals were not sta• tistically different from controls or untreated diabetic rats by ANOV A with Duncans multiple range tests. B Bar chart showing sciatic nerve levels of SP NPY. and NGF. Control versus diabetic p 0.01 for SP NPY and NGF 2.5 GLA1\ATA increased sciatic nerve levels of SP and NPY p 0.05 although they remained significantly different from controls p 0.05 and p 0.01 respectively. GLA/V\TA 2.5 also

slide 202:

increased NGF levels so that these values were not different from controls.

slide 203:

NeurotrophicSupport in ExperimentalDiabetic Neuropathy 163 EFFECT OF THIOCTYL--y-LINOLENICACID ON THE ACTIVATION OF STRESS-ACTIVATED PROTEIN KINASES Mitogen-activated protein MAP kinases are praline-directed serine/threo• nine kinases that are activated by dual phosphorylation in response to a wide variety of extracellular stimuli. Three distinct groups of MAP kinases have been identified in mammalian cells extracellular signal-regulated kinase ERK c-jun N-terminal kinase JNK and p38MAP kinase p38 see Refs. 97 and 98 for review. MAP kinase activation is achieved through kinase cas• cades which serve as information relays connecting cell surface receptors to specific transcription factors and other regulatory proteins thus allowing extracellular signals to regulate the expression of specific genes 99. Recently among this large family of MAP kinases a family of stress• activated protein kinases SAPKs including JNK and p38 have been deline• ated and characterized see Ref. 100 for review. JNK exits in three forms in mammalian cells: JNKI JNK2 and JNK3 of 46 54 and 56 kDa molecular weight respectively. JNK has been found to be activated by a variety of differ• ent stimuli including inflammatory cytokines such as interleukin- I and tumor necrosis factor a y-irradiation ultraviolet irradiation and oxidative stress IOI. Once activated JNK phosphorylates a number of different substrates including c-jun and ATF2. Both of these transcription factors can form part of the AP- I transcription factor complex which is known to regulate the tran• scription of many different genes reviewed in Ref. 98. Because oxidative stress as part of the dysmetabolism of diabetes mellitus seemed an excellent candidate for activation of SAPKs we included measurement of activation of these molecules in the present study. The expression of the subtypes of JNK was investigated using antibodies raised against either the native protein JNK-FL of the phosphorylated acti• vated form pJNK in the DRG from 8-week diabetic rats and diabetic rats treated with 2.5 GLAMTA. Western blots were analyzed by densitometry and levels of protein compared by normalizing to controls. Eight weeks of diabetes resulted in a significant increase in the level of the p54 form of activated JNK p 0.05 compared with controls. This in• crease in activation of p54 JNK as measured by Western blotting with phospho• specific antibodies was reversed by 2.5 GLNV\TA Fig. 3 Total levels of the full-length JNK protein were unchanged by diabetes or GLAMT A treatment. Levels of the phosphorylated transcription factors ATF2 and c-jun were investigated using antibodies specific to the phosphorylated forms of these transcription factors and were also unchanged by diabetes or GLNATA treat• ment.

slide 204:

o JNK-F 164 Hounsom and Tomlinso n p54/56- .. p46- .. Control Diabetic Diabetic +GLAMTA Native JNK p54/56- p46- Phospho-JNK .ll 2.5 · • JNK-P L 2 2 :: C:- 1.5 · -e 1. 0.5 Control Diabetic Diabetic + GLAMTA Figure 3 Western blots from lumbar DRG exposed to antibodies against a nonphos• phorylated epitope of JNK JNK-FL or a phosphorylated epitope pJNK. Bar chart shows diabetes-induced increase in p54/p56 JNK phosphorylation p 0.05 controls can be reversed by. GLAMTA. There were no changes in the levels of the native pro• tein JNK-FL. The reduction in the activation of JNK by GLAMTA may reflect a re• duction in neuronal stress. TA treatment is known to increase levels of GSH in diabetic sciatic nerve 19 and GSH is a critical regulator for the induction of SAPKs including JNK 102. However it is not yet known whether the conjugate has similar effects on glutathione metabolism but this warrants closer investigation.

slide 205:

NeurotrophlcSupport in ExperimentalDiabetic Neuropathy 165 VIII. ROLE FOR GLA/\/\TA IN THE TREATMENT OF DIABETIC NEUROPATHY This is the first instance of a treatment that is capable of attenuating both electrophysiological and neurochemical deficits in the nerves of diabetic rats. The effect of the GLA/\ATA conjugate is also remarkable when related to the current effects of TA and previously reported effects of EPO. Approximate calculations suggest that the dose of GLA /\ATA used here could deliver about 14 mg/kg/day TA and 18 mg/kg/day GLA. TA must be given at doses of at least 50 mg/kg/day to influence nerve conduction 19 and in the present and in previous 20 studies had little effect on sciatic nerve NGF levels at 100 mg/kg/day. Assuming an approximate content of 10 GLA in EPO 83 a daily consumption of about 180 mg oil would be required to match the current dose of the conjugate. A previous study demonstrated no effect of EPO at about 3.5 g/day per rat equivalent to about 10 g/kg/day on the deficit in sciatic nerve SP in diabetic rats 82. It is therefore clear that the properties of the conjugate are significantly greater than those of its constituent mole• cules. We suspected that a membrane localization might be important and that was why BHT a lipid-soluble antioxidant was included in this study but it showed no efficacy indicating that simple membrane sequestration of TA does not provide an explanation for the efficacy of the conjugate. Furthermore although NGF treatment of diabetic rats does not affect nerve conduction deficits the conjugate had multiple protective effects on different fiber groups-viz increasing nerve NGF levels stimulating NPY expression by a different mechanism and boosting deficient conduction velocities. Clearly this molecule shows clinical potential. REFERENCES I. Diabetes Control and Complications Trial Research Group. The effect of inten• sive treatment of diabetes on the development and progression of long-term complications in insulin-dependentdiabetes mellitus. N Engl J Med 1993 329: 977-986. 2. Brown MJ Asbury AK. Diabetic neuropathy. Ann Neurol 1984 15:2-12. 3. Dyck PJ Bushek W Spring EM Karnes JL Litchy WJ OBrien PC Service FJ. Vibratory and cooling detection thresholds compared with other tests in diagnosing and staging diabetic neuropathy. Diabetes Care 1987 10:432-440.

slide 206:

166 Hounsomand Tomlinson 4. Thomas PK Tomlinson DR. Diabetic and hypoglycaemic neuropathy. In: Dyck PJ Thomas PK Griffin JW Low PA Poduslo JF ed. Peripheral Neuropathy. 3rd ed. Philadelphia: W.B. Saunders 1992:1219-1250. 5. Biessels GJ Van Dam PS. Diabetic neuropathy pathogenesis and current treat• ment perspectives. Neurosci Res Commun 1997 20: 1-10. 6. Yagihashi S. Pathology and pathogenetic mechanisms of diabetic neuropathy. Diabetes Metab Rev 1996 11: 193-225. 7. Dyck PJ Giannini C. Pathologic alterations in the diabetic neuropathies of hu• mans: a review. J Neuropathol Exp Neurol 1996 55: 1181-1193. 8. Tesfaye S Malik R Ward JD. Vascular factors in diabetic neuropathy. Diabeto• logia 1994 37:847-854. 9. Williamson JR Chang K Frangos M Hasan KS ldo Y Kawamura T et al. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993 42: 801-813. 10. Horrobin DF. The roles of essential fatty acids in the development of diabetic neuropathy and other complications of diabetes mellitus. Prostaglandins Leukot Essent Fatty Acids 1988 31:181-197. 11. Cameron NE Cotter MA Basso M Hohman TC. Comparison of the effects of inhibitors of aldose reductase and sorbitol dehydrogenase on neurovascular function nerve conduction and tissue polyol pathway metabolites in streptozo• tocin-diabetic rats. Diabetologia 1997 40:271-281. 12. Sasaki H Schmelzer JD Zollman PJ Low PA. Neuropathology and blood flow of nerve spinal roots and dorsal root ganglia in longstanding diabetic rats. Acta Neuropathol Berl 1997 93:118-128. 13. Kihara M Low PA. Impaired vasoreactivity to nitric oxide in experimental diabetic neuropathy. Exp Neurol 1995 132:180-185. 14. Tomlinson DR Femyhough P Diemel LT. Role of neurotrophins in diabetic neuropathy and treatment with nerve growth factors. Diabetes 1997 46:543- 549. 15. Dyck PJ. Nerve growth factor and diabetic neuropathy. Lancet 1996 348: 1044-1045. 16. Tomlinson DR Femyhough P Diemel LT. Neurotrophins and peripheral neu• ropathy. Philos Trans R Soc Loud B 1996 351:455-462. 17. Thomas PK. Growth factors and diabetic neuropathy. Diabet Med 1994 11: 732-739. 18. Packer L. Antioxidant properties of lipoic acid and its therapeutic effects in prevention of diabetes complications and cataracts. Ann NY Acad Sci 1994 738:257-264. 19. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H et al. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care 1995 18:1160-1167.

slide 207:

Neurotrophic Support in Experimental Diabetic Neuropathy 167 20. Garrett NE. Malcangio M Dewhurst M. Tomlinson DR. u-Lipoic acid corrects neuropeptide deficits in diabetic rats via induction of trophic support. Neurosci Lett 1997 222: 191-194. 21. Ebendal T. Function and evolution in the NGF family and its receptors. J Neu• rosci Res 1992 32:461-470. 22. Hendry IA Stoeckel K. Thoenen H Iversen LL. The retrograde axonal trans• port of nerve growth factor. Brain Res 1974 68: 103-121. 23. Schmidt RE Plurad SB Saffitz JE Grabau GG Yip HK. Retrograde axonal transport of 11511-nerve growth factor in rat Ilea mesentric nerves-effect of streptozocin diabetes. Diabetes 1985 34: I 230-1240. 24. Richardson PM Verge Issa VMK Riopelle RJ. Distribution of neuronal recep• tors for nerve growth factor in the rat. J Neurosci 1986 6:2312-2321. 25. DiStefano PS Friedman B Radziejewski C Alexander C Boland P Schick CM et al. The neurotrophins BDNF NT-3. and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron 1992 8:983-993. 26. Meakin SO. Suter U Drinkwater CC Welcher AA Shooter EM. The rat trk protooncogene product exhibits properties characteristic of the slow nerve growth factor receptor. Proc Natl Acad Sci USA 1992 89:2374-2378. 27. Martin-Zanca D Oskarn R. Mitra G Copeland T Barbacid M. Molecular and biochemical characterization of the human trk proto-oncogene. Mot Cell Biol 1989 9:24-33. 28. Klein R Nanduri V Jing SA Lamballe F Tapley P Bryant S et al. The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 1991 66:395-403. 29. Klein R Lamballe F Bryant S Barbacid M. The trkB tyrosine protein kinase is a receptor for neurotrophin-4. Neuron 1992 8:947-956. 30. Lamballe F. Tapley P Barbacid M. trkC encodes multiple neurotrophin-3 re• ceptors with distinct biological properties and substrate specificities. EMBO J 1993 12:3083-3094. 31. Lindsay RM Harmar AJ. Nerve growth factor regulates expression of neuro• peptide genes in adult sensory neurons. Nature l 989337:362-364. 32. Fernyhough P Diemel LT Brewster WJ Tomlinson DR. Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor rnRNA in streptozotocin-diabetic rats effects of insulin treat• ment. Neuroscience 1994 62:337-344. 33. Femyhough P Diemel LT Hardy J Brewster WJ Mohiuddin L Tomlinson DR. Human recombinant nerve growth factor replaces deficient neurotrophic support in the diabetic rat. Eur J Neurosci 1995 7: I 107-1110. 34. Jakobsen J Brimijoin S Skau K Sidenius P Wells D. Retrograde axonal trans• port of transmitter enzymes fucose-labeled protein and nerve growth factor in streptozotocin-diabetic rats. Diabetes 1981 30:797-803.

slide 208:

168 Hounsomand Tomlinson 35. Schmidt RE Grabau GG Yip HK. Retrograde axonal transport of 125Inerve growth factor in ilea mesenteric nerves in vitro: effect of streptozotocin diabe• tes. Brain Res 1986 378:325-336. 36. Hellweg R Hartung H-D. Endogenous levels of nerve growth factor NGF are altered in experimental diabetes mellitus: a possible role for NGF in the pathogenesis of diabetic neuropathy. J Neurosci Res 1990 26:258-267. 37. Hellweg R Wehrle M Hartung H-D Stracke H Hock C Federlin K. Diabetes mellitus-associated decrease in nerve growth factor levels is reversed by alloge• neic pancreatic islet transplantation. Neurosci Lett 1991 125:1-4. 38. Hanaoka Y Ohi T Furukawa S Furukawa Y Hayashi K Matsukura S. Effect of 4-methylcatechol on sciatic nerve growth factor level and motor nerve con• duction velocity in experimental diabetic neuropathic process in rats. Exp Neu• rol 1992 115:292-296. 39. Faradji V Sotelo J. Low serum levels ofnerve growth factor in diabetic neurop• athy. Acta Neurol Scand 1990 81 :402-406. 40. Delcroix J Tomlinson DR Femyhough P. Diabetes and axotomy-induced deficits in retrograde axonal transport of nerve growth factor correlate with decreased levels of p 75LNTR protein in lumbar dorsal root ganglia. Mol Brain Res 1997 51 :82-90. 41. Doherty P Seaton P Flanigan TP Walsh FS. Factors controlling the expression of the NGF receptor in PC12 cells. Neurosci Lett 1988 92:222-227. 42. Maeda K Femyhough P Tomlinson DR. Effects of treatment of diabetic rats with human recombinant nerve growth factor on sciatic nerve conduction Doppler flux and substance P levels. Diabet Nutr Metab 1997 10:3-8. 43. Perez-Polo JR. The nerve growth factor receptor. In: Horrocks LA Neff NH Yates AJ Handjiconstantinou M eds. Trophic Factors and the Nervous System. New York: Raven Press 1990:107-118. 44. Gaetani P Lombardi D. Brain damage following subarachnoid hemorrhage: the imbalance between anti-oxidant systems and lipid peroxidative processes. J Neurosurg Sci 1992 36: 1-10. 45. Kitagawa K Matsumoto M Oda T Niinobe M Hata R Handa N et al. Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death. Neuroscience 1990 35:551-558. 46. Jenner P Dexter DT Sian J Schapira AH Marsden CD. Oxidative stress as a cause of nigral cell death in Parkinsons disease and incidental Lewy body disease. Ann Neurol 1992 32suppl:S82-S87. 47. Romero FJ Monsalve E Hermenegildo C Puertas FJ Higueras V Nies E. et al. Oxygen toxicity in the nervous tissue: comparison of the antioxidant defense of rat brain and sciatic nerve. Neurochem Res 1991 16: 157-161. 48. Jackson GR Apffel L Werrbach-Perez K Perez-Polo JK. Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hy• drogen peroxide resistance. J Neurosci Res 1990 25:360-368. 49. Tiffany-Castiglioni E Perez-Polo JR. Stimulation of resistance to 6-hydroxy-

slide 209:

Neurotrophic Support in Experimental Diabetic Neuropathy 169 dopamine in a human neuroblastoma cell line by nerve growth factor. Neuro• sci Lett 1981 26:157-161. 50. Tiffany-Castiglioni E Perez-Polo JR. The role of nerve growth factor in vitro in cell resistance to 6-hydroxydopamine toxicity. Exp Cell Res 1979 121:I 79- 189. 51. Sampath D Perez-Polo R. Regulation of antioxidant enzyme expression by NGF. Neurochem Res 1997 22:351-362. 52. Sampath D Jackson GR Werrbach-Perez K Perez-Polo JR. Effects of nerve growth factor on glutathione peroxidase and catalase in PC 12 cells. J Neuro• chem 1994 62:2476-2479. 53. Navcilhan P Neveu I. Jehan F Baudet C Wion D Brachet P. Reactive oxygen species influence nerve growth factor synthesis in primary rat astrocytes. J Neu• rochem 1994 62:2178-2186. 54. Wolff SP. Diabetes mellitus and free radicals. Br Med Bull 1993 49:642-652. 55. Wolff SP Jiang ZY Hunt JV. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Rad Biol Med 1991 10:339-352. 56. Stith BJ Proctor WR. Microinjection of inositol I 2-cyclic-45-trisphosphate inositol 1345-tetrakisphosphate and inositol 145-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium. J Cell Biochem 1989 40:321-330. 57. Nickander KK McPhee BR Low PA Tritschler H. Alpha-lipoic acid: antioxi• dant potency against lipid peroxidation of neural tissues in vitro and implica• tions for diabetic neuropathy. Free Rad Biol Med 1996 21:631-639. 58. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes 1991: 40:873-877. 59. Cameron NE Cotter MA Archibald V Dines KC. Maxfield EK. Anti-oxidant and pro-oxidant effects on nerve conduction velocity endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats. Diabeto• logia 1994 37:449-459. 60. Karasu c Dewhurst M Stevens EJ Tomlinson DR. Effects of anti-oxidant treatment on sciatic nerve dysfunction in streptozotocin-diabetic rats compari• son with essential fatty acids. Diabetologia 1995 38:129-134. 61. Cameron NE Cotter MA Maxfield EK. Anti-oxidant treatment prevents the development of peripheral nerve dysfunction in streptozotocin-diabetic rats. Diabetologia 1993 36:299-304. 62. Love A Cotter MA Cameron NE. Effects of a-tocopherol on nerve conduction velocity and regeneration following a freeze lesion in immature diabetic rats. Naunyn Schmiedebergs Arch Pharmacol 1997 355:126-130. 63. Bravenboer B Kappelle AC Hamers FPT Van Buren T Erkelens DW Gispen WH. Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. Diabetologia 1992 35: 813-817. 64. Cameron NE. Cotter MA. Neurovascular dysfunction in diabetic rats. Potential

slide 210:

170 Hounsom and Tomlinson contribution of autoxidation and free radicals examined using transition metal chelating agents. J Clin Invest 1995 96: 1159-1163. 65. Reed LJ DeBusk BG Gunsalius IC Hornberger CSJ. Crystalline alpha-lipoic acid: a catalytic agent associated with pyruvate dehydrogenase. Science 1951 114:93-94. 66. Reed LJ. Multienzyme complex. Ace Chem Res 1974 7:40-46. 67. Packer L Witt EH Tritschler HJ. n-Lipoic acid as a biological antioxidant. Free Rad Biol Med 1995 19:227-250. 68. Busse E Zimmer G Schopohl B Kornhuber B. Influence of alpha-lipoic acid on intracellular glutathione in vitro and in vivo. Arzneimittelforschung 1992 42:829-831. 69. Natraj CY Gandhi VM Menon KKG. Lipoic acid and diabetes: effects of dihy• drolipoic acid administration in diabetic rats and rabbits. J Biosci 1984 6:37- 46. 70. Wagh SS Natraj CV Menon KKG. Mode of action of lipoic acid in diabetes. J Biosci 1987 11 :59-74. 71. Kahler W Kuklinski B Ruhlmann C Plotz C. Diabetes mellitus-a free radi• cal-associated disease. Results of adjuvant antioxidant supplementation. Z Gesamte Inn Med 1993 48:223-232. In German. 72. Ziegler D Hanefeld M Ruhnau KJ Meissner HP Lobisch M Schutte K et al. Treatment of symptomatic diabetic peripheral neuropathy with the anti• oxidant a-lipoic acid-a 3-week multicentre randomized controlled trial ALADIN study. Diabetologia 1995 38: 1425-1433. 73. Haugaard N Haugaard ES. Stimulation of glucose utilization by thioctic acid in rat diaphragm incubated in vitro. Biochim Biophys Acta 1970 222:583- 586. 74. Bashan N Burdett E Klip A. Effect of thioctic acid on glucose transport. 3rd Int Thoctic Acid Workshop 1993 3:218-223. 75. Grunert R. The effect of D L-alpha-lipoic acid on heavy metal intoxication in mice and dogs. Arch Biochem Biophys 1960 86: 190-195. 76. Matsugo S Yan LJ Han D Trischler HJ Packer L. Elucidation of antioxidant activity of alpha-lipoic acid toward hydroxyl radical. Biochem Biophys Res Commun 1995 208:161-167. 77. Ou P Tritschler HJ Wolff SP. Thioctic lipoic acid: a therapeutic metal-chelat• ing antioxidant Biochem Pharmacol 1995 50:123-126. 78. Brownlee M. Glycation products and the pathogenesis of diabetic complica• tions. Diabetes Care 1992 15: 1835-1843. 79. Dimpfel W Spuler M Pierau FK Ulrich H. Thioctic acid induces dose-depen• dent sprouting of neurites in cultured rat neuroblastoma cells. Dev Pharmacol Thcr 1990 14: 193-199. 80. Kemplay S Martin P Wilson S. The effects of thioctic acid on motor nerve terminals in acrylamide-poisoned rats. Neuropathol Appl Neurobiol 1988 14: 275-288. 81. Murase K Hattori A Kohno M Hayashi K. Stimulation of nerve growth factor

slide 211:

Neurotrophic Support in Experimental Diabetic Neuropathy 171 synthesis/secretion in mouse astroglial cells by coenzymes. Biochem Mo Biol Int 1993 30:615-62 l. 82. Tomlinson DR Robinson JP Compton AM Keen P. Essential fatty acid treat• ment-effects on nerve conduction polyol pathway and axonal transport in streptozotocin diabetic rats. Diabetologia 1989 32:655-659. 83. Lockett MJ Tomlinson DR. The effects of dietary treatment with essential fatty acids on sciatic nerve conduction and activity of the Na+ /K+ pump in strcptozo• tocin-diabetic rats. Br J Pharmacol 1992 105:355-360. 84. Stevens EJ Carrington AL Tomlinson DR. Prostacyclin release in experimen• tal diabetes: effects of evening primrose oil. Prostaglandins Leukot Essen Fatty Acids 1993 49:699- 706. 85. Stevens EJ Lockett MJ Carrington AL Tomlinson DR. Essential fatty acid treatment prevents nerve ischaernia and associated conduction anomalies in rats with experimental diabetes mellitus. Diabetologia 1993 36:397-401. 86. Carrington AL Lockett MJ Tomlinson DR. The effects of dietary supplemen• tation with evening primrose oil on resistance to anoxic conduction block in nerves from control and streptozotocin-diabetic rats abstr. Diabet Med 1992 9suppl l:35A. 87. Stevens EJ Lockett MJ Carrington AL Tomlinson DR. Essential fatty acid treatment prevents nerve ischaemia and associated conduction anomalies in ex• perimental diabetes abst. Br J Pharmacol 1993 108:38P. 88. Lundberg JM Terenius L Hokfelt T Martling CR Tatemoto K Mutt V et al. Neuropeptide Y NPY-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol Scand 1982 116:477-480. 89. Tatemoto K Carlquist M Mutt V. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982 296:659-660. 90. Wakisaka S Kajander KC Bennett GJ. Increased neuropeptide Y NPY-like immunoreactivity in rat sensory neurons following peripheral axotomy. Neu• rosci Lett 1991 124:200-203. 91. Ohara S Tantuwaya V DiStefano PS Schmidt RE. Exogenous NT-3 mitigates the transganglionic neuropeptide Y response to sciatic nerve injury. Brain Res 1995 699:143-148. 92. Verge VMK Richardson PM Wiesenfeld-Hallin Z Hokfelt T. Differential in• fluence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J Neurosci 1995 15:2081- 2096. 93. Allen JM Tischler AS Lee YC Bloom SR. Neuropeptide Y NPY in PCl2 phaeochromocytoma cultures: responses to dexamethasone and nerve growth factor. Neurosci Lett 1984 46:291-296. 94. Higuchi H Nakano K Miki N. Identification of NGF-response element in the rat neuropeptide Y gene and induction of the binding proteins. Biochem Bio• phys Res Commun 1992 189:1553-1560.

slide 212:

172 Hounsom and Tomlinson 95. Balbi D Allen JM. Role of protein kinase C in mediating NGF effect on neuro• peptide Y expression in PC 12 cells. Brain Res Mol Brain Res 1994 23:310- 316. 96. Zhang X Wiesenfeld-Hallin Z Hokfelt T. Effect of peripheral axotomy on expression of neuropeptide Y receptor mRNA in rat lumbar dorsal root ganglia. Eur J Neurosci 1994 6:43-57. 97. Kiefer F Tibbles LA Lassam N Zanke B lscove N Woodgett JR. Novel components of mammalian stress-activated protein kinase cascades. Biochem Soc Trans 1997 25:491-498. 98. Whitmarsh AJ Davis RJ. Transcription factor AP-1 regulation by mitogen• activated protein kinase signal transduction pathways. J Mol Med 1996: 74: 589-607. 99. Su B Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 1996 8:402-411. 100. Woodgett JR Avruch J Kyriakis J. The stress activated protein kinase pathway. Cancer Surv 1996 27:127-138. IO I. Minden A Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta Rev Cancer 1997 1333:F85-Fl04. I 02. Wilhelm D Bender K Knebel A Angel P. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction path• ways including Jun N-terminal protein kinases and p38 kinase by alkylating agents. Mol Cell Biol 1997 17:4792-4800.

slide 213:

12 Clinical Trials of o-Lipoic Acid in Diabetic Polyneuropathy and Cardiac Autonomic Neuropathy To Kill Diabetes Forever Click Here Dan Ziegler Diabetes Research Institute Heinrich-Heine-University Diissekiort Germany Polyneuropathy involving the somatic and autonomic nervous system is re• sponsible for substantial morbidity and increased mortality among diabetic patients. Near normoglycemia is now generally accepted as the primary ap• proach to prevention of diabetic neuropathy 12. However in diabetic pa• tients with advanced stages of peripheral neuropathy relatively long periods of near-normal glycemic control for several months or even years may be needed to retard the progression of nerve dysfunction 3. Because normogly• cemia is not achievable in most diabetic patients the effects of several medical treatments derived from the pathogenetic concepts of diabetic neuropathy have been evaluated in numerous randomized clinical trials during the past two decades. However due to various reasons none of these compounds has been marketed as yet in the major European countries or in the United States. None• theless in symptomatic diabetic neuropathy additional pharmacological treat• ment of painful neuropathic symptoms is frequently required to maintain the patients quality of life. Although treatment of pain with antidepressants is effective it may be of limited value because of frequent adverse reactions 4. Other symptomatic approaches including anticonvulsants mexiletine and

slide 214:

topical capsaicin either have not been unequivocally effective have shown only partial effects or caution has been expressed as to potential neurotoxic 173

slide 215:

174 Ziegler side effects in view of longer term treatment 4. Furthermore these medica• tions are designed to modulate symptoms without influencing the underlying neuropathy. Cardiovascular autonomic neuropathy CAN is a serious complication of diabetes that is associated with a poor prognosis and may result in severe clinical symptoms 5. Although CAN is appreciated as a clinical entity since 1945 in the past it has received less attention than peripheral sensorimotor neuropathy one reason being that noninvasive quantitative and reliable meth• ods for assessment of cardiovascular autonomic function are available since only the last two decades. After the introduction of cardiovascular reflex tests based on changes in heart rate variability HRV and blood pressure regulation into clinical routine it became evident that CAN may be frequently detected at early stages in asymptomatic diabetic patients 5. The question of long-term primary prevention of CAN has been previ• ously addressed only in trials that studied the role of near nonnoglycemia in Type I diabetic patients. These studies have shown that the development of abnormalities in HRV can be prevented or retarded by intensive insulin ther• apy 16. Secondary intervention trials in patients with advanced CAN have demonstrated that its progression can be delayed during long-term near nor• moglycemia but periods of more than 2 years are needed 35. I. ROLE OF OXIDATIVE STRESS IN DIABETIC NEUROPATHY: CLINICAL AND EXPERIMENTAL EVIDENCE A growing body of evidence suggests that oxidative stress resulting from en• hanced free radical formation and/or defects in antioxidant defense is impli• cated in the development of various disorders including neurodegenerative diseases 7 and diabetic complications 89. Increased free radical formation and changes in hemostatic variables related to endothelial damage have been found in Type 2 diabetic patients with microalbuminuria 10. Impaired endo• thelium-dependent vasodilation is improved by administration of vitamin C suggesting that nitric oxide inactivation by increased oxygen free radical activ• ity contributes to abnormal vascular reactivity in diabetes 11 . Furthermore impaired cellular scavenging activity against oxidative stress 12 and elevated levels of plasma hydroperoxides in conjunction with a trend to lower vitamin E levels 13 have recently been demonstrated in patients with Type 2 diabetes. In experimental diabetic neuropathy oxygen free radical activity in the sciatic nerve is increased 9. Treatment with n-lipoic acid a potent lipophilic

slide 216:

o-Lipolc Acid in Diabetic Neuropathy 175 free radical scavenger 14 results in prevention of neurovascular abnormali• ties associated with experimental diabetic neuropathy 15. It has been demon• strated that reduced digital nerve conduction velocity NCV nerve blood flow and glutathione levels in diabetic rats are normalized and in vitro lipid peroxidation of neural tissue reduced by u-lipoic acid in a dose-dependent manner 15 16 suggesting that the improvement in neurovascular changes were induced by improving oxygen free radical scavenging activity. One mechanism of reduced nerve blood flow is the inhibitory effect of superoxide anion on nitric oxide synthase. Because nitric oxide synthase is reduced in experimental diabetic neuropathy 17 n-lipoic acid might prevent this inhibi• tion by reducing oxidative stress 15. A recent study has also demonstrated that treatment with o-Iipoic acid may correct neuropeptide deficits in diabetic rats indicating that the compound may boost neurotrophic support 18. Ad• ministration of low doses of an o-lipoic acid-gamma-linolenic acid conjugate corrects the nerve conduction and nerve blood flow deficits 19 and sciatic nerve contents of nerve growth factor substance P and neuropeptide Y 20 in diabetic rats suggesting a marked synergistic action of these compounds. These experimental findings provide the rationale for a potential therapeutic value of a-lipoic acid in diabetic patients with neuropathy. II. MULTICENTER CONTROLLED CLINICAL TRIALS Randomized clinical trials RCTs are a widely accepted means of applying experimental methods to a clinical setting and have been advocated as the gold standard for comparing and evaluating different treatments 21 . However the quality of the RCTs that evaluated the effects of medical treatment in diabetic polyneuropathy was poor. Cavaliere et al. 22 assessed the quality of scientific evidence for the efficacy of various pathogenetically oriented treatment ap• proaches for diabetic polyneuropathy examined in RCTs published between 1981 and 1992. They used a quality system covering the internal scientific validity the ability to demonstrate a treatment effect if it really exists and external validity the possibility of generalizing the study results to patients seen in clinical practice. The analysis based on 38 RCTs in total revealed a devastating picture: The methods of randomization were unspecified and a detailed a priori estimate of the sample size needed to detect a treatment differ• ence was not reported in 95 of the RCTs respectively. Only 11 of the RCTs had sufficient statistical power to detect a clinically meaningful differ• ence 22. More generally spoken a recent analysis by Freiman et al. 23 revealed that only 4 of 71 controlled clinical trials 5.6 that reported nega-

slide 217:

176 Ziegler Table 1 Multicenter Randomized Double-Blind Placebo-Controlled Trials Using u-Lipoic Acid in Diabetic Peripheral and Cardiac Autonomic Neuropathy ALADIN Study DEKAN Study" ALADIN 3 Study Number Design 328 Four parallel groups 73 Two parallel groups 509 Three parallel groups Dose l 200/600/l 00 mg aJP 800 mg a/P PO 600 IV/1800 mg Duration of I.V. 3 wk 4 mo POa-a/a-P/P-P 3 wk+ 6 mo treatment Effect Symptoms+ NDS+ HRV+ TSS TSS NIS+ a a-Lipoic acid P placebo + improvement IV intravenous PO orally. "Diabetologia 38 1995. b Diabetes Care 20 1997. Diabetes Care 22 1999. tive results p 0.1 in major medical journals between 1960 and 1977 in• cluded a sample size large enough to have a 90 chance of detecting a 25 difference in treatment effects. In another analysis the situation did not im• prove as the number was 4 of 65 trials 6.2 23. Thus as recently stated by Altman and Bland 24 absence of evidence is not evidence of absence. In other words to interpret most published RCTs as providing evidence of an ineffectiveness of the respective treatment in diabetic neuropathy would be clearly misleading because the likelihood to detect differences in the parame• ters of nerve function between the treatment groups was too low in view of the small sample sizes. Adequate designs for RCTs in diabetic neuropathy have to consider the following aspects: type and stage of neuropathy homogeneity of the study population outcome measures neurophysiological markers intermediate clin• ical end points ultimate clinical outcomes quality of life natural history sample size study duration reproducibility of neurophysiological and inter• mediate end points regression to the mean true and perceived placebo effects measures of treatment effect and the generalizability of the overall trial result to individual patients 25. The results of three multicenter RCTs that evalu• ated the effects of o-lipoic acid in Type 2 diabetic patients with polyneuropa• thy are summarized and discussed below Table I. A. ALADIN Study The improvement in neuropathic symptoms during a 3-week period of intrave• nous treatment with 600 mg o-lipoic acid/day as compared with vitamin 81

slide 218:

o:-Lipoic Acid in Diabetic Neuropathy 26 in conjunction with the recent experimental evidence 15 formed the rationale for a large-scale 3-week multicenter randomized double-blind pla• cebo-controlled trial Alpha-Lipoic Acid in Diabetic Neuropathy: ALADIN 27. Assuming from previous studies a placebo response of about 30 and a drug response of about 60 the a priori estimate of the required sample size yielded n 67 patients per group with a 0.05 and O. l for the two-tailed log-rank test. The efficacy and safety of intravenous infusion of o-lipoic acid were evaluated in 328 NIDDM outpatients with symptomatic peripheral neuropathy who were randomly assigned to treatment using three doses a-lipoic acid 1200 mg/day 600 mg/day 100 mg/day or placebo. Neuropathic symptoms pain burning paresthesias and numbness were assessed by the total symp• tom score TSS at baseline and each visit days 2-5 8-12 and 15-19 before infusion. In addition the Hamburg Pain Adjective List HPAL a multidimen• sional specific pain questionnaire and the Neuropathy Symptom and Disabil• ity Scores NOS were assessed at baseline and at day 19 27. According to the protocol 260 patients n 65 1200 mg/day: 11 63 600 mg/day n 66 100 mg/day n 66 placebo completed the study. No significant differences were noted for the mean changes in HbA1 and blood glucose levels between the four groups studied. TSS in the feet decreased from baseline to day 19 rnean z SD by -4.5 :± 3.7 -58.6 points in o-lipoic acid 1200 -5.0 :± 4.1 -63.5 points in o-lipoic acid 600 -3.3 :± 2.8 -43.2 points in a-Iipoic acid 100 and -2.6 :± 3.2 -38.4 points in placebo o-lipoic acid 1200 vs. placebo p 0.003 n-Iipoic acid 600 vs. placebo p 0.00 I. The response rates after 19 days defined as an improve• ment in the TSS of at least 30 were 70.8 in a-lipoic acid 1200 82.5 in u-lipoic acid 600 65.2 in o-lipoic acid 100 and 57.6 in placebo a• lipoic acid 600 vs. placebo p 0.002. The total scale of the HPAL was significantly reduced in o-lipoic acid 1200 and o-lipoic acid 600 as compared with placebo after 19 days both p 0.0 l . Detailed analysis of pain revealed that both the affective and sensory components of pain representing pain ex• perience and pain perception could be improved. NOS decreased by - 1.8 :± 0.3 points in a-lipoic acid 1200 by -1.5 :± 0.3 points in o-lipoic acid 600 by -0.9 :± 0.3 in o-lipoic acid 100 and by -1.0 :± 0.2 in placebo after 19 days p 0.03 for a-lipoic acid 1200 vs. placebo. The rates of adverse events were 32.6 in n-Iipoic acid 1200 18.2 in a-lipoic acid 600 13.6 in a• lipoic acid 100 and 20.7 in placebo. These findings demonstrate that paren• teral treatment with o-lipoic acid over 3 weeks using a dose of 600 mg/day in NIDDM patients is associated with a significant reduction of various symp• toms of peripheral neuropathy including pain paresthesias and numbness as

slide 219:

178 Ziegler compared with placebo. Furthermore it is evident that a dose of 100 mg/day does not exert an effect superior to that seen with placebo. An increase in the dosage to 1200 mg/day is associated with an enhanced rate of adverse events rather than with maximized efficacy. The increased risk of gastrointestinal side effects associated with 1200 mg/day precludes from using this dose 27. It may be argued that nerve conduction studies have not been used in this study as objective measures of neuropathy. However electrophysiological changes are not relevant in patients with pain. In addition in a short-term study of this kind a significant difference between the groups treated with a• lipoic acid and the placebo group regarding NCV would not appear likely to occur. Previous studies using drugs such as the aldose reductase inhibitors have shown that NCV was either unchanged 28 or only a minimal increase was seen within several weeks of treatment 29 that was subject to substantial criticism as to whether it represented a clinically meaningful degree of change or merely a physiological variation 30. By contrast neuropathic symptoms have been shown susceptible to intervention within a few weeks. Painful symptoms but not motor and sensory NCV were improved after 4 weeks of treatment with sorbinil as compared with placebo 28 and withdrawal of tolrestat resulted in a rapid worsening of pain scores 31 . It may also be argued that the exclusion of 5 J patients due to failures to adhere to the protocol potentially could have introduced bias. However an additional analysis of the results of the ALADIN Study based on the intention to treat revealed no appreciable differences in the outcome of the parameters studied when compared with the per-protocol analysis indicting that the ad• herence to the study protocol did not introduce bias. In summary it has been demonstrated that in diabetic subjects a relief of neuropathic symptoms can be achieved by short-term intravenous with n-lipoic acid. B. DEKAN Study An effective treatment aimed at improving or retarding the progression of reduced HRV the hallmark and earliest sign of CAN might potentially favor• ably influence the poor prognosis among these patients. The efficacy and safety of o-lipoic acid were studied in a randomized double-blind placebo-controlled multicenter trial Deutsche Kardiale Auto• nome Neuropathie: DEKAN Study in NIDDM patients with CAN 32. Inclu• sion criteria were age 18 and :570 years NIDDM treated with diet oral antidiabetic agents and/or insulin spectral power of HRV in the low-fre• quency band 0.05-0.15 Hz and/or high-frequency band 0.15-0.5 Hz below

slide 220:

o-Llpoic Acid in Diabetic Neuropathy 179 the 2.5 centile of age-related normal ranges 33. Eligible patients were ran• domly assigned to treatment with an oral dose of 800 mg/day 200 mg four times daily o-Iipoic acid n 39 or placebo n 34 for 4 months. Parame• ters of HRV at rest including the coefficient of variation CV root mean squared successive difference RMSSD and spectral power in the low-fre• quency 0.05-0.15 Hz and high-frequency 0.15-0.5 Hz bands and the QTc interval were assessed at baseline 2 weeks and at the end of each month of study using a validated computer system 33. Seventeen patients o-lipoic acid n 10 placebo n 7 dropped out of the study but only 3 o-lipoic acid n I placebo n 2 of these dropouts were due to adverse reactions. Mean blood pressure heart rate and HbA1 levels did not differ between the groups during the study. RMSSD increased from baseline to 4 months by 1.5 -37.6-77.1 ms median min-max in the group treated with o-lipoic acid and decreased by -0.1 -19.2-32.8 ms in the placebo group p 0.05 for u-lipoic acid vs. placebo. Power spectrum in the low-frequency band increased by 0.06 -0.09-0.62 bpm2 beats per minute squared in n-lipoic acid whereas it declined by -0.0 I -0.48-1.86 bpm in placebo p 0.05 for o-lipoic acid vs. placebo. Furthermore there was a trend toward a fa• vorable effect of cx-lipoicacid versus placebo for the CV and for the high• frequency band power spectrum. QTc interval was shortened insignificantly by -8.8 -87.9-46.5 ms in o-lipoic acid and remained unchanged in placebo 0.0 -118- 77.91. No differences between the groups were noted regarding the rates of adverse events. These findings suggest that oral treatment with cx• hpoic acid for 4 months using a well-tolerated dose of 800 mg/day in NIDDM patients is associated with a significant two indices or borderline two indi• ces improvement in HRV but not QTc interval as compared with placebo 32. Apart from the DEKAN study the effects of antioxidants on CAN have not been previously examined in a controlled clinical trial. However a recent study has demonstrated that glutathione a physiological antioxidant normal• izes reduced HRV during the squatting test baroreflex changes induced by acute hyperglycemia in healthy nondiabetic subjects 34. Presumably such an effect is mediated by a reduction in free radical activity. In the isolated perfused diabetic rat heart treatment with vitamin E completely prevented a progressive loss of histofluorescent nerve fibers in the myocardium and intra• neural catecholamines 35. There are no studies using o-lipoic acid in experi• mental diabetic cardiac neuropathy but the compound normalizes reduced glucose uptake and glucose utilization and consequently oxygen uptake myo• cardial ATP levels and cardiac output in the isolated diabetic rat heart model

slide 221:

180 Ziegler 36. However it is not known whether and to which degree these experimen• tal findings relate to the chronic process that characterizes human diabetic autonomic neuropathy. C. ALADIN 3 Study This was a randomized double-blind placebo-controlled multicenter trial in• cluding type 2 diabetic outpatients with symptomatic distal symmetric poly• neuropathy. Patients were allocated to three parallel groups receiving n-Iipoic acid or placebo: group l 11 167 o-lipoic acid 600 mg intravenously for 3 weeks followed by ce-lipoic acid 600 mg three times a day orally for 6 months group 2 11 174 o-lipoic acid 600 mg intravenously for 3 weeks followed by placebo orally for 6 months group 3 n 168 placebo intrave• nously for 3 weeks followed by placebo orally for 6 months. At baseline there were no significant differences between the groups regarding the demographic variables such as age sex body mass index duration of diabetes and HbA1c. After 3 weeks the Neuropathy Impairment Score NIS decreased signifi• cantly from baseline by -4.34 :::: 0.35 :::: SEM in the patients treated with a-lipoic acid and by -3.49 :::: 0.58 points in the placebo group p 0.016 for u-Iipoic acid vs. placebo. The NIS of the lower limbs NISLL declined by -3.32 :::: 0.26 on a-lipoic acid and -2.79 :::: 0.42 points on placebo p 0.055. After completion of the 6-month oral phase the reduction in the NIS was -5.82 :::: 0.73 in group l -5.76 :::: 0.69 in group 2 and -4.37 :::: 0.83 points in group 3 p 0.095 for group I vs. 3 whereas the reduction in NISLL was -4.39 :::: 0.51 in group l -4.20 :::: 0.52 in group 2 and -3.37 :::: 0.54 points in group 3 p 0.086 for group l vs. 3. Regarding the TSS no significant differences were noted between the groups at baseline and after 6 months. These results indicate a trend toward an improvement of neuropathic deficits but not symptoms after 6 months of oral treatment with n-Iipoic acid 600 mg three times a day after a 3-week intravenous phase in type 2 diabetic patients with polyneuropathy. The percentages of adverse events were compa• rable in the three groups throughout the study. 111. FUTURE ASPECTS Dyck et al. 37 recently calculated the sample size needed in a clinical trial based on the 2-year follow-up of the Rochester Diabetic Neuropathy Study. The estimates were derived from the changes of a composite score including NIS of the lower limbs plus seven tests NISLL+7: VPT great toe R-R

slide 222:

a-Llpoic Acid in Diabetic Neuropathy 181 variation to DB peroneal CMAP MNCV and MNDL tibial MNDL and sural SNAP that among other measures performed best at showing monotone worsening over time. Assuming that a treatment effect of two NIS points is clinically meaningful a 2-year study would need 68 patients in each treatment arm to have a power of 0.90 at the two-sided 0.05 level. If the effect of treat• ment is to halt the progression of neuropathy without improving it a study of 3.7 years would be required. A 4-year study would require 45 patients per arm to achieve power of 0.90 to detect a treatment effect that inhibits progres• sion of neuropathy and a clinically meaningful effect could be expected after approximately 2.4 years. Thus a conservative estimate would yield 70-100 patients per arm for a period of at least 3 years to achieve a high probability of detecting a clinically meaningful effect 37. A 4-year RCT evaluating the effects of o-lipoic acid in diabetic polyneuropathy Neurological Assessment of Thioctic Acid in Diabetic Neuropathy l Study has been designed on the basis of these estimates. The design of this trial is summarized in Table 2. In conclusion it is conceivable that the initial diabetes-related changes in the nerve are mediated by oxidative stress that on a long-term basis could result in progressive neuronal damage and therefore would be of pathogenetic relevance. Studies in proof of the promising results reported herein are needed. An ongoing pivotal long-term trial of oral treatment with c-lipoic acid de• signed along the recent guidelines of the Peripheral Nerve Society 38 aimed at slowing the progression of clinical neuropathy using a reliable clinical end Table 2 Outline of the NATHAN 1 Neurological Assessment of Thioctic Acid in Diabetic Neuropathy Study Design: Randomized double-blind placebo-controlled multicenter trial Subjects: Two parallel groups of type I or type 2 diabetic patients 11 500 enrolled Medication: Thioctic acid 600 mg or placebo tablets once daily orally Duration: Screening 2 wk placebo run-in 6 wk treatment 192 wk follow-up 4 wk interim analysis at 96 wk Inclusion criteria: Stage I or 2a polyneuropathy NJSLL + 7 : 97.5 centile TSS s: 5 Primary outcome measure: NISLL + 7 tests score VDT HBDB: peroneal CMAP MNCV and MNDL tibial MNDL sural SNAP Secondary outcome measures: NSC TSS CDT HP other NIS and NC VDT: vibration detection threshold HBDB: heart beat to deep breathing CMAP: compound muscle action potential MNCV: motor nerve conduction velocity MNDL: motor nerve distal latency SNAP: sensory nerve action potential NSC: neuropathy symptoms and changes CDT: coding detection threshold HP: heat as pain NC: nerve conduction.

slide 223:

182 Ziegler point addresses the question as to whether the observed improvement in neuro• pathic symptoms and autonomic dysfunction can be translated into long-term effects on objective neurophysiological parameters and neuropathic deficits. ACKNOWLEDGMENTS The ALADIN and DEKAN studies were supported by ASTA Medica AG Frankfurt am Main Germany. REFERENCES I. The Diabetes Control and Complications Trial Research Group. The effect of intensive diabetes therapy on the development and progression of neuropathy. Ann Intern Med 1995 122:561-568. 2. Diabetes Control and Complications Trial DCCT Research Group. Effect of intensive diabetes treatment on nerve conduction in the Diabetes Control and Complications Trial. Ann Neural 1995 38:869-880. 3. Kennedy WR Navarro X Goetz FC Sutherland DER. Najarian JS. Effects of pancreatic transplantation on diabetic neuropathy. N Engl J Med 1990 322: 1031-1037. 4. Ziegler D. Diagnosis and management of diabetic peripheral neuropathy. Dia• betic Med 1996 13:S34-S38. 5. Ziegler D. Diabetic cardiovascular autonomic neuropathy: prognosis. diagnosis and treatment. Diabetes Metab Rev 1994 10:339-383. 6. Ziegler D Piolot R Gries FA. The natural history of diabetic neuropathy is gov• erned by the degree of glycaemic control. A l O-year prospective study in IDDM. Diabetologia 1996 39suppl l:A35. 7. Jenner P. Oxidative damage in neurodegenerative disease. Lancet 1994: 344: 796-798. 8. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991 40:405-412. 9. Cameron NE. Cotter MA. The relationship of vascular changes to metabolic fac• tors in diabetes mellitus and their role in the development of peripheral nerve complications. Diabetes Metab Rev 1994 I 0: 189-224. 10. Collier A Rumley A Rumley AG Paterson JR Leach JP Lowe GOO Small M. Free radical activity and hemostatic factors in NIDDM patients with and without microalbuminuria. Diabetes 1992: 41 :909-913. 11. Ting HH Timimi FK. Boles KS Creager SJ Ganz P Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with non-insulin• dependent diabetes mellitus. J Clin Invest 1996 97:22-28.

slide 224:

o-Lipolc Acid in Diabetic Neuropathy 183 12. Yoshida K Hirokawa J Tagami S Kawakami Y Urata Y Kondo T. Weakened cellular scavenging activity against oxidative stress in diabetes mellitus: regula• tion of glutathione synthesis and efflux Diabetologia 1995 38:201-210. 13. Nourooz-Zadeh J Tajaddini-Sarmadi J McCarthy S Betteridge DJ Wolff SP. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: 1054- I 058. 14. Suzuki YJ Tsuchiya M Packer L. Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species. Free Rad Res Commun 1991 15:255-263. 15. Nagamatsu M. Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care 1995 18: 1160-1167. 16. Nickander KK McPhee BR Low PA Tritschler H. Alpha-lipoic acid: antioxi• dant potency against lipid peroxidation of neural tissues in vitro and implications for diabetic neuropathy. Free Rad Biol Med 1996 21:631-639. 17. Kihara M Low PA. Impaired vasoreactivity to nitric oxide in experimental dia• betic neuropathy. Exp Neurol 1994 132: 180-185. 18. Garrett NE Malcangio M. Dewhurst M Tomlinson DR. a-Lipoic acid corrects neuropeptide deficits in diabetic rats via induction of trophic support. Neurosci Lett 1997 222:191-194. 19. Cameron NE Cotter MA Horrobin DH Tritschler HJ. Effects of a-lipoic acid on neurovascular function in diabetic rats: interaction with essential fatty acids. Diabetologia 1998 41 :390-399. 20. Hounsom L Horrobin DF Tritschler H Corder R Tomlinson DR. A lipoic acid• gamma linolenic acid conjugate is effective against multiple indices of experi• mental diabetic neuropathy. Diabetologia 1998 41 :839-843. 21. Pringle M Churchill R. Randomized controlled trials in general practice. Br Med J 1995 311:1382-1383. 22. Cavaliere D Scorpiglione N Belfiglio M Carinci F Cubasso D Labbrozzi D Mari E. Massi Benedetti M Pontano C Tognoni G Nicolucci A. Quality assess• ment of randomised clinical trials on medical treatment of diabetic neuropathy. Diab Nutr Metab 1994 7:287-294. 23. Freiman JA Chalmers TC Smith H Kuebler RR. The importance of beta the type II error and sample size in the design and interpretation of the randomized controlled trial: survey of two sets of "negative" trials. In: Bailar JC Mosteller F eds. Medical Uses of Statistics. Boston: NEJM Books 1992:357-373. 24. Altman DG Bland JM. Absence of evidence is not evidence of absence. Br Med J 1995: 311:485. 25. Ziegler D. The design of clinical trials for treatment of diabetic neuropathy. Neurosci Res Commun 1997 21:83-91. 26. Ziegler D Mayer P Miihlen H Gries FA. Effekte einer Therapie mit u-Lipon• saure gegeniiber Vitamin 81 bei der diabetischen Neuropathie. Diab Stoffw 1993 2:443-448.

slide 225:

184 Ziegler 27. Ziegler D Hanefeld M Ruhnau KJ MeiBner HP Lobisch M Schiitte K Gries FA The ALADIN Study Group. Treatment of symptomatic diabetic peripheral neuropathy with the antioxidant n-lipoic acid. A 3-week randomized controlled trial ALADIN Study. Diabetologia 1995 38:1425-1433. 28. Young RJ Ewing DJ Clarke BF. A controlled trial of sorbinil an aldose reduc• tase inhibitor in chronic painful diabetic neuropathy. Diabetes 1983 32:938- 942. 29. Judzewitsch RG Jaspan JB Polansky KS Weinberg CR Halter JB Halar E Pfeifer MA Vukadinovic C Bernstein L Schneider M Liang K-Y Gabbay KH Rubenstein AH Porte D. Aldose reductase inhibition improves nerve conduction velocity in diabetic patients. N Engl J Med 1983 308: 119-125. 30. Young RR Shahani BT. Nerve conduction velocity in diabetes. N Engl J Med 1983 308: 190-191. 31. Santiago JV Sonksen PH Boulton AJM Macleod A Beg M Bochenek W Graepel GJ Gonen B The Tolrestat Study Group. Withdrawal of the aldose reductase inhibitor tolrestat in patients with diabetic neuropathy: effect on nerve function. J Diab Comp 1993 7:170-178. 32. Ziegler D Conrad F Ulrich H Reichel G Schatz H Gries FA the DEKAN Study Group. Effects of treatment with the antioxidant n-lipoic acid on cardiac autonomic neuropathy in NIDDM patients. A 4-month randomized controlled multicenter trial DEKAN Study. Diabetes Care 1997 20:369-373. 33. Ziegler D Laux G Dannehl K Spiiler M Miihlen H Mayer P Gries FA. Assess• ment of cardiovascular autonomic function: age-related normal ranges and repro• ducibility of spectral analysis vector analysis and standard tests of heart rate variation and blood pressure responses. Diabetic Med 1992 9: 166-175. 34. Marfella R Verrazzo G Acampora R La Marca C Giunta R Lucarelli C Pao• lisso G Ceriello A Giugliano D. Glutathione reverses systemic hemodynamic changes induced by acute hyperglycemia in healthy subjects. Am J Physiol 1995 268:EI 167-EI 173. 35. Rosen P Ballhausen T Bloch W Addicks K. Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995 38:1157-1168. 36. Strodter D Lehmann E Lehmann U Tritschler H-J Bretzel RG Federlin K. The influence of thioctic acid on metabolism and function of the diabetic heart. Diabetes Res Clin Pract 1995 29: 19-26. 37. Dyck PJ Davies JL Litchy WJ OBrien PC. Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort. Neurology 1997 49:229-239. 38. Peripheral Nerve Society. Diabetic polyneuropathy in controlled clinical trials: consensus report of the Peripheral Nerve Society. Ann Neurol 1995 38:478- 482.

slide 226:

13 Oxidative Stress NF-KB Activation and Late Diabetic Complications Click Here For Best Diabetes Treatment Peter P. Nawroth Valentin Borcea Angelika Bierhaus Martina Joswig and Stephan Schiekofer University of Heidelberg Heidelberg Germany Hans J. Tritschler ASTA Medica AWO GmbH Frankfurt Germany Oxidative stress is widely believed to play a central role in the pathogenesis of late diabetic complications. Recently the understanding of oxidative stress in diabetes has been improved by the availability of assays exactly determining defined products of reactive oxygen species. These studies have revealed oxi• dative stress to occur before diabetic complications are present further sup• porting the concept that oxidative stress is pivotal for the development of diabetic complications. Studies looking at the oxidative stress-activated tran• scription factor NF-KB help to understand the cellular consequences of oxida• tive stress at the molecular level. However the occurrence of oxidative stress and oxygen species-mediated secondary end products are not sufficient proof for the hypothesis of oxidative stress-dependent diabetic complications. It has to be demonstrated that antioxidant therapy does not only reduce plasmatic markers of oxidative stress and subsequent NF-KB activation but also late diabetic complications.

slide 227:

185

slide 228:

186 Nawrothet al. I. OXIDATIVE STRESS IN PATIENTS WITH DIABETES MELLITUS Several studies have shown that increased production of reactive oxygen spe• cies and antioxidant depletion occurs in patients with diabetes mellitus 1-11 . Oxidative stress may lead to endothelial cell damage and vascular dysfunction through various mechanisms 12-32. Lately considerable effort has been devoted to gain insights into the role of oxidative stress in the development and progression of late micro• and macrovascular complications in diabetes 1723272831-34. Although hyperglycemia is an acknowledged pathogenic factor in diabetic complica• tions it is not known through which mechanism an excess of glucose results in tissue damage. Accumulating data support the hypothesis that oxidative stress might play an important role in the pathogenesis of late diabetic compli• cations. Several pathways are leading to oxidative stress associated with acute or chronic hyperglycemia such as the polyol pathway prostanoid synthesis glucose autoxidation and protein glycation by increasing the production of free radicals 35-39. A close relationship of oxidative stress with glycemic control has been described showing a significant positive correlation between malondialdehyde MDA and both fasting blood sugar and glycosilated hemo• globin 40. Moreover many injurious effects of hyperglycemia on endothelial functions such as delayed cell replication impaired endothelial cell-dependent relaxation and the activation of NF-KB are reversed by antioxidants 3841. Several studies indicate that not only increased production of free radi• cals but also the depletion of antioxidative capacities may play an important role in the pathogenesis of late diabetic complications 4243. In this regard a prospective study has described an association between low lipid-standard• ized u-tocopherol levels and the incidence of type 2 diabetes suggesting a link between hyperglycemia-induced depletion of antioxidants and the pathol• ogy of diabetes 44. Recently it has been shown that oxidative stress appears to be primarily related to the underlying metabolic disorder occurring before manifestation of late diabetic complications consistent with the idea that oxi• dative stress is an early event in the pathology of diabetes and its complications 45. The imbalance between lipid peroxidation products and antioxidant ca• pacities in diabetic patients has been demonstrated using a precise technique for measurement of plasma lipid hydroperoxides. A significantly higher ratio of hydroperoxides to cholesterol-standardized o-tocopherol has been found in diabetics compared with control subjects 45. Further it has been described that endothelial dysfunction is associated with oxidant injury and tubular dam-

slide 229:

Late Diabetic Complications 187 age and may precede microalbuminuria in development of diabetic nephropa• thy 46. Free radicals produced by the system myeloperoxidase/hydrogen peroxide/halogen derivatives activate proteinases which break down collagen and other components of the extracellular matrix present in the basal mem• brane of the glorneruli and in the mesangium. It has been shown that hydroxyl radicals may depolarize glomerular heparan sulfate in vitro and in experimen• tal nephrotic syndrome leading to loss of glomerular basement membrane integrity and albuminuria 47. Thus oxygen radicals and proteinases can cause and amplify glomerular damage. II. HYPOTHESIS OF ADVANCED GLYCATION END PRODUCTS AND ITS RECEPTOR A. Advanced Glycation End Products Advanced glycation end products AGEs are a heterogeneous group of irre• versible adducts resulting from nonenzymatic glycation and oxidation of pro• teins lipids and nucleic acids. Glucose and other reducing sugars react in a nonenzymatic reaction Maillard reaction with the N-terminal residues and/ or e-amino groups of proteins initially forming a Schiff base. Rearrangement of this aldimine leads after a short time to the formation of more stable but still reversible Amadori adducts. The open chain of the resulting ketoamin can react with other amino groups. Oxidation dehydration and condensation reactions finally lead to the production of irreversible crosslinks which are proteinase resistant. The formation of AGEs in vitro and in vivo depends on the turnover rate of the modified substrate sugar concentration and time. Recent studies have shown that AGEs can be formed not only at long-living proteins but occur also on short-living proteins 48 peptides 48 lipids 49 and nucleic acids 50-52. AGE formation and protein crosslinking alter the structural and func• tional properties of proteins lipid components and nucleic acids. AGEs have also been shown to induce cellular signaling activation of transcription fac• tors and consequently gene expression in vitro and in vivo 32. They have been suggested to represent general markers of oxidative stress and long-term damage to proteins and to induce pathogenic changes in endothelial cells. Thus AGEs are not only markers but also mediators of chronic vasuclar dis• eases and late diabetic complications.

slide 230:

188 Nawroth et al. B. Formation of AGEs in Diabetes AGE formation proceeds slowly under normal glycemic conditions but is en• hanced in the presence of hyperglycemia oxidative stress and/or conditions in which protein and lipid turnover are prolonged. For example N-epsilon• carboxymethyllysine CML one of the various AGE structures postulated to date has been found to be a product of both glycoxidation combined non• enzymatic glycation and oxidation and lipid peroxidation reactions 53. CML and pentosidine have been shown to accumulate in diabetic kidneys in colocal• ization with a marker of lipid peroxidation MDA suggesting an association of local oxidative stress with the etiology of diabetic glomerular lesions 54. Evidence for an age-dependent increase in CML accumulation in distinct lo• calizations and acceleration of this process in diabetes has been provided by immunolocalization of CML in skin lung heart kidney intestine interverte• bral discs and particularly in arteries 55. In diabetic kidneys AGEs were preferentially localized in vascular lesions 56 renal cortex 57 expanded mesangial areas 58 and the glomerular basement membrane 56-59. An increased CML content in serum proteins of diabetic patients 55 and a corre• lation of serum AGE levels with the progressive loss of kidney function was found 60. The increased formation of tissue AGEs has been described to precede and to correlate with early manifestations of renal and retinal compli• cations in patients with diabetes 61 . Increased levels of AGE-modified low-density lipoprotein LDL with a markedly impaired clearance have been found in the plasma of diabetic patients suggesting a pathway for pathogenic modification of LDL 62. The mediating role of AGEs in development of late diabetic complications Table I 4963-70 has been studied in animal models by short- and long-term administration of AGEs. Short-term administration of AGEs led to increased vascular permeability and leakage impaired endothelial relaxation subendo• thelial mononuclear recruitment activation of NF-KB and subsequent VCAM-1 gene expression 71-73. Long-term administration of AGEs re• sulted in arteriolar basement thickening and complex vascular dysfunction 74 and in glomerular basement thickening mesangial expansion glomerulo• sclerosis and proteinuria 68. C. AGE-RAGE Interactions The principal means through which AGEs exert their cellular effects is via specific cellular receptors Table 2. One of them the receptor for AGE RAGE a 35-kDa protein is also expressed by endothelial cells 237576.

slide 231:

Late Diabetic Complications 189 Table 1 The Role of AGEs in Diabetic Complications Formation of HbA" as a marker of production of AGEs: poor glycemic control increases formation of AGEs and AGE-dependent cell activation 374163 Toxic effects of AGEs on retinal endothelial cells 64 and positive correlation between accumulation of AGEs expression of vascular endothelial growth factor and nonproliferative and proliferative diabetic retinopathy 65 Inhibition of development of experimental diabetic retinopathy by aminoguanidine treatment 49. Excessive deposition of intra- and extracellular AGEs in human diabetic peripheral nerve 66 Inhibition of AGE formation prevents diabetic peripheral nerve dysfunction 67 Accumulation of AGEs in the kidney of diabetic patients 5660 Injection of AGE-albumin in normal rats induces symptoms of diabetic nephropathy 68 Blocking of AGE binding to RAGE reduces albuminuria 69 Inhibitor AGEs reduces urinary ablumin excretion mesangial expansion and glomerular basement membrane thickening 70 Table 2 AGE Binding Proteins and Their Localization AGE binding proteins Localization AGE-R1 OST-48 AGE-R1 80KH AGE-R Galectin-3 or GBP-35 RAGE Lactoferrin lysozyme Fructosylline-specific binding protein Macrophage scavenger receptor Monocytes/rnacrophagesendothelial cells T lymphocytes mesangial cells neurons Monocytes/macrophages. endothelial cells T lymphocytes fibroblasts mesangial cells neurons Monocytes/rnacrophages endothelial cells T lymphocytes Endothelial cells monocytes/ macrophages smooth muscle cells mesangial cells neu• rons T lymphocytes erythrocytes Endothelial cells Monocytes Macrophages

slide 232:

190 Nawroth et al. An induction of endothelial RAGE expression has been shown on vessels from patients with arteriosclerosis diabetes uremia and vasculitis 77- 79. Binding of AGEs to their cellular binding sites results in generation of oxygen free radicals and depletion of antioxidants such as glutathione and ascorbate 3280. The consequently enhanced cellular oxidative stress leads to acti• vation of the redox-sensitive transcription factor NF-KB in endothelial cells smooth muscle cells mesangial cells and monocytes/macrophages 23253278-81 . Ill. ACTIVATION OF NF-KB The multiprotein complex NF-KB resides as an inactive form in the cytoplasm associated with its inhibitor IKB Table 3. NF-KB translocates to the nucleus after phosphorylation and proteolitic degradation of IKB. NF-KB activation is modulated by redox reactions that increase the cytosolic phosphorylation and degradation of IKB and requires a thioredoxin-dependent status in the nucleus 82-85. NF-KB-dependent genes and their products include lKBa RAGE cytokines tumor necrosis factor-a interleukin-6 and -8 adhesion molecules VCAM-1 ICAM- 1 ELAM receptors for coagulation factors such as the procoagulant tissue factor endothelin- 1 inducible nitric oxide synthase induc• ible cyclooxygenase heme oxygenase type 1 and 5-lipoxygenase 237786. Because transcription of IKBa is autoregulated by NF-KB 87 activation of NF-KB terminates itself 8688 leading to a short-living acute cellular re• sponse. Recent studies showed that IKB mediates a more sustained activation of NF-KB that lasts up to 48 h 8990. Activation of NF-KB and induction of increased binding activity of NF• KB are believed to have a pivotal role in the pathogenesis and progression of chronic diseases such as diabetes and atherosclerosis 39869192. Accumu- Table 3 Proteins of the NF-KB and IKB Families Proteins of the NF-KB family Proteins of the IKB family P50 p50 P52/p49 p I 00 P65 relA c-rel relB IKBa IKB IKNy IKBE IKB-R

slide 233:

Late Diabetic Complications 191 lating data indicate a close link between hyperglycemia oxidative stress for• mation of AGEs and induction of NF-KB to the etiology of late diabetic com• plications. Increased glucose concentration has been shown to induce NF-KB activation in endothelial cells 38 and to increase NF-KB binding activity in peripheral blood mononuclear cells isolated from diabetic patients with poor glycemic control 37 suggesting that NF-KB activation is an early event in response to elevations in glucose contributing to diabetes-induced endothelial cell injury. AGEs interacting with endothelial cell RAGE have been identified as relevant mediators of NF-KB activation by generating intracellular oxidative stress 39417680. Recently it has been shown that the binding of AG Es or amyloid-f peptides to RAGE leads to perpetuated NF-KB activation in vitro and in vivo resulting in a I-week translocation of NF-KB p50/p65 from the cytoplasm into the nucleus 39. The AGE-RAGE-mediated NF-KB activation was initiated by the degradation of both lKBcx. and IKB. The key event in maintaining the activation of NF-KB is the induction of de novo synthesis of p65-mRNA leading to a constantly growing pool of free NF-KBp65. Thus AGEs a.re capable of activating NF-KB in vitro and in vivo pointing to a central role of AGE-mediated NF-KB activation in late diabetic complications. IV. INHIBITION OF DIABETIC COMPLICATIONS BY ANTIOXIDANT TREATMENT In patients with diabetes mellitus oxidative stress is increased by enhanced production of free radicals and by antioxidant depletion resulting in an in• creased susceptibility to oxidative damage and possibly development of late diabetic complications. Endogenous antioxidant proteins such as superoxide dismutase glutathione peroxidase and metal-binding proteins may protect the body against the effect of prooxidant reactions. Multiple antioxidants includ• ing cx.-lipoic acid vitamins C and E urate carotenoids flavonoids the amino acid methionine and protein-bound zinc and selenium are interacting addi• tively in these biological systems. In vitro and in vivo studies using antioxi• dants support the concept of radical-mediated diabetic complications. A. Vitamin E Vitamin E is the most abundant antioxidant in LDL. In vitro it scavenges peroxyl radicals 10000-fold avidly and then these react with fatty acids. But

slide 234:

192 Nawroth et al. in LDL vitamin E is located in the more rigid outer layer of the particle whereas the free radicals seem to accumulate in the more fluid core and might therefore not be scavenged effectively by the antioxidant. However vitamin E has been shown to inhibit lipid peroxidation and to reduce protein glycation in diabetic patients 93-95. Intervention studies have shown that vitamin E treatment could prevent early changes of diabetic glomerular dysfunction in diabetic rats through acti• vation of diacylglyceral kinase decreasing diacylglycerol and protein kinase C levels 9697. Further vitamin E has been demonstrated to improve sig• nificantly diabetes-induced abnormal contractility and endothelial dysfunction 98 and to exhibit cardiovascular protection in diabetes 99. Thus these stud• ies support the concept of oxidative stress-mediated diabetic complications. B. Vitamin C Vitamin C is the most effective water-soluble antioxidant in the organism. It acts as a potent electron donor which is then reduced back to ascorbic acid primarily by glutathione. Ascorbate is the first antioxidant consumed in plasma exposed in vitro to aqueous peroxyl radicals followed by sulfhydryl groups urate and vitamin E. Vitamin C supplementation has been shown to regenerate vitamin E and to increase glutathione levels 94. Treatment with vitamin C improved endothelium-dependent vasodilata• tion in patients with insulin-dependent diabetes mellitus 100 and decreased albuminuria glomerular transforming growth factor-B and glomerular size in diabetic rats 101 . These data are in support for a role of water-soluble antiox• idants in diabetic complications. C. o-Lipolc Acid o-Lipoic acid occurs naturally in physiological systems as a cofactor for enzy• matically catalyzed acyl transfer reactions. It has powerful antioxidant actions in vitro and in vivo 102103. n-Lipoic acid exists in oxidized and reduced forms and regenerates NAO+ from NADH 104-106. It acts as a universal antioxidant both in the membrane and the aqueous phase by reducing peroxyl ascorbyl and chromanoxyl radicals 107 and by decreasing microsomal lipid peroxidation 108. n-Lipoic acid participates in establishing a cellular antiox• idant network raising intracellular glutathione levels reducing the oxidized amino acid cysteine to cystine and regenerating other important antioxidants such as the vitamins C and E. On the cellular level n-lipoic acid has also been shown to prevent single oxygen-induced DNA damage 104109. Also

slide 235:

Late Diabetic Complications 193 of interest and related to diabetic complications are the findings regarding the effects of n-lipoic acid with respect to the glucose homeostasis and the production of AGEs 110. a-Lipoic acid has been found to reduce glycemia and to stimulate glucose uptake and transport activity in skeletal muscle both human and experimental diabetes 111 112. One of the earliest events in atherogenesis is the adhesion of monocytes to the endothelium and its migration into the arterial intima. Endothelin-f which is increased in diabetes and is believed to be relevant for the progression of nephropathy 113 has been shown to increase monocyte chemotaxis in a dose-dependent manner 97. n-Lipoic acid inhibits migration I 14. rz-Lipoic acid has also been shown to be an effective inhibitor of aldose reductase 115. Aldose reductase inhibitors have been suggested to prevent or reduce the dif• ferent components of vascular dysfunction cataract neuropathy and nephrop• athy in animal models of diabetes. Several intervention studies have been performed to establish the role of n-lipoic acid as a powerful antioxidant in diabetes. Therapeutic effects of rx-lipoic acid in the prevention of diabetic retinopathy and cataract have been described 116. The neuroprotective effect of ce-Ilpoic acid in the treatment of symptomatic diabetic peripheral neuropathy by reducing oxidative stress and improving nerve blood flow and distal nerve conduction is well docu• mented 117-119. The increased blood flow is consistent with the vascular protective effect of o-lipoic acid. Recently it has been reported that n-lipoic acid completely prevented the AGE-dependent depletion of glutathione and ascorbate in vitro and re• duced in a time- and dose-dependent manner the AGE albumin-mediated acti• vation of NF-KB in endothelial cells as long as c-Iipoic acid was added at least 30 min before AGE albumin stimulation Fig. l 41. It was shown that the inhibition of NF-KB activation was not due to physical interactions with protein DNA binding because o-lipoic acid did not prevent binding activity of recombinant NF-KB when it was included directly into the binding reaction. Furthermore it was demonstrated by Western blots that n-Iipoic acid inhibited the release and translocation of NF-KB from the cytoplasm into the nucleus. In addition rx-lipoic acid reduced the NF-lB-mediated transcription of tissue factor and endothelin-I both of them being relevant for endothelial cell dys• function in diabetes 41. Ongoing studies demonstrated that n-lipoic acid• dependent downregulation of NF-KB is also evident in monocytes of diabetic patients under o-lipoic acid therapy Fig. 1 3741. Thus n-lipoic acid re• duces oxidative stress-dependent NF-KB activation in vitro and in vivo. It is of interest that these effects are present even in patients with poor glycemic control.

slide 236:

- cellalnd-- • AGMllumln 194 a•lf Polc acid reduces NF-xB DNA-bindingectl vlty In vitro In vivo Nawroth et al. Figure 1 Inhibition of NF-KB DNA binding activity by n-Iipoic acid in vitro left and in vivo right. The effects of cc-lipoic acid on the development of late diabetic compli• cations have been studied with respect to the progressive endothelial cell dam• age and alburninuria in patients with diabetes mellitus 120. The progression of endothelial cell damage has been evaluated in a pilot study over 18 months that assessed the course of plasma thrombomodulin as a marker of endothelial

slide 237:

e E Late Diabetic Complicatio ns 195 120 - 110 .5 ::s "C Control group alpha Lipoic acid group 0 100 J--_- .. .. .... E 0 .c L----- 90 I- ------ I 0 6 12 18 Months Figure 2 Progression of thrombomodulin in 205 patients with diabetes mellitus studied over 18 months according to the use of n-lipoic acid expressed as relative values of study entry values. -- n 151 patients without n-lipoic acid treat• ment --- n 54 patients treated with 600 mg/day a-lipoic acid. injury. It has been shown that o-Iipoic acid significantly reduced the time• dependent increase of plasma thrombomodulin that was seen in the control patients Fig. 2. Treatment with o-lipoic acid was found to be the only factor significantly predicting a decrease of the urinary albumin concentration and a decrease of plasma thrombomodulin in multiple regression analysis 120. V. SUMMARY There is increasing evidence that oxidative stess plays a major role in the development of late diabetic complications. Oxidative stress generated by hy• perglycemia AGEs or other factors of cellular activation results in activation of NF-KB activation correlates in diabetic patients with glucose control and can be reduced by treatment with o-lipoic acid. The vasculoprotective action of o-lipoic acid supports the hypothesis shown in Figure 3.

slide 238:

196 Nawroth et al. Hyperglycemia Antioxidant Prostanold Polyol Glucose Protein AGE-RAGE Production of depletion synthesis pathway autoxldatlon glycatlon Interactions free radicals Oxidative stress NF-KB activation Nephropathy Retlnopathy Neuropathy Vasculopathy Figure 3 Possible linkage between hyperglycemia-induced oxidative stress subse• quent activation of NF-KB and development of secondary complications in patients with diabetes mellitus. REFERENCES I. Nourooz Zadeh J Tajaddini Sarmadi J. Mcflarthy S Betteridge DJ Wolff SP. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: 1054-1058. 2. Rabini RA Fumelli P Galassi R et al. Increased susceptibility to lipid oxida• tion of low-density lipoproteins and erythrocyte membranes from diabetic pa• tients. Metabolism 1994 43:1470-1474. 3. Ghiselli A Laurenti 0 De-Mattia G Maiani G. Ferro-Luzzi A. Salicylate hy• droxylation as an early marker of in vivo oxidative stress in diabetic patients. Free Radie Biol Med 1992 13:621-626. 4. Karpen CW Cataland S ODorisio TM Panganamala RV. Production of 12- hydroxyeicosatetraenoic acid and vitamin E status in platelets from type I hu• man diabetic subjects. Diabetes 1985 34:526-531. 5. Griesmacher A Kinderhauser M Andert S Schreiner W Toma C Knoebl P

slide 239:

Late Diabetic Complications 197 Pietschmann P Prager R. Enhanced serum levels of TBARS in diabetes melli• tus. Am J Med 1995 98:469-475. 6. Srivastava S Joshi CS Sethi PP Agrawal AK Srivastava SK Seth PK. Altered platelet functions in non-insulin-dependent diabetes mellitus NIDDM. Thromb Res 1994 76:451-461. 7. Gopaul NK Anggard EE Mallet Al Betteridge DJ Wolff SP Nourooz-Zadeh J. Plasma 8-epi-PGF2 alpha levels are elevated in individuals with non-insulin• dependent diabetes mellitus. FEBS Lett 1995 368:225-229. 8. Asayama K Uchida N Nakane T et al. Antioxidants in the serum of children with insulin-dependent diabetes mellitus. Free Radie Biol Med 1993 15:597- 602. 9. De-Mattia G Laurenti 0 Bravi C Ghiselli A Iuliano L Balsano F. Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of dia• betic patients. Metabolism 1994 43:965-968. 10. Sinclair AJ Taylor PB Lunec J Girling AJ Barnett AH. Low plasma ascorbate levels in patients with type 2 diabetes mellitus consuming adequate dietary vita• min C. Diabet Med 1994 11 :893-898. 11. Nourooz-Zadeh J Halliwell B Tritschler H Betteridge DJ. Decreased lipid standardised cc-tocopherol in non-insulin dependent diabetes mellitus. Diab Stoffw 1997 6suppl 2:20-23. 12. Zhao B Zhang Y Liu B Nawroth P Dierichs R. Endothelial cells injured by oxidized low density lipoprotein. Am J Hematol 1995 49:250-252. 13. Zhang W Khanna P Chan LL Campbell G Ansari NH. Diabetes-induced apoptosis in rat kidney. Biochem Mo Med 1997 61:58-62. 14. Messent M Sinclair DG Quinlan GJ Mumby SE Gutteridge JM Evans TW. Pulmonary vascular permeability after cardiopulmonary bypass and its relation• ship to oxidative stress. Crit Care Med 1997 25:425-429. 15. Holman RG Maier RV. Oxidant-induced endothelial leak correlates with de• creased cellular energy levels. Am Rev Respir Dis 1990 141:134-140. 16. Rosen P Ballhausen T Bloch W Addicks K. Endothelial relaxation is dis• turbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995 38: 1157-1168. 17. Giugliano D Ceriello A Paolisso G. Diabetes mellitus hypertension and car• diovascular disease: which role for oxidative stress Metabolism 1995 44:363- 368. 18. Mertsch K Grune T Siems WG Ladhoff A Saupe N Blasig IE. Hypoxia and reoxygenation of brain endothelial cells in vitro: a comparison of biochemical and morphological response. Cell Mo Biol Noisy-le-grand 1995 41 :243- 253. 19. Ciolino HP Levine RL. Modification of proteins in endothelial cell death dur• ing oxidative stress. Free Radie Biol Med 1997 22:1277-1282. 20. Halliwell B. Free radicals proteins and DNA: oxidative damage versus redox regulation. Biochem Soc Trans 1996 24: 1023-1027. 21. Pieper GM Siebeneich W Roza AM Jordan M Adams MB. Chronic treatment

slide 240:

198 Nawrothet al. in vivo with dimethylthiourea a hydroxyl radical scavenger prevents diabetes• induced endothelial dysfunction. J Cardiovasc Pharmacol 1996 28:741- 745. 22. Shi MM Godleski JJ Paulauskis JD. Regulation of macrophage inflammatory protein- I alpha mRNA by oxidative stress. J Biol Chem 1996 271 :5878-5883. 23. Hori 0 Yan SD Ogawa S et al. The receptor for advanced glycation end• products has a central role in mediating the effects of advanced glycation end• products on the development of vascular disease in diabetes mellitus. Nephrol Dial Transplant 1996 I I suppl 5: 13- I 6. 24. Lewis MS Whatley RE. Cain P Mcintyre TM Prescott SM Zimmerman GA. Hydrogen peroxide stimulates the synthesis of platelet-activating factor by en• dothelium and induces endothelial cell-dependent neutrophil adhesion. J Clin Invest 1988 82:2045-2055. 25. Aghajanian AA Oguogho A Sinzinger H. lsoprostanes a new substance group in angiology-of future significance Vasa 1997 26:65-69. 26. Rattan V Shen Y Sultana C Kumar D Kalra VK. Diabetic RBC-induced oxidant stress leads to transendothelial migration of monocyte-like HL-60 cells. Am J Physiol 1997 2732 Pt l:E369-375. 27. Chappey 0 Dosquet C Wautier MP Wautier JL. Advanced glycation end products oxidant stress and vascular lesions. Eur J Clin Invest 1997 27:97- 108. 28. Tesfamariam B. Free radicals in diabetic endothelial cell dysfunction. Free Radie Biol Med I 994 16:383-391. 29. Ceriello A Giacomello R Stel G et al. Hyperglycemia-induced thrombin for• mation in diabetes. The possible role of oxidative stress. Diabetes 1995 44: 924-928. 30. Ceriello A Curcio F. dello-Russo P et al. The defence against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor I over-production. Blood Coagul Fibrinolysis 1995 6:133-137. 31. Brownlee M. Advanced glycation end products in diabetic complications. Curr Opin Endocrinol Diabetes 1996 3:291-297. 32. Schmidt AM Hori 0 Brett J Yan SD Wautier JL Stern D. Cellular receptors for advanced glycation end products. Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions. Arte• rioscleros Thrombus 1994 14:1521-1528. 33. Baynes JW Thorpe SR. The role of oxidative stress in diabetic complications. Curr Opin Endocrinol 1996 3:277-284. 34. Giugliano D Ceriello A Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996 19:257-267. 35. Ceriello A. Acute hyperglycaemia and oxidative stress generation. Diabet Med 1997 14suppl 3:S45-49. 36. Reddi AS Bollineni JS. Renal cortical expression of mRNAs for antioxidant enzymes in normal and diabetic rats. Biochem Biophys Res Commun 1997 235:598-601. 37. Hofmann AM Schiekofer S Kanitz M et al. Insufficient glycemic control in-

slide 241:

Late Diabetic Complications 199 creases NF-8 binding activity in peripheral blood mononuclear cells isolated from patients with diabetes mellitus. Diabetes Care in press. 38. Pieper GM Riaz-ul-Haq Activation of nuclear factor-kappaB in cultured endo• thelial cells by increased glucose concentration: prevention by calphostin C. J Cardiovasc Pharmacol 1997 30:528-532. 39. Bierhaus A Klevesath M Schwanninger M. et al. Perpetuated NF-KB activa• tion: A receptor mediated transcription dependent pathway. Exp Med in press. 40. Noberasco G Odetti P Boeri D Maiello M Adezati L. Malondialdehyde MDA level in diabetic subjects. Relationship with blood glucose and glyco• sylated hemoglobin. Biomed Pharmacother 1991 45:193-196. 41. Bierhaus A. Chevion S Chevion M et al. Advanced glycation end product• induced activation of NF-KBis suppressed by o-Iipoic acid in cultured endothe• lial cells. Diabetes 1997 46: 1481-1490. 42. Sundararn RK Bhaskar A Vijayalingam S Viswanattham M Mohan R. Shan• mugasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes with and without complications. Clin Sci Colch 1996 90:255-260. 43. Maxwell SR Thomason H Sandler D et al. Antioxidant status in patients with uncomplicated insulin-dependent and non-insulin-dependentdiabetes mellitus. Eur J Clin Invest 1997 27:484-490. 44. Salonen JT Nyyssonen K Tuomainen TP et al. Increased risk of non-insulin dependent diabetes mellitus at low plasma vitamin E concentrations: a four year follow up study in men. BMJ 1995 311: 1124-1127. 45. Nourooz-Zadeh J Rahimi A Tajaddini-Sarmadi J. et al. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabeto• logia 1997 40:647-653. 46. Yaqoob M. Patrick AW McClelland P et al. Relationship between markers of endothelial dysfunction oxidant injury and tubular damage in patients with insulin-dependent diabetes mellitus. Clin Sci Colch 1993 85:557-562. 47. Raats CJ Bakker MAH van den Born J Berden JHM. Hydroxyl radicals de• polimerize heparan sulfate in vitro and in experimental nephrotic syndrome. J Biol Chem 1997 42:26734-26741. 48. Vlassara H. Advanced glycation end-products and atherosclerosis. Ann Med 1996 28:419-426. 49. Hammes HP Martin S Federlin K Geisen K Brownlee M. Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc Natl Acad Sci USA 1991 88:11555-11558. 50. Bucala R Model P Cerami A. Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression. Proc Natl Acad Sci USA 1984 81:05-109. 51. Gugliucci A Bendayan M. Histones from diabetic rats contain increased levels of advanced glycation end products. Biochem Biophys Res Commun 1995 212:56-62. 52. Papoulis A Youssef A Bucala R. Identification of N2-l-carboxyethyl gua-

slide 242:

200 Nawroth et al. nine CEG as a guanine advanced glycosylation end product. Biochemistry 1995 34:684-655. 53. Fu MX Requena JR Jenkins AJ Lyons TJ Baynes JW Thorpe SR. The ad• vanced glycation end product N epsilon-carboxymethyllysine is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 1996 271: 9982-9986. 54. Horie K Miyata T Maeda K et al. Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomer• ular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy. J Clin Invest 1997 100:2995-3004. 55. Schleicher ED Wagner E Nerlich AG. Increased accumulation of the glycoxi• dation product Nepsilon-carboxymethyllysine in human tissues in diabetes and aging. J Clin Invest 1997 99:457-468. 56. Nishino T Horii Y Shiiki H et al. Immunohistochemical detection of advanced glycosylation end products within the vascular lesions and glomeruli in diabetic nephropathy. Hum Pathol 1995 26:308-313. 57. Mitsuhashi T Nakayama H Itoh T et al. Immunochemical detection of ad• vanced glycation end products in renal cortex from STZ-induced diabetic rat. Diabetes 1993 42:826-832. 58. Shikata K Makino H Sugimoto H et al. Localization of advanced glycation endproducts in the kidney of experimental diabetic rats. J Diabetes Complica• tions 1995 9:269-271. 59. Gugliucci A Bendayan M. Reaction of advanced glycation endproducts with renal tissue from normal and streptozotocin-induced diabetic rats: an ultrastruc• tural study using colloidal gold cytochemistry. J Histochem Cytochem 1995 43:591-600. 60. Makita Z Radoff S Rayfield EJ et al. Advanced glycosylation end products in patients with diabetic nephropathy N Engl J Med 1991 325:836-842. 61. Beisswenger PJ Makita Z Curphey TJ et al. Formation of immunochemical advanced glycosylation end products precedes and correlates with early mani• festations of renal and retinal disease in diabetes. Diabetes 1995 44:824- 829. 62. Bucala R Makita Z Vega G et al. Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc Natl Acad Sci USA 1994 91:9441-9445. 63. Makita Z Vlassara H Rayfield E. Hemoglobin-AGE: a circulating marker of advanced glycosylation. Science 1992 258:651-653. 64. Chibber R Molinatti PA Rosatto N Lambourne B Kohner EM. Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy. Diabetologia 1997 40: 156- 164. 65. Murata T Nagai R Ishibashi T Inomuta H Ikeda K Horiuchi S. The relation• ship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas. Diabetologia 1997 40:764-769.

slide 243:

Late Diabetic Complications 201 66. Sugimoto K Nishizawa Y Horiuchi S Yagihashi S. Localization in human diabetic peripheral nerve of Nepsilon-carboxymethyllysine-proteinadducts an advanced glycation endproduct. Diabetologia I 997 40: I 380- I 387. 67. Cameron NE Cotter MA Dines K Love A. Effects of aminoguanidine on peripheral nerve function and polyol pathway metabolites in streptozotocin• diabetic rats. Diabetologia 1992 35:946-950. 68. Vlassara H Striker LJ Teichberg S Fuh H Li YM Steffes M. Advanced glyca• tion end products induce glomerular sclerosis and albuminuria in normal rats. Proc Natl Acad Sci USA 1994 91: 11704-11708. 69. Wautier JL Zoukourian C Chappey 0 et al. Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glyca• tion end products blocks hyperpermeability in diabetic rats. J Clin Invest 1996 97:238-243. 70. Yamauchi A Takei I. Makita Z et al. Effects of aminoguanidine on serum advanced glycation endproducts urinary albumin excretion mesangial expan• sion and glomerular basement membrane thickening in Otsuka Long-Evans Tokushima fatty rats. Diabetes Res Clin Pract 1997 34: 127-133. 71. Esposito C Gerlach H Brett J Stern D Vlassara H. Endothelial receptor-medi• ated binding of glucose-modified albumin is associated with increased mono• layer permeability and modulation of cell surface coagulant properties. J Exp Med 1989 170: 1387-1407. 72. Schmidt AM Hori 0 Chen JX et al. Advanced glycation endproducts inter• acting with their endothelial receptor induce expression of vascular cell adhe• sion molecule- I VCAM-1 in cultured human endothelial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. J Clin In• vest 1995 96:1395-1403. 73. Vlassara H Fuh H Donnelly T Cybulsky M. Advanced glycation endproducts promote adhesion molecule VCAM-1 ICAM-1 expression and atheroma for• mation in normal rabbits. Mol Med 1995 I :447-456. 74. Vlassara H Fuh H Makita Z K.rungkrai S Cerami A Bucala R. Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications. Proc Natl Acad Sci USA 1992 89:12043-12047. 75. Yan SD Stern D Schmidt AM. Whats the RAGE The receptor for advanced glycation end products RAGE and the dark side of glucose. Eur J Clin Invest 1997 27:179-181. 76. Bierhaus A Hofmann MA Ziegler R Nawroth PP. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. Cardiovasc Res 1998 37:586-600. 77. Bierhaus A Illmer T Kasper M. Advanced glycation end product AGE-medi• ated induction of tissue factor in cultured endothelial cells is dependent on RAGE. Circulation 1997 96:2262-2271. 78. Bierhaus A Ritz E Nawroth PP. Expression of receptors for advanced glyca• tion end-products in occlusive vascular and renal disease. Nephrol Dial Trans• plant 1996 11 suppl 5:87-90.

slide 244:

202 Nawroth et al. 79. Abel M Ritthaler U Zhang Y. Expression of receptors for advanced glycosyl• ated end-products in renal disease. Nephrol Dial Transplant 1995 I 0: 1662- 1667. 80. Yan SD Schmidt AM Anderson GM. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem 1994 269:9889-9897. 81. Yan SD Yan SF Chen X et al. Non-enzymatically glycated tau in Alzheimers disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid beta-peptide. Nat Med 1995 I :693-699. 82. Flohe L Brigelius-Flohe R Saliou C Traber MG Packer L. Redox regulation of NF-kappa B activation. Free Radie Biol Med 1997 22: 1115-1126. 83. Jin DY Chae HZ Rhee SG Jeang KT. Regulatory role for a novel human thioredoxin peroxidase in NF-kappa B activation. J Biol Chem 1997 272: 30952-30961. 84. Piette J Piret B Bonizzi G. Multiple redox regulation in NF-kappa B transcrip• tion factor activation. Biol Chem 1997 378:1237-1245. 85. Alkalay I Yaron A Hatzubai A Orian A Ciechanover A Ben-Beriah Y. Stim• ulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B in• hibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 1995 92: 10599-10603. 86. Baeuerle PA Baltimore D. NF-KB: ten years after. Cell 1996 87:13-20. 87. de-Martin R Vanhove B Cheng Q Hofer E Csizmadia V Winkler H Bach FH. Cytokine-inducible expression in endothelial cells of an I kappa B alpha• like gene is regulated by NF kappa B. EMBO J 1993 12:2773-2779. 88. Read MA Whitley MZ Williams AJ Collins T. NF-kappa-B and 1-kappa-B• alpha: an inducible regulatory system in endothelial activation. J Exp Med 1994 179:503-512. 89. Thompson JE Phillips RJ Erdjument-Bromage H Tempst P Ghosh S. I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell 1995 80:573-582. 90. Johnson DR Douglas I Jahnke A Ghosh S Pober JS. A sustained reduction in IkappaB-beta may contribute to persistent NF-kappa B activation in human endothelial cells. J Biol Chem 1996 271: 16317-16322. 91. Collins T. Endothelial nuclear factor-kappa B and the initiation of the athero• sclerotic lesion. Lab Invest 1993 68:499-508. 92. Hofmann AM Schiekofer S Klevesath MS et al. Peripheral blood mononu• clear cells isolated from patients with diabetic nephropathy demonstrate in• creased activation of the oxidative-stress sensitive transcription factor NF-KB Diabetologia in press. 93. Jain SK Mc Vie R Jaramillo JJ et al. The effect of modest vitamin E supple• mentation on lipid peroxidation products and other cardiovascular risk factors in diabetic patients. Lipids 1996 31 suppl:S87-90. 94. Stall W Sies H. Antioxidant defense: vitamins E and C and carotenoids. Diabe• tes. 1997 46suppl 2:514-18.

slide 245:

Late Diabetic Complications 203 95. Ceriello A Giugliano D Quatraro A Donzella C Dipalo G Lefebvre PJ. Vita• min E reduction of protein glycosylation in diabetes. New prospect for preven• tion of diabetic complications Diabetes Care 1991 14:68- 72. 96. Ishii H Koya D King GL. Protein kinase C activation and its role in the devel• opment of vascular complications in diabetes mellitus. 1 Mo Med 1998 76: 21-31. 97. Koya D Lee IK Ishii H Kanoh H King GL. Prevention of glomerular dysfunc• tion in diabetic rats by treatment with n-alpha-tocopherol. 1 Am Soc Nephrol 1997 8:426-435. 98. Karasu C Ozansoy G Bozkurt 0 Erdogan D Omeroglu S. Antioxidant and triglyceride-lowering effects of vitamin E associated with the prevention of abnormalities in the reactivity and morphology of aorta from streptozotocin• diabetic rats. Antioxidants in Diabetes-Induced Complications ADIC study group. Metabolism 1997 46:872-879. 99. Gazis A Page S Cockcroft J. Vitamin E and cardiovascular protection in diabe• tes. BMJ 1997 314:1845-1846. I 00. Timi mi FK Ting HH Haley EA Roddy MA Ganz P Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with insulin-depen• dent diabetes mellitus. 1 Am Coll Cardiol 1998 31 :552-557. 101. Craven PA DeRubertis FR Kagan VE Melhem M Studer RK. Effects of supplementation with vitamin C or E on albuminuria glomerular TGF-beta and glornerular size in diabetes. 1 Am Soc Nephrol 1997 8:1405-1414. 102. Chevion S Hofman M Ziegler R Chevion M Nawroth PP. The antioxidant properties of thioctic acid: characterization by cyclic voltammetry. Biochem Mo Biol Int 1997 41:317-327. 103. Packer L Witt EH Tritschler HJ. Alpha-lipoic acid as a biological antioxidant. Free Radie Biol Med 1995 19:227-250. 104. Scott BC Aroma EI Evans PJ et al. Lipoic and dihydrolipoic acids as antioxi• dants. A critical evaluation. Free Radie Res 1994 20: 119-133. 105. Roy S Sen CK Trirschler HJ Packer L. Modulation of cellular reducing equiv• alent homeostasis by alpha-lipoic acid. Mechanisms and implications for diabe• tes and ischemic injury. Biochem Pharmacol 1997 53:393-399. 106. Suzuki YJ Tsuchiya M Packer L. Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species. Free Radie Res Commun 1991 15:225-236. 107. Kagan VE. Shvcdova A. Serbinova E et al. Dihydrolipoic acid-a universal antioxidant in the membrane and the aqueous phase. Reduction of peroxyl ascorbyl and chromanoxyl radicals. Biochem Pharmacol 1992 44:1637-1649. I 08. Scholich H Murphy ME Sies H. Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol. Bio• chim Biophys Acta 1989 100I :256-261. 109. Devasagayam TP Subramanian M Pradhan DS Sies H. Prevention of singlet oxygen-induced DNA damage by lipoate. Chem Biol Interact 1993 86: 79- 92.

slide 246:

204 Nawroth et al. 110. Suzuki YJ Tsuchiya M Packer L. Lipoate prevents glucose-induced protein modifications. Free Radie Res Commun 1992 17:211-217. 111. Khamaisi M Potashnik R Tirosh A et al. Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats. Metabolism 1997 46:763-768. 112. Estrada DE Ewart HS Tsakiridis T et al. Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioctic acid: participation of elements of the insulin signaling pathway. Diabetes 1996 45: 1798-1804. 113. Bruzzi I Remuzzi G Benigni A. Endothelin: a mediator of renal disease pro• gression. J Nephrol 1997 I 0: 179-183. 114. Achmad TH Rao GS. Chemotaxis of human blood monocytes toward endo• thelin-I and the influence of calcium channel blockers. Biochem Biophys Res Commun 1992 189:994-1000. 115. Ou P Nourooz-Zadeh J Tritschler HJ Wolff S. Activation of aldose reductase in rat lens and metal-ion chelation by aldose reductase inhibitors and lipoic acid. Free Radie Res 1996 25:337-346. 116. Packer L. Antioxidant properties of lipoic acid and its therapeutic effects in prevention of diabetes complications and cataracts. Ann NY Acad Sci 1994 738:257-264. 117. Ziegler D Hanefeld M Ruhnau KJ et al. Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidantn-lipoic acid. A 3-week multicentre randomized controlled trial ALADIN Study. Diabetologia 1995 38: 1425- 1433. 118. Nagamatsu M Nickander KK Schmelzer JD et al. Lipoic acid improves blood flow reduces oxidative stress and improves distal nerve conduction in experi• mental diabetic neuropathy. Diabetes Care 1995 18: 1160-1167. 119. Packer L Tritschler HJ Wessel K. Neuroprotection by the metabolic antioxi• dant alpha-lipoic acid. Free Radie Biol Med 1997 22:359-378. 120. Borcea V Isermann B Henkels M et al. Effect of the antioxidant alpha-lipoic acid on the progression of endothelial cell damage and albuminuria in patients with diabetes mellitus. Submitted.

slide 247:

14 Role of Oxidative Stress and Antioxidants on Adhesion Molecules and Diabetic Microangiopathy Click Here If You Also Want To Be Free From Diabetes Klaus Kusterer Jorg Bojunga Gerald Bayer Thomas Konrad Eva Haak Thomas Haak and Klaus H. Usadel University of Frankfurt Frankfurt Germany Hans J. Tritschler ASTA Medica A WO GmbH Frankfurt Germany Each cell can mobilize an armory of antioxidant defense systems. Under nor• mal metabolic conditions the production of free radicals and the antioxidant capacity are balanced. Hyperglycemia in diabetes mellitus is associated with an increased production of free radicals. Furthermore observational studies indicate lower levels of antioxidants like vitamin E vitamin C carotene ascor• bate and thiols in patients with diabetes mellitus 12. Imbalance between free radical production and the antioxidant defense system leads to oxidative stress. In diabetic patients oxidative stress can be demonstrated by increased levels of lipid peroxidation products 3-8. There is a body of evidence that vascular and neurological complications in patients with diabetes mellitus are a consequence of oxidative stress 9-12. I. FREE RADICALS AND DIABETES MELLITUS

slide 248:

Various sources of free radicals are considered in patients with diabetes melli• tus. Free radicals are produced during autooxidation 10. Furthermore glu- 205

slide 249:

206 Kusterer et al. Glucose + Protein Schiff Base Rearrangements Amadori-type Advanced Glycosylation End Products AGE Free Radicals Specific Receptors RAGE Figure 1 The production of AGEs. The interaction of AGEs with their specific re• ceptors RAGEs generates free radicals. cose is known to form glycosylation products with protein Fig. 1. These Schiff bases rearrange and form more stable Amadori-type glycosylation end products 13. Some of these early glycosylation end products on collagen or proteins of the vessel wall undergo a complex series of chemical rearrange• ments to form irreversibleadvanced glycosylationend products AGE. Recep• tors specific for AGEs RAGE have been identified on endothelial cells monocytes neurons and smooth muscle cells 14-20. AGE-RAGE inter• action induces free radicals 18. The sorbitol pathway is another mechanism involved in glucotoxicity 21 Fig. 2. Glucose is reduced to sorbitol by aldose reductase. Then sorbitol is oxidized by sorbitol dehydrogenase to fructose. The second reaction is cou• pled with the reduction of NAD+ to NADH Fig. 1. The pathophysiological consequences are similar to changes during ischemia and the increased NADH/NAD+ ratio has been termed pseudohypoxia 11. During hyperglyce• mia the sorbitol pathway activity is increased. The increased ratio of NADH/

slide 250:

Stress and Antioxidants 207 D-Glucose Al dosered11c · tase Sorbitol Sorbi to/dehydro · gena.e D-Fructose NADPH NAO NADH Figure 2 Sorbitol oxidation increases cytosolic NADH/NAD+ which is linked to hyperglycemic pseudohypoxia I I. NAO+ enhances the synthesis of prostaglandins which leads to free radical production. Further the oxidation of NADH to NAO+ in the electron transport chain in the mitochondria involves increased production of superoxide radical Fig. 3. The superoxide radical is normally catalyzed by superoxide dismutase to hydrogen peroxide. The iron-dependent Fenton reaction leads to the aggres• sive hydrogen peroxide radical that further attacks the side chains of lipids Hyperglycemia Sorbitol pathway activity• NADH/NAD+ f- Prostaglandin synthesis f- NADH .... NAD+ \ /xidation inmitochondria o· 2 Figure 3 Hyperglycemia activates the sorbitol pathway. Sorbitol oxidation increases cytosolic NADH/NAD+. Increased NADH/NAD+ ratios increase prostaglandin syn•

slide 251:

thesis leading to free radical production. In the mitochondria the superoxide radical is generated by oxidation of NADH in the electron transport chain.

slide 252:

I 208 Kusterer et al. Lipid peroxidation Figure 4 The superoxide radical is rapidly dismutized to H202 by superoxide dismu• tase. The iron-dependent Fenton reaction produces a hydrogen peroxide radical which induces lipid peroxidation. resulting in lipid peroxidation products 22 Fig. 4. Patients with diabetes mellitus have increased lipid peroxidation products 3-8. II. ENDOTHELIUM-DEPENDENT VASODILATION In physiological terms hyperglycemia increases blood pressure and leads to endothelial dysfunction with impaired vascular reactivity 23. Hypoxia is ac• companied with an influx of calcium which might activate nitric oxide NO synthase followed by vasodilation and hyperemia 11 Fig. 5. In diabetes increased free radicals might quench NO leading to ischemia 1124. In vitro acetylcholine-induced vasodilation of vasculature from diabetic animals is im• paired 2526. The rate of NO synthesis in vivo compared with the rate of NO quenching is unclear. We measured the blood flow of the arteria iliaca in diabetic and nondiabetic rats. The NO-mediated stimulation by acetylcholine was impaired in diabetic rats but in contrast to in vitro experiments treatment

slide 253:

ca2+ Bradyllnin Stress and Antioxidants 209 Blood flow Endo• thelial Cell Shear stress Figure 5 The increase of cytosolic calcium activates nitric oxide synthase. with antioxidants did not restore the impaired endothelium-dependent vasodi• lation unpublished results. Ill. ISCHEMIA-REPERFUSION IN DIABETES MELLITUS Microvascular dysfunction has been studied extensively in animal models. One of the most widely used models is streptozotocin-induced diabetes in the rat. In this model rats are treated with a single toxic dose of streptozotocin which destroys the islets of Langerhans 27. The effect of hyperglycemia on the microvasculature is then evaluated after 4-12 weeks 2128-37. Gly• cernic control by insulin treatment can prevent the microvascular dysfunction. In diabetic humans metabolic status is not stable and episodes of normogly• cernia are followed by phases of hyper- and hypoglycemia. Thus blood sugar levels vary considerably and in consequence to the pathophysiological changes presented above the status of oxidative stress changes and blood flow will go up and down. Therefore it is reasonable to assume that under realistic conditions a diabetic patient undergoes episodes of ischemia and reperfusion

slide 254:

210 Kusterer et al. during the lime until manifestation of microangiopathy. The lime intervals of the increased blood sugar levels are sufficient to induce leukocyte adherence lo the vascular endothelium. We therefore propose an additional pathophysiological approach to the understanding of microangiopathy Fig. 6. A patient with diabetes mellitus has frequent episodes of ischemia followed by reperfusion. During ischemia there is a loss of energy-rich phosphates leading to an accumulation of hypo• xanthine 38. In the endothelial cell xanthine dehydrogenase is converted to xanthine oxidase. During reperfusion superoxide radical and hydrogen perox- Changing blood sugar level Changing blood flow l Ischemia ATP Degradalion .. Hypoxanthine Xanthine dehydrogenase Xanthine oxidase Reperfusion Xomhine oxidase Hypoxanthine . 02- + H202 + Urate 02 Figure 6 Changes in blood sugar are associated with changes in blood How. Frequent episodes of hyperglycemia in a diabetic patient will induce frequent episodes of

slide 255:

ischemia-reperfusion. Ischemia-reperfusion generates free radicals and induces leuko• cyte adherence to vascular endothelium followed by tissue injury.

slide 256:

Stress and Antioxidants 211 ide are produced by the oxidation of hypoxanthine. In addition ischemia• reperfusion induces the adherence of leukocytes to the endothelium. The ad• herent leukocytes migrate to the tissue of the vessel wall and release their inflammatory mediators including free radicals. Free radicals are produced by leukocytes by the following reaction: 202 + NADPH 202 + NADP+ + H+ cxidase To test this hypothesis we first evaluated the effect of lipoic acid on leukocyte adherence induced by ischemia-reperfusion. Lipoic acid was cho• sen because its beneficial effect in diabetic neuropathy has been demonstrated in animals and multicenter trials of clinically manifested polyneuropathy. Li• poic acid is an effective radical scavenger 39. Nerve conductance and blood flow of the nerves improved by the treatment with lipoic acid 37. In multicen• ter clinical trials symptoms in diabetic patients with polyneuropathy improved 40. Ischemia-reperfusion experiments were performed in groups of rats re• ceiving either solvent propylenglycol or 25 50 or 100 mg/kg intravenous IV lipoic acid respectively 30 min before the beginning of the ischemia. lschemia of 30 min was produced by means of a plastic ring in mesentery vessels. With in vivo microscopy leukocyte adherence was measured during I 0 20 and 30 min of reperfusion. Leukocyte adherence was reduced dose dependently by lipoic acid. Diabetic animals were treated either with propylen• glycol or lipoic acid at 100 mg/kg intraperitoneally IP 5 days a week for l month. After I month of treatment with propylenglycol IP the acute pretreat• ment with lipoic acid I 00 mg/kg IV did not prevent the increased leukocyte adhesion in diabetic rats. Treatment with lipoic acid 100 mg/kg/day IP from the induction of diabetes combined with an IV bolus 10 min before the ex• periment reduced ischemia-reperfusion-induced leukocyte adhesion from 7550 ± l073/mm2 vein cross-section to 1774 ± 840 p 0.001. In conclusion the therapeutic agent for polyneuropathy lipoic acid reduces reperfusion injury. Thus ischemia-reperfusion caused by changing blood sugar levels might contribute to the pathogenesis of diabetic polyneu• ropathy and the benefical effects of lipoic acid in diabetic polyneuropathy might be partially explained by the inhibition of leukocyte adherence. IV. ADHESION MOLECULES The migration of leukocytes from the bloodstream into inflamed tissue re• quires a cascade of events in the microcirculation 4 l . The sequence of bind-

slide 257:

receptors LFA-1 VLA-4 LFA-1 212 Kusterer et al. --- rolling - c - ell-activa - tion adhesion migration leukocyte receptors L-Selectin cytokine• MAC-1 endothelial PNAd cytokines ICAM-1 VCAM-1 ICAM-1 ligand CD34 GlyCAM MAdCAM chemotactic factors ICAM-2 MAdCAM-1 ICAM-2 Figure 7 The migration of leukocytes from the bloodstream to the tissue starts with rolling of the leukocytes which is mediated by seleetins. Chemoattractants or cytokines increase the expression of adhesion molecules on the endothelium or increase the avid• ity of the integrins on the leukocytes leading to adhesion of leukocytes to the endothe• lium and finally to emigration from the bloodstream. ing events starts with leukocytes rolling along the endothelium Fig. 7. This first step is mediated by selectins 42-47. Upon activation by inflammatory signals such as chernoattractants rolling progresses to firmer adhesion due to interaction of the integrins on leukocytes with adhesion molecules of the immunoglobulin family such as ICAM-1 or VCAM-1 on the endothelium. Monoclonal antibodies that interfere with different steps of the cell-binding cascade have shown a beneficial effect on ischernia-reperfusion injury 48. Enhanced expression of ICAM-1 and P-selectin has been demonstrated in the diabetic human retina and choroid 49. This increased expression of cell adhesion molecules may contribute to the retinal and choroidal microangi• opathy in diabetic patients. A potential mechanism for the accelerated vasculo• pathy has been proposed recently 14. The interaction of AGEs with their endothelial receptor induced the expression of VCAM-1 in cultured human endothelial cells and in mice 14. AGE-RAGE interaction generates free radicals that activate NF-KB. NF-KB regulates in addition to other genes the expression of VCAM-1. The incubation of human umbilical vein endothelial cells with AGE increased the endothelial expression of VCAM-1. Preincuba• tion of the cells with lipoic acid suppressed VCAM-1 expression to baseline

slide 258:

Stress and Antioxidants 213 levels 50. The AGE-induced endothelial binding of monocytes was also re• duced by Iipoic acid 50. Soluble adhesion molecules are detectable only in small quantities in the serum of healthy individuals 5152. However increased serum levels of soluble adhesion molecules have been described in different pathologic situations including ischemia-reperfusion injuries 5354 insulin-dependent IDDM 55 and non-insulin-dependent diabetes mellitis NIDDM 56. In IDDM circulating ICAM-1 and VCAM-1 but not ELAM- I was elevated. The increased levels were found in IDDM patients with and without microangiopa• thy. In first-degree relatives of NIDDM the levels of E-selectin relates to vascular risk in contrast to soluble VCAM-1 which showed no difference compared with control subjects 56. Thus elevated levels of circulating adhe• sion molecules can be used as a risk marker for developing microangiopathy. The pathophysiological role of the circulating adhesion molecules is unclear. It might be that they just indicate endothelial cell stimulation by oxidative stress or that they are a reaction to protect the endothelium from further leuko• cyte attack. The second hypothesis might be reasonable because we could recently demonstrate that leukocyte adhesion during ischemia-reperfusion can be reduced by the application of naturally occurring soluble recombinat ICAM-1 57. V. CONCLUSION In line with many other investigators oxidative stress is an important patho• genic factor for the development of microangiopathy in diabetic patients. In addition to the well-characterized soruces of free radicals we propose a new concept for free radical production: Changes of metabolic control are coupled to episodes of ischemia-reperfusion with subsequent free radical production and leukocytes adherence. Lipoic acid prevents increased leukocyte adherence in diabetic and nondiabetic rats which might contribute to its beneficial effect in diabetic neuropathy. REFERENCES I. Godin TV Wohaieb SA Garnett ME Goumeniouk AD. Antioxidant enzyme alterations in experimental and clinical diabetes. Mo Cell Biochem 1988 84: 223-231.

slide 259:

214 Kustereret al. 2. Packer L Witt EH. Tritschler HJ. o-Lipoic acid as a biological antioxidant. Free Radie Biol Med 1995 19:227-250. 3. Jennings PE Jones AF Florkowski CM Lunec J Barnett AH. Increased diene conjugates in diabetic subjects with microangiopathy. Diabetic Med 1987 4: 452-456. 4. Valazques E Winocour HP Kesteven P Alberti KGMM Laker MF. Relation of lipid peroxides to macrovascular disease in type 2 diabetes. Diabetic Med 1991 8:752-758. 5. MacRury SM Gordon D Wilson R Bradley H Gemmell CG Paterson JR et al. A comparison of different methods of assessing free radical activity in type 2 diabetes and peripheral vascular disease. Diabetic Med 1993 10:331- 335. 6. Griesmacher A Kinder-Hauser M Andert S Schreiner W Toma C Knoebel P et al. Enhanced serum level of TBARS in diabetes mellitus. Am J Med 1995 98:469-475. 7. Sundaram RK Bhaskar A Vijayalingam S Viswanatthan M Mohan R Shanrnu• gasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes with and without complications. Clin Sci 1996 90:255-260. 8. Nourooz-Zadeh J Tajaddini-Samadi J McCarthy S Betteridge DJ. Elevated lev• els of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: I 054- 1058. 9. Giugliano D Ceriello A Paolisso G. Oxidative stress in diabetic vascular compli• cations. Diabetes Care 1996 19:257-267. 10. Baynes JW. Perspectives in diabetes. Role of oxidative stress in development of complications in diabetes. Diabetes 1991 40:405-412. 11. Williamson JR Chang K Frangos M Hasan KS Ido Y Kawamura T et al. Perspectives in diabetes hyperglycemic pseudohypoxia and diabetic complica• tions. Diabetes 1993 42:801-813. 12. Nourooz-Zadeh J Rahimi A Tajaddini-Sarmadi J Tritschler H Rosen P Halli• well B et al. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997 40:647-653. 13. Brownlee M Cerami A Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988 318:1315-1321. 14. Schmidt AM Hori 0 Chen JX. Li JF Crandall J Zhang J et al. Advanced glycation endproducts interacting with their endothelial receptor induce expres• sion of vascular cell adhesion molecule- I VCAM-1 in cultured human endothe• lial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. J Clin Invest 1995 96:1395-1403. 15. Ritthaler U Deng Y Zhang Y Greten J Abel M Sido B et al. Expression of receptors for advanced glycation end products in peripheral occlusive vascular disease. Am J Pathol 1995 146:688-694. 16. Schmidt AM Mora R Cao R Yan SD Brett J Ramakrishnan R et al. The endothelial cell binding site for advanced glycation end products consist of a

slide 260:

Stress and Antioxidants 215 complex: an integral membrane protein and a lactoferrin-like polypeptide. J Biol Chem 1994 269:9882-9888. 17. Wu VY Cohen MP. Identification of aortic endothelial cell binding proteins for Amadori adducts in glycated albumin. Biochem Biophys Res Commun 1993 193:1131-1136. 18. Stitt AW Li YM Gardiner TA Bucala R Archer DB Vlassara H. Advanced glycation end products AGEs co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats. Am J Pathol 1997 150:523- 531. 19. Yang Z. Makita Z Hori Y Brunelle S Cerami A Sehajpal P et al. Two novel rat liver membrane proteins that bind advanced glycosylation end products: rela• tion to macrophage receptor for glucose-modified proteins. J Exp Med 1991 174:515-524. 20. Yan SD Stern D Schmidt AM. Whats the RAGE The receptor for advanced glycation end products RAGE and the dark side of glucose. Eur J Clin Invest 1997 27:179-181. 21. Tilton RG Chang K Neyengaard JR Van den Enden M ldo Y Williamson JR. Inhibition of sorbitol dehydrogenase. Effects on vascular and neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 1995 44:234-242. 22. Farber JL Kyle ME Coleman JB. Biology of disease: mechanisms of cell injury by activated oxygen species. Lab Invest 1990 62:670-679. 23. Tooke JE. Perspectives in Diabetes Microvascular function in human diabetes. A physiological perspective. Diabetes 1995 44:721- 726. 24. Stevens MJ Dananberg J Feldman EL Lattimer SA Karnijo M Thomas TP et al. The linked roles of nitric oxide aldose reductase and Na K +-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat. J Clin Invest 1994 94:853-859. 25. Rosen P Ballhausen T Bloch W Addicks K. Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995 38:1157-1168. 26. Cameron NE Cotter MA. Impaired contraction and relaxation in aorta from streptozotocin-diabetic rats: role of polyol pathway. Diabetologia 1992 35: 1011-1019. 27. Brosky G Logothetopoulos J. Streptozotocin diabetes in the mouse and guinea pig. Diabetes 1969 18:606-611. 28. Cameron NE Cotter MA. Dines KC Maxfield EK. Pharmacological manipula• tion of vascular endothelium function in non-diabetic and streptozotocin-diabetic rats: effects on nerve conduction hypoxic resistance and endoneurial capillariza• tion. Diabetologia 1993 36:516-522. 29. Low PA Nickander KK. Oxygen free radical effects in sciatic nerve in experi• mental diabetes. Diabetes 1991 40:873-877. 30. Low PA Ward K Schmelzer JD Brimijoin S. Ischemic conduction failure and energy metabolism in experimental diabetic neuropathy. Am J Physiol 1985 248:E457-E462.

slide 261:

216 Kusterer et al. 31. Cotter MA Love A Watt MJ Cameron NE Dines KC. Effects of natural free radical scavangers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia 1995 38:1285-1294. 32. Cameron NE Cotter MA Dines KC Maxfield EK. Anti-oxidant and pro-oxidant effects on nerve conduction velocity endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabeticrats. Diabetologia 1994 37:449-459. 33. Cameron NE Cotter MA. Neurovascular dysfunction in diabetic rats. Potential contribution of autoxidation and free radicals examined using transition metal chelating agents. J Clin Invest 1995 96: 1159-1163. 34. Cameron NE Cotter MA Maxfield EK. Anti-oxidant treatment prevents the de• velopment of peripheral nerve dysfunction in streptozotocin-diabeticrats. Diabe• tologia 1993 36:299-304. 35. Cameron NE Cotter MA Hohman TC. Interactions between essential fatty acid prostanoid polyol pathway and nitric oxide mechanisms in the neurovascular deficit of diabetic rats. Diabetologia 1996 39: 172-182. 36. Bravenboer B Kappelle AC Hamers FPT van Buren T Erkelens DW Gispen WH. Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. Diabetologia 1992 35: 813-817. 37. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H et al. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care 1995 18:1160-1167. 38. McCord JM. Oxygen-derivedfree radicals in postischemic tissue injury. N Engl J Med 1985 312:159-163. 39. Matsugo S Yan L-J Han D Tritschler HJ Packer L. Elucidation of antioxidant activity of rx-lipoic acid toward hydroxyl radical. Biochem Biophys Res Com• mun 1995 208:161-167. 40. Ziegler D Hanefeld M Ruhnau KJ Meiner HP Lobisch M Schutte K et al. Treatment of symptomatic diabetic perpheral neuropathy with the anti-oxidant a• lipoic acid. A 3-week multicentre randomized controlled trial ALADIN study. Diabetologia 1995 38: 1425-1433. 41. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigra• tion: the rnultistep paradigm. Cell 1994 76:301-314. 42. Lawrence MB Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 1991 65:859-873. 43. Ley K Tedder TF. Leukocyte interactions with vascular endothelium. New in• sights into selectin-mediated attachment and rolling. J lmmunol 1995 155:525- 528. 44. Ley K Bullard DC Arbones ML Bosse R Vestweber D Tedder TF et al. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 1995 181 :669-675. 45. Morita Y Clemens MG Miller LS Rangan U Kondo S Miyasaka M et al.

slide 262:

Stress and Antioxidants 217 Reactive oxidants mediate TNF-a-induced leukocyte adhesion to rat mesenteric venular endothelium. Am J Physiol 1995 269:Hl833-Hl842. 46. Tedder TF Steeber DA Chen A Engel P. The selectins: vascular adhesion mole• cules. FASEB 1 1995 9:866-873. 47. McEver RP Moore KL. Cummings RD. Leukocyte trafficking mediated by selectin-carbohydrate interactions. 1 Biol Chem 1995 270: 11025-1 I 028. 48. Kubes P Jutila M. Payne D. Therapeutic potential of inhibiting leukocyte rolling in ischeruia/reperfusion. J Clin Invest 1995 95:2510-2519. 49. Mcl.eod Lefer DJ Merges C Lutty GA. Enhanced expression of intracellular adhesion molecule- I and P-selectin in the diabetic human retina and choroid. Am J Pathol 1995: 147:642-653. 50. Kun T Forst T. Wilhelm A Tritschler H. Pfuetzner A Harzer 0 et al. Alpha lipoic acid reduces expression of vascular cell adhesion molecule- I and endothe• lial adhesion of human monocytes after stimulation with advanced glycation endproducts. Clin Sci 1999: 96:75-82. 5 l. Rothlcin R Mainolfi EA. Czajkowski M Marlin SD. A form of circulating CAM- I in human serum. J lmmunol 1991: 147:3788-3793. 52. Seth R. Raymond FD. Makgoba MW. Circulating ICAM-1 isoforms: diagnostic prospects for intlammatory and immune disorders. Lancet 1991 338:83-84. 53. Haught WH Mansour M. Rothlein R Kishimoto TK Mainolfi EA. Hendricks JB. et al. Alterations in circulating intercellular adhesion molecule- I and L-selec• tin: further evidence for chronic inflammation in ischernic heart disease. Am Heart J 1996: 132: 1-8. 54. Gearing AJH Newman W. Circulating adhesion molecules in disease. lmmunol Today 1993 14:506-512. 55. Fasching P. Veit M. Rohac M Streli C. Schneider B Waldh1iusl W et al. Ele• vated concentrations of circulating adhesion molecules and their association with microvascular complications in insulin-dependent diabetes mellitus. J Clin Endo• crinol Metab 1996: 81 :4313-4317. 56. Bannan S. Mansfield MW Grant PJ. Soluble vascular cell adhesion molecule• I and E-selectin levels in relation to vascular risk factors and to E-selectin geno• type in the first degree relatives of NIDDM patients and in NIDDM patients. Diabetologia 1998: 41 :460-466. 57. Kusterer K Bojunga J. Enghofer M Heidenthal E. Usadel KH. Kolb H et al. Soluble intercellular adhesion molecule I reduces leukocyte adhesion to vascular endothelium in ischernia-reperfusion injury in mice. Am J Physiol 1998 275: 0377-0380

slide 263:

This Page Intentionally Left Blank

slide 264:

15 Molecular Basis of o-Tocopherol Action and Its Protective Role Against Diabetic Complications To Stop Diabetes In Few Days Click Here Angelo Azzi Roberta Ricciarelli and Sophie Clement University of Bern Bern Switzerland Nesrin Ozer Marmara University Istanbul Turkey Atherosclerosis is a pathology frequently associated with diabetes 1-4 . Mi• gration and proliferation of smooth muscle cells smc from the media to the intima of the arterial wall take place early at the beginning of the atheroscle• rotic process 5. Proliferation of smc is accompanied by an increased expression of extra• cellular matrix protein which contributes to the development of diabetic vas• cular complications 6-8. Several studies have shown beneficial effects of antioxidant vitamins in particular vitamin E in protection against the vascular complications seen in diabetic patients 9-12. In recent years the mechanism of action of vitamin E has been discussed 1314 because its simple role as an antioxidant and protective component of the cell membrane 15 is becoming insufficient to explain the action of this compound. Vitamin E deficiency leads to premature cell aging and to the binding of immunoglobulin G to erythrocytes 16 and it has been shown to play an

slide 265:

important role in porphyrin metabolism 17. Moreover reports suggest an effect of vitamin Eon the arachidonic acid cascade related to the biosynthesis of prostaglandins 18. A specific neuropathology ataxia with vitamin E defi- 219

slide 266:

220 Azzi et al. ciency A VED has also been found to be due to familiar vitamin E deficiency 19-21. In our laboratory we found that rx-tocopberol the most active form of vitamin E inhibits vascular smooth muscle cell proliferation 22-25. smc are under the control of several growth factors that activate a cascade of ki• nases and phosphatases ending with the activation of transcription factors such as AP-I and NF-KB and those proteins involved in the cell cycle pro• gression in particular protein kinase C PKC 26. First described 20 years ago as a proteolytically activated serine/threo• nine kinase PKC still justifies the attention of researchers providing new surprises. The enormous number of weekly publications requires a continuous effort to summarize and simplify the knowledge on the field. Since its original discovery PKC has expanded into a family of closely related proteins which can be subdivided on the basis of certain structural and biochemical properties. The original members of the PKC family are the Ca2-dependent or conven• tional c a 1 11 and y isoforms. Later with the discovery of Ca2+ -indepen• dent PKC isoenzymes the group of a new or novel n isoforms has been classified including e TJ and e PKCs. Finally there are the so-called atypical a t A and L isoforms which take an intermediate position between the nPKC and aPKC isoforms. For a complete description of the structures there are several recent reviews 27-29. PKC has played an important role in the field of cancer research since the discovery that the tumor-promoting phorbol ester class of compounds caused PKC activation. For the first time a connection between the process of signal transduction and tumor promotion could be made. Since then PKC isoforms have been implicated in several pathways not always related to carci• nogenesis. Today the observation that individual isoenzymes are located in different subcellular compartments and undergo different regulation strongly suggests that each isoform has a unique individual role. For example PKCµ has been found to be associated with the B-cell antigen receptor complex 30 PKCTJ is thought to mediate transcriptional activation of the human transglu• taminase I gene 31 and PKC8 has been described to stimulate the transcrip• tion factor complex AP-1 in T lymphocytes 32. Implication of specific PKC isoenzymes has also been shown in pathological conditions such as Alzhei• mers 33-36 atherosclerosis 37-39 and diabetes 40-42. In our laboratory we observed the inhibition of PKC activity and smc proliferation by physiological concentrations of n-tocopherol. By using isoform-specific inhibitors and activators we found that PKCa is the selec• tive target of a-tocopherol action. -Tocopherol an antioxidant almost as po• tent as o-tocopherol did not show any effect on cell proliferation of PKC

slide 267:

Molecular Basis of o-Tocopherol Action 221 activity suggesting that the mechanism of action of n-tocopherol is not related to its antioxidant properties. Our results could offer a model to explain the beneficial effects of vitamin E on diabetic vascular complication. I. METHODS Rat A7r5 aortic vascular smc were obtained from American Type Culture Collection. Purified PKCa and protein phosphatase type 2A were from UBI New York. Tissue culture media and polyclonal antibodies to PKCa 8 and E isoforms were from Life Technologies Inc. Grand Island NY. Fetal calf serum FCS was from PAA Linz Austria. Anti-rat PKC i polyclonal anti• body purified PKCa and trimeric PP2A were from Upstate Biotechnology Lake Placid NY. Polyclonal anti-PKCµ antibody caliculin A okadaic acid phorbol 12-myristate 13-acetate and Go 6976 were from LC Laboratories Woburn MA. Ly379196 was a gift of Eli Lilly Indianapolis IN. y-32P ATP 3000 Ci/rnmol 32Pi monoclonal anti-PKCa clone MC5 ECL detec• tion system and ECL Hyperfilm were from Amersham International Buck• inghamshire UK. Anti-protein kinase Ca rabbit polyclonal used for the ki• nase reactions of the immunoprecipitated protein was from Oxford Biomedical Research Inc. Oxford Ml. Phosphorylase-B phosphorylase kinase strepto• lysin-O Histone III-SS and phorbol dibutyrate were from Sigma St. Louis MO. Ci-ceramide was from BioMol Hamburg Germany. Phenylmethylsul• fonylfluoride PMSF leupeptin pepstatin and aprotinin were from Bohringer Mannheim Germany. Protein A-Sepharose 48 was from Pharmacia Biotech Inc. PKCI antisense oligodeoxynucleotide was from MWG Biotech Ebers• berg Germany. The peptide PLSRTLSV AAKK used as substrate for assay PKC activity was synthesized by Dr. Servis Epalinges Switzerland. Myelin basic protein 4-14 fragment was produced by Bachem Switzerland. RRR• c-tocopherol and RRR--tocopherol are obtained from Henkel LaGrange IL. Tocopherols are adsorbed to FCS before the addition to the cells as described 43. Protein concentration was determined using a Pierce kit ac• cording to the manufacturers procedures. A. Cell Cultures Rat A 7r5 cells are maintained in Dulbeccos modified Eagles medium DMEM containing 1.0 g/L glucose 60 U/mL penicillin 60 mg/mL strepto• mycin and supplemented with 10 v/v FCS. Cells in a subconfluent state were made quiescent by incubation in DMEM containing 0.2 FCS for 48

slide 268:

222 Azzi et al. h. Cells were then washed with phosphate-buffered saline PBS and treated as indicated in the figure legends. Cell viability determined by Trypan blue exclusion method was 90-95 in all experiments. Cells are used between passages 7 and 15. B. Animals Thirty male albino rabbits aged 2-4 months were assigned randomly to one of the following four groups. All rabbits were fed 100 g/day vitamin E-free diet as described 4445. One group of rabbits was only fed the diet without addition of treatments. The second group received daily injections of 50 mg/ kg vitamin E. The diet of the third group contained 2 cholesterol and the fourth received the same diet with daily injections of 50 mg/kg vitamin E. After 4 weeks the thoracic aortas were removed and media strips were minced homogenized and cytosolic and membrane extracts were prepared for analysis of PKC activity 46. C. Cell Proliferation Quiescent A 7r5 cells were restimulated to grow by addition of I 0 FCS. a• Tocopherol was added to cells at the indicated concentrations. Cell number was determined 30 h later by using a hemocytometer. To measure DNA syn• thesis cells were pulsed for I h with 3Hthymidine I µCi/well during the S phase 11 h after entry into the cycle. After labeling cells were processed as described 22 and radioactivity was determined. D. PKC Activityin Permeabilized Cells Quiescent A7r5 cells were subjected to different treatments as indicated in the figure legends. During the last hour of the preincubation period cells were treated with I 00 nM phorbol 12-myristate 13-acetate. Aliquots of cells were resuspended in a reaction buffer containing 5.2 mM MgCl2 94 mM KCl 12.5 mM HEPES pH 7.4 12.5 mM EGTA and 8.2 mM CaCl2 and assays were started by adding y-32PATP 9 cpm/pmol final concentration 250 µM peptide substrate final concentration 70 µM and streptolysin-0 0.3 IU. Samples were incubated at 37 °C for IO min quenched and analyzed as de• scribed previously 2247.

slide 269:

Molecular Basis of a-Tocopherol Action 223 E. lmmunoprecipitation of PKC lsoforms After treatment cells were harvested in 1 mL lysis buffer 50 mM Tris pH 7.5 150 mM NaCl 1 v/v Triton X-100 I mM EGTA 2 mM EDTA chymostatin leupeptin antipain pepstatin 5 mg/L each I mg/L E64 and 1 mM PMSF. Cell lysates were forced through a 25-gauge syringe 15 times and cleared by centrifugation at 15800 X g for 10 min. Immunoprecipitation was carried out on equal amounts of protein with the indicated anti-PKC anti• body 3 µg incubated for 1-3 h at 4 °C followed by adsorption to protein A-Sepharose beads IO mg for 1 h at 4 °C. Precipitated samples were recov• ered by centrifugation and proteins were either resolved by SDS-PAGE or used in autophosphorylation and kinase reactions. F. Western Blot Analyses Immunocomplexes were dissolved in Laernmlis sample buffer and separated by electrophoresis on a 10 polyacrylamide gel followed by electrotransfer to poly vinylidene difluoride PVDF membranes DuPont NEN Research Products. Membranes were incubated at room temperature with 0.1 mg/mL anti-PKC isoforms. Proteins were detected with the ECL system Amersham. G. Autophosphorylatioo nf PKC lsoforms Immunoprecipitated PKCs bound to protein A-Sepharose beads were washed three times with lysis buffer and once with the same buffer containing 0.4 M NaCl and without EDTA/EGTA. Samples were incubated in 40 mL of a mix• ture containing 5 µCi of y-32PATP 10 mM ATP 400 µM MgCl2 5 mM CaC2 400 µM phosphatidylserine I 00 nM phorbol 12 I-dibutyrate I mM sodium orthovanadate and 20 mM Tris pH 7.4 at 37 °C for 10 min. The reactions were terminated by addition of 10 µL of boiling SDS sample buffer and electrophoresed on a 10 polyacrylamide gel. Gels were stained using the SYPRO protein gel stain kit Molecular Probes blotted and radioactivity in the membranes was detected by using a BioRad GS-250 Molecular Imager. Alternatively gels were dried down for autoradiography on Kodak X-Omat S films. Quantification was done by using a BioRad GS-700 imaging densi• tometer. H. Activityof lmmunoprecipitated PKCa Confluent A7r5 cells after the treatment described in the figure legends were lysed in a buffer containing 150 mM NaCl 50 mM Tris-HCI pH 8.0 I

slide 270:

224 Azzi et al. Nonidet P-40 0.5 deoxycholate 0.1 SDS 10 mM NaF protease inhibitor cocktail Bohringer I mM Na3 V04 and 1 mM PMSF. Extracts were pre• pared by passing the lysates through a 25-gauge needle 15 times and cleared by centrifugation at 15800 X g for 10 min. Anti-protein kinase Ca antibody 3 µg was added to the supematants for 1 h at 4 °C and afterward protein A-Sepharose was added for an additional hour. The resulting immunocom• plexes were collected by centrifugation washed in lysis buffer and finally in kinase buffer 50 mM Tris-HCI pH 7.4 10 mM NaF 0.5 mM EDTA 0.5 mM EGTA 2 mM MgCl2 1 mM PMSF and protease inhibitor cocktail. Kinase reactions with the immunocomplexes were carried out in a 40-µL final volume of an activation buffer containing 20 mM Tris-HCI pH 7.4 10 mM MgCl2 10 mM ATP 2.5 µCi y-32P ATP 600 Ci/mmol 0.4 mg/mL histone III-S 1.2 mM CaCl2 40 mg/mL phosphatidylserine and 3.3 mM dioleylglycerol. Reactions were terminated by adding 20 µL boiling SDS sample buffer and frozen until use. Samples were loaded in a 10 SDS-PAGE and blotted on a PVDF membrane for 1 h at 100 mA. Histone phosphorylation was detected by using a phosphorimager and the signals were quantified by densitometric scanning and normalized in respect to the amount of immunoprecipitated PKCa which was detected by immunoblots using the MC5 anti-PKC anti• body. I. In Vivo Labeling of Cells Quiescent A7r5 cells 6 X 105 were incubated in phosphate-free DMEM Arnimed with 0.25 mCi/mL 32Pi during 14 hat 37 °C. Then cells were stimu• lated with 10 dialyzed FCS Sigma in the presence of the indicated agents and further incubated for 7 h. During the last hour cells received 100 nM phorbol 12-myristate 13-acetate PMA. Cells were then washed exhaustively with PBS lysed in SDS buffer and subjected to immunoprecipitation for PKCa. PKCa was resolved on SOS-PAGE and its phosphorylation was ana• lyzed on a BioRad Molecular Imager GS-250. Staining with the SYPRO kit has been used to control protein loading of the gels. J. Protein Phosphatase ActivityAssay Purified PP2A 25 ng resuspended in 10 µL assay buffer Tris 50 mM EDTA 1 mM pH 7.6 was preincubated with a solution of either n-tocopherol 50 µM or -tocopherol 50 µM for 10 min at 30 °C. Control reactions contained vehicle ethanol 0.1 alone. Assays were started by addition to the mixtures of 32Pphosphorylase-a solution 8.5 mg protein 9 X 104 dpm and further incubated for 10 min at 30 °C. Reactions were stopped by adding 120 µL ice• cold trichloroacetic acid 10 and 150 mg albumin in 20 µL H20. Samples

slide 271:

- o- cc 40 Molecular Basis of o-Tocopherol Action 225 were left on ice and centrifuged 2 min at 12000 X g. The clear supernatant was counted in a liquid scintillation counter. K. PKCodProtein Phosphatase Assay PK Ca 16 nM was incubated for IO min at 30°C with PP2A or pp I at the indicated concentrations in 40 µL activation buffer containing 10 mM MOPS 3-N-morpholinopropanesulfonic acid pH 7.2 0.5 mM DTT 14-dithio• DL-threitol 100 µM MBPw myelin basic protein peptide fragment 4-14 0.25 mM ATP 20 mM MgC12 5 µg phosphatidylserine 5 µg diacylglicerides and 5 µCi 32P-ATP. Reaction was stopped with 20 µL 25 TCA trichloro• acetic acid. Aliquots of 50 µL were spotted onto 3 X 3 cm P81 Whatman filters washed twice with 0.75 phosphoric acid and then washed once with acetone. Radioactivity was counted in a liquid scintillation analyzer. II. o:-TOCOPHEROLIS A SPECIFIC INHIBITOR OF CELL PROLIFERATION n-Tocopherol at concentrations between 10 and 50 µM was shown to inhibit rat A 7r5 smc proliferation Fig. I whereas -tocopherol appeared ineffective 48. When o-tocopherol and -tocopherol were added together no inhibition of cell proliferation was seen. Both compounds were transported equally in 0 c :: c .. o .. o 0 .._ .... 0 0 .... ·- 8 Q .... . : 0 10 so 60 -·--1 - i I •.. :2-. Eo - ..c I- :i: 2:l .•...... e. 10 20 30 40 d-a-Tocopherol concentration µM

slide 272:

Figure 1 Inhibition of SMC proliferation. Quiescent cells were restimulated with FCS in the presence of the indicated concentrations of e-tocopherol. For DNA synthe• sis determination 3Hthimydine was given to the cells in the S-phase. The control represents 84332 :::: 5150 cpm.

slide 273:

226 Azzi et al. cells and did not compete with each other for uptake 48. The oxidized prod• uct of n-tocopherol o-tocopheryl quinone and several other water- and Iipid• soluble antioxidants were not inhibitory indicating that the effects of a• tocopherol were not related to its antioxidant properties 48. Inhibitory effects of o-tocopherol were also observed in primary human aortic smc Balb/3T3 mouse fibroblasts and NB-2a mouse neuroblastoma but not in Chinese hamster ovary CHO Saos-2 human osteosarcoma cells or P388 mouse macrophages. Ill. a-TOCOPHEROL IS A SPECIFIC INDIRECT INHIBITOR OFPKC A. e-Tocopherol Inhibits PKC Activity During the transition G0 G1 phase of the cell cycle an o-tocopherol-sensi• tive increase in PKC activity not paralleled by changes in the mRNA levels of the PKCcx 8 E and 1 isoforms was observed Fig. 2 Ricciarelli R. et al. 1997 unpublished results. Similarly no changes in the protein levels of the major isoform PKCcx expressed during the transition were observed in the presence or absence of o-tocopherol Fig. 2. Maximal PKC inhibition by n-tocopherol was found 6- 7 h after the entry of cells into the G1 phase Fig. 2 inset and this inhibition was only observed when o-tocopberol was added at the time of restimulation 22. PKC activity has been also measured in homogenates of aortas from rabbits fed different dietary supplements. As shown in Table 1 an approximate 50 reduction in PKC activity is observed in vitamin E-treated animals com• pared with controls vitamin E-poor diet. With cholesterol supplementation PKC activity increases to 10.2 -absorbency units/min/mg protein which is significantly reduced by vitamin E treatment. B. PKC lsoforms in A7r5 smc The finding that PKC is involved in the u-tocopherol inhibition of smc prolif• eration 22 prompted the question of which isoforms isare affected by cx• tocopherol. In A 7r5 cells the presence of PKCcx 8 E 1 and u was documented Fig. 3 lane A. To determine the specificity of the reaction the corresponding competitor peptides were used Jane B. The PKC isoform was determined in a separate experiment as well and shown to be present in smc. It was also

slide 274:

I """ \ i 0 f Molecular Basis of e-Tocopherol Action 227 1.6 --- ·- ----- - -- -- -------- ----------------------1 1.41 control activity LJ--- J 1.2\ /// - - - /\ .l I /.· / \ · " _ _....-----....- -- 1 ·-- ·- ·- 7- _-----· - :..- :o---- C- .\--- - . \Kea protein J / ------- " --- \ Western blot 0.81- _..£.--o ·r----- " \ o.i/ :1: o g- 1o1 x - rJ- --- 0 : .2 0 .D -·I /:cs--- ------1.:r--· - -- J 0.4 g \ - """ . ---·--·-:::-iJ .2 :S ·• ·a·-.-T912 activity +u-tocopherol - Tmic h ------------------·L- -· l J _ --·-· 0 2 4 6 8 10 12 Timeh Figure 2 Inhibition of PKC activity by a-tocopherol is a function of the cell cycle. Quiescent A 7r5 cells were stimulated for different times with FCS in the absence or presence of 50 µM a-tocopherol. Al the indicated points PM A-stimulated PKC activity in permeabilized cells was measured. PKCa levels were analyzed by Western analysis and the signals scanned by densitometry. Data are expressed as arbitrary units of ab• sorbency. The inset shows the percentage of PKC inhibition by o-tocopherol al differ• ent incubation limes. Data are representative of four independent experiments. found that Jong PMA treatment downregulated the PKCa 8 E lane C and P and µ isofonns not shown. C. o-Tocopherol Selectively InhibitsPKCa Nanomolar concentrations of Go 6976 have been shown to inhibit PKCa and p whereas even micromolar concentrations have no effect on the activity of PKCo E or t 49. As can be seen in Figure 4 when PKCa and p isoforms were inhibited by Go 6976 the residual activity was not sensitive to u-tocopherol indicating

slide 275:

that the PKCo E t and µ isoforms were not involved in the n-tocopherol• induced PKC inhibition. Differently after PKCP activity was inhibited by the specific inhibitor Ly379l96 42 the effect of u-tocopherol was still present

slide 276:

228 Azzi et al. Table 1 PKC Activity from smc Homogenates Obtained from Differently Treated Rabbits Treatment PKC activity Control 8.4 :::: I. I Vitamin E 4.5 :::: 2.5 Cholesterol Cholesterol + vitamin E 10.2 :::: 2.4 4.5 :::: l.Ot p 0.0 I compared with control group. t p 0.02 with respect to cholesterol group. Aortic media are minced and homogenized. and nuclei are sedimented by centrifugation. Supematants are centri• fuged again at 100.000 x g to obtain cytosolic fractions. Pellets are used for preparation of membrane fractions. Protein kinase C activity is measured in both frac• tions. Since the homogenates did not show significant mernbrune/cytosol distribution changes. only the values of total PKC activity are reported. Results are expressed as mean :::: SD n 5. Statistical analysis was performed by one-way analysis of variance ANOV A. indicating that this isoform of PKC is not involved in cc-tocopherol inhibition Table 2. Further evidence was obtained by the experiments reported in Figures 5 and 6. Figure 5 shows that after the inhibition of PKCcx and p by Go 6976 and t by PKC specific antisense oligonucleotide the remaining PKC iso• forms o E and u were not affected by n-tocopherol. Finally when PKCcx p o E and µ isoforms were downregulated the residual PKC was not sensi• tive to u-rocopherol either in the absence or presence of the activator C2- ceramide 50 Fig. 6. Taken together the above experiments suggested that PKCcx is the spe• cific target for e-tocopherol. To further substantiate this finding immunopre• cipitation of the different PKC isoforms and the determination of the kinase and autophosphorylating activity were carried out. D. Effect of o-Tocopherol on PKC lsoforms Autophosphorylation PKC autophosphorylation in immunoprecipitates has been found to correlate with its enzymatic activity and has been taken as a reliable indication of PKC activity 28.

slide 277:

-63k0 - Molecular Basis of e-Tocopherol Action 229 a-PKC N-84k0 -52k0 A B C -PKC - -8410 -63k0 3-PKC ::: -52kD A B C s-PKC -B4k0 - •• -63k0 .......... -52k0 A B C µ-PKC -112kD - 84kD • A B C a-PKC - - 52k0 -112 kO - 84k0 - 63kD - 52k0 - 63k0 - 52k0 Figure 3 Characterization of PKC isoforms in A7r5 cells. Cells I x I 06 at 90 confluence were harvested. PKC isoforms were immunoprecipitated with the corre• sponding antibodies and subjected to SDS-PAGE and immunoblotting as described in Methods. A control B plus competitor peptide C cells treated for 24 h with I µM PMA. Cells were incubated in the absence or presence of u-tocopherol or • tocopherol for 7 h during the GI phase. Then extracts were prepared and immunoprecipitation of the individual PKC isoforms was performed. Auto• phosphorylation activity and protein amounts were determined for each iso• form In Figure 7 the effects on PKCa and PKCI are shown. The bar graphs correspond to the PKC activity and values are normalized with respect to the protein content. As can be seen only PKCa from a-tocopherol-treated cells

slide 278:

was less active relative to its control. The activity of all other PKC isoforms was not affected by the treatment of cells with o-tocopherol data not shown.

slide 279:

230 Azzi et al. Protein kinase C activity pmol/min/106 cells 0 50 100 150 200 Figure 4 Selective inhibition of PKCa by u-tocopherol. Quiescent A7r5 cells were restimulated for 7 h with FCS in the absence or presence of 50 µM n-tocopherol. Go 6976 20 nM was added to the permeabilized cells where indicated and PMA-stimu• lated PKC activity was measured as described in Methods. E. e-Tocopherol Selectively InhibitsPKCa Activity and Phosphorylation State To establish whether the incubation of cells with n-tocopherol resulted in a change in the phosphorylation state of PKCa an in vivo labeling reaction was carried out. Cells were labeled with 32Pi overnight and after stimulation with Table 2 Effect of the PKC P-Specific Inhibitor Ly379 I 96 on the n-Tccopherol-Induced Inhibition of PKC Treatment cpm Change Inhibition PMA 13428 100 0 n-tocopherol 8928 67 33 Ly379196 15280 113 -13 Ly379196 + n-tocopherol 9228 69 31 Quiescent A7r5 cells were restimulated for 7 h with FCS in the absence or presence of 50 µM n-tocopherol. Ly379 l 96 20 nM was added to the permeabilized cells where indicated and PMA-stimulated PKC activity was measured as described in Method. PMA phorbol 12-myristate 13-acetate.

slide 280:

a-tocopherol PMA anti-£ +Go 6976 anti -£ +Go 6976 Molecular Basis of a-Tocopherol Action 231 Protein kinase C activity pmol/min/10 6 cells 0 50 100 150 200 250 300 a-tocopherol Figure 5 Selective inhibition of PKCa. by n-tocopherol. Quiescent cells were treated for 24 h with I mM phosphorothioate antisense oligodeoxynucleotide designed to hy• bridize PKCI mRNA anti-Z Then cells were restimulated with FCS for 7 h in the absence or presence of 50 µM u-tocopherol. Go 6976 was added to the PKC assay as described above. pmol/min/1 O 6 cells 0 10 20 30 40 50 60 70 Figure 6 Effect of n-tocopherol on PKCI. Quiescent cells were treated for 24 h with 1 µM PMA downregulation DR and afterward treated for 7 h with 50 µM a• tocopherol. C2-ceramide 20 µM was added to the PKC assay in permeabilized cells as described.

slide 281:

I I ·111 10 5 0 0 232 Azzi et al. Autophosphorylation of a-PKC 111 • 1 32PJ 1 1 - - - 1 1 wb 1 : I c a c a Autophosphorylation of s-PKC 32P 75 . 1 --1 1 wb 1 2 . c a c a Figu re 7 Autophosphorylation activity of different PKC isofonus. Quiescent cells were restimulated for 7 h with FCS in the absence C or presence of either 50 µM c-tocopherol a or -tocopherol . PMA 100 nM was added for the last hour of the preincubation period. Then cell extracts were prepared and PKC isoforms were immunoprecipitated with the indicated antibodies. Autophosphorylation reaction of the individual isofonus was performed as described in Methods. Samples were electropho• resed and blotted and the calculated ratio between incorporated radioactivity 32P and the protein levels Western blot is represented in the bar graphs for each condition. Data are representative of three independent experiments. FCS for 7 h in the presence or absence of n-tocopherol PKCa was immuno• precipitated blotted and the 32P incorporation measured. Figure 8 shows a bar graph presentation of the radioactivity intensities integrated and normal• ized with respect to the protein levels of each sample. Relative to the control N the PMA-treated cells P showed a significant increase in 32P incorpora• tion into PKCa. Cells pretreated with n-tocopherol column a showed a large inhibition of PKCa phosphorylation whereas cells preincubated with • tocopherol column showed much less inhibition. The inhibitory effect of o-tocopherol was reversed by two potent protein phosphatase inhibitors oka• daic acid 2 nM not shown or calyculin A 2 nM column a + C. Figure 9 shows the PKCa activity measured after immunoprecipitation of the enzyme

slide 282:

c 120 - .. . I l Molecular Basis of cx-Tocopherol Action 233 14q·--- ......... i I ... i " .. .. i . 0 I... o ......... a.. lOQ sd 69 N 41· I . . . 2J: . -----· . L LL_ . J N p a a+C Figure8 Effect of o.-tocopherol and -tocopherol on PKCa phosphorylation state. Quiescent A7r5 cells were incubated in phosphate-free DMEM medium ICN for 48 h. They received 0.25 mCi/ml P for the last 14 h. Cells were restimulated for 7 h with FCS in the absence P or presence of either 50 LM u-tocopherol a or -tocopherol . PMA I 00 nM was added for the last hour to all samples except N. Caliculin A 2 nM was added to cells for I h where indicated a+ C. Cell extracts were prepared and immunoprecipitated with anti-PKCa. Proteins were resolved by SDS-PAGE and radioactivity Jip and protein levels were quantified as described in Methods. The bar graph represents the ratio between cpm incorporated to PKCa and the protein levels. Data are representative of three independent experiments. from cells preincubated with o-tocopherol or -tocopherol at the late GI phase of the cell cycle. As can be seen o-tocopherol inhibited PKCa activity more strongly compared with -tocopherol. In the case of PKC no significant PKC activity changes were observed if the cells were preincubated with either rx-tocopherol or -tocopherol. F. Protein Phosphatase PP2A Is Activated by o-Tocopherol and Can Dephosphorylate PKCa The role of a phosphatase PP2A on the deactivation of PKCa has been postu• lated on the basis of previous experiments 22. To establish if a direct effect of PP2A on PKC took place the following experiment was carried out Fig.

slide 283:

lO. The two enzymes were preincubated together and then the activity of PKCa was measured. It was observed that PP2A produced a deactivation of PKCa. The inhibition of approximately 50 in PKCa activity obtained at a ratio I PP2A/ 16 PKC molecules indicates a catalytic role of PP2A on PKC inactivation.

slide 284:

234 Azzi et al. 70 60 50 40 0 30 20 10 0 I I Figure 9 Determination of PKCa. activity after its immunoprecipitation from cells treated with n-tocopherol or P-tocopherol. Cells were stimulated for 7 h with FCS in the absence Control or presence of either 50 µM a.-tocopherol or P-tocopherol as indicated. During the last hour of preincubation they received 100 nM PMA. Then extracts were prepared PKCa. was immunoprecipitated and a kinase reaction using Histone III-SS was performed as described in Methods. Proteins were resolved by electrophoresis and radioactive bands were quantified with a BioRad Molecular Ana• lyst software. Protein levels were estimated by staining the gel with the SYPRO kit or by immunoblots with the MCS monoclonal antibody. The ratio between radioactivity incorporated into the substrate and the amount of PKCa. precipitated was expressed as arbitrary units of the densitometric scanning of the bands. Data are representative of three independent experiments. The inhibition by n-tocopherol of PKCa at a cellular level may thus be related to a possible activation of PP2A by a-tocopherol. This hypothesis was investigated in the experiment outlined in Figure 11. Purified PP2A was incu• bated with 32Pphosphorylase-a as a substrate its activation by n-tocopherol -tocopherol and a mixture of the two was analyzed. u-Tocopherol produced almost a twofold activation. -Tocopherol was slightly inhibitory and the mixture of both tocopherols was without significant effect. It thus appears that one of the cellular targets of o-tocopherol may be PP2A.

slide 285:

------- - 40 60 50 -... d- -" s::: .9 :E 30 u 20 11.. 10 0 0.16mU 0.13 mu 0.06 mu PP2A concentration Figure 10 Inhibition of PKCcx activity by PP2A. Purified PKCcx 16 nM was incu• bated for IO min at 30°C with PP2A in 40 µL activation buffer as described in Methods. PKCcx activity was determined using a peptide substrate as described previously. Data are representative of three independent experiments. 70 60 50 40 · 30 20 10 Figure 11 Effect of rx-toccpherol and f-tocopherol on PP2A activity. Phosphatase activity using pure PP2A was assayed in the presence of different u-tocopherol or f• tocopherol concentrations as indicated. PP2A activity stimulation was calculated with respect to control samples. The background in the absence of the enzyme represented less than 3.5 of the initial total counts. Data are representative of three separate experiments.

slide 286:

236 Azzi et al. IV. CONCLUSIONS Cell proliferation especially of smc and mesangial cells plays an important role in the pathogenesis of diabetic complications. Increase in smc prolifera• tion rate is of primary importance in the progression of atherosclerosis. In• creased growth of mesangial cells a cell type similar to srnc takes place during nephrosclerosis. PKC has been shown to be involved in both diseases. Smooth muscle cells from aortas of rabbits fed a high cholesterol diet show increased PKC activity. o-Tocopherol treatment of the animals has been shown to dimin• ish such activity. In vitro smooth muscle cells are subject to a fine control by ce-tocoph• erol. Their proliferation is strongly diminished by o-tocopherol in a specific way. Many other cell types are in fact insensitive to n-tocopherol. o-Tocoph• erol is also unique in its action in smooth muscle cells because other tocopher• ols tocotrienols and general antioxidants are not as potent as o-tocopberol. Some of them such as probucol are not effective at all. It is thus conceivable that the action of rx-tocopherol is not mediated by its radical scavenging func• tion. Data on the competition of n-tocopherol and P-tocopherol suggest the existence of a receptor with the capacity of recognizing o-tocopherol as an agonist and P-tocopherol as an antagonist. The finding that a phosphatase PP2A is modulated in an opposite way to proliferation by several tocopherols may indicate a role of this enzyme in the cascade of events at the basis of a• tocopherol inhibition of cell proliferation. In the studies reported above it has also been clarified that PKC in smooth muscle cells is inhibited by o-tocoph• erol. Also such an event is o-tocopherol specific with a specificity pattern similar to that of cell proliferation. PKC inhibition takes place only at cellular level and is associated with a diminution of PKC phosphorylation. This finding parallels the observed activation pattern of a phosphatase. Consequently the entire picture relative to the understanding of smc inhibition may be under• stood in the following way. In the hypothesis that a receptor for o-tocopherol exists this may produce PP2A increase expression. Alternatively the latter enzyme could be by itself the receptor distinguishing between n-tocopherol and P-tocopherol. In either case the increased PP2A activity results in a de• phosphorylation of PKC and in a reversible diminution of its activity. The role of rx-tocopherol has also been described in terms of inhibition in mesan• gia\ cells of PKC activity via diminution of diacylglycerol the physiological activator of PKC. Such a decrease would be the consequence of the specific activation by o-tocopherol of the enzyme diacylglycerol kinase.

slide 287:

Molecular Basis of a-Tocopherol Action 237 The two described mechanisms of cell proliferation control by o-tocoph• erol are both centered on PKC regulation. In one 2248 emphasis is given to the postranslational modifications of PKC mediated by an n-tocopherol• sensitive protein phosphatase. In the other 42 the role of n-tocopherol would be to subtract PKC major activator by biochemical conversion. Both mecha• nisms may coexist or acquire a major role in one or the other cell type. ACKNOWLEDGMENTS Supported by the Swiss National Science Foundation and by F. Hoffmann• La Roche AG. We thank Mrs. Maria Feher for her expert help with cellular cultures. REFERENCES I. Ybarra J Lopez-Talavera JC. Non insulin dependent diabetes mellitus lipid me• tabolism and atherosclerosis editorial. Med Clin Bare 1998 110:19-21. 2. Valek J. Prevention of atherosclerosis in diabetics. Cas Lek Cesk 1997 136: 523-526. 3. Steiner G. Diabetes mellitus dyslipoproteinaemias and atherosclerosis. Diabeto• logia 1997 40:Sl47-Sl48. 4. Visseren FL Bouter KP Pon MJ Hoekstra JB Erkelens DW Diepersloot RJ. Patients with diabetes mellitus and atherosclerosis: a role for cytomegalovirus Diabetes Res Clin Pract 1997 36:49-55. 5. Bornfeldt KE Raines EW Nakano T Graves LM Krebs EG Ross R. Insulin• like growth factor-I and platelet-derived growth factor-BB induce directed mi• gration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest 1994 93: 1266-1274. 6. Gilbert RE Cox A Wu LL Allen TJ Hulthen UL Jerums G Cooper ME. Ex• pression of transforming growth factor-beta l and type IV collagen in the renal tubulointerstitium in experimental diabetes: effects of ACE inhibition. Diabetes 1998 47:414-422. 7. Park IS Kiyomoto H Abboud SL Abboud HE. Expression of transforming growth factor-beta and type IV collagen in early streptozotocin-induced diabetes. Diabetes 1997 46:473-480. 8. Sumida Y Ura H Yano Y Misaki M Shima T. Abnormal metabolism of type• IV collagen in normotensive non-insulin-dependent diabetes mellitus patients. Honn Res 1997 48:23-28.

slide 288:

238 Azzi et al. 9. Karasu C Ozansoy G Bozkurt 0 Erdogan D. Omeroglu S. Antioxidant and triglyceride-lowering effects of vitamin E associated with the prevention of abnormalities in the reactivity and morphology of aorta from streptozotocin• diabetic rats. Antioxidants in Diabetes-Induced Complications ADIC Study Group. Metabolism 1997 46:872-879. 10. Gazis A Page S Cockcroft J. Vitamin E and cardiovascular protection in diabe• tes editorial. Br Med J 1997 314:1845-1846. 11. Siman CM Eriksson UJ. Vitamin E decreases the occurrence of malformations in the offspring of diabetic rats. Diabetes 1997 46: 1054-1061. 12. Viana M Herrera E Bonet B. Teratogenic effects of diabetes mellitus in the rat. Prevention by vitamin E. Diabetologia 1996 39: I 041-1046. 13. Shklar G Schwartz JL. Vitamin E inhibits experimental carcinogenesis and tu• mour angiogenesis. Eur J Cancer B Oral Oncol 1996 328: 114-1 19. 14. Pentland AP Morrison AR Jacobs SC Hruza LL Hebert JS Packer L. Tocoph• erol analogs suppress arachidonic acid metabolism via phospholipase inhibition. J Biol Chem 1992 267: 15578-15584. 15. Lucy JA. Functional and structural aspects of biological membranes: a suggested structural role for vitamin E in the control of membrane permeability and stabil• ity. Ann NY Acad Sci 1972 203:4-11. 16. Gillilan RE Mondell B Warbasse JR. Quantitative evaluation of vitamin E in the treatment of angina pectoris. Am Heart J 1977 93:444-449. 17. Briggs M. Are vitamin E supplements beneficial Med J Aust 1974 1:434- 437. 18. Dip lock AT Xu GL Yeow CL. Okikiola M. Relationship of tocopherol structure to biological activity tissue uptake and prostaglandin biosynthesis. Ann NY Acad Sci 1989 570:72-84. 19. Hentati A Deng HX Hung WY Nayer M Ahmed MS He X Tim R Stumpf DA Siddique T. Ahmed. Human alpha-tocopherol transfer protein: gene structure and mutations in familial vitamin E deficiency. Ann Neurol 1996 39:295-300. 20. Doerflinger N Linder C Ouahchi K Gyapay G Weissenbach J Le Paslier D Rigault P Bela S Ben Hamida C Hentati F et al. Ataxia with vitamin E defi• ciency: refinement of genetic localization and analysis of linkage disequilibrium by using new markers in 14 families. Am J Hum Genet 1995 56:1116- 1124. 21. Ouahchi K Arita M Kayden H Hentati F Ben Hamida M Sokol R Arai H Inove K Mandel JL Koenig M. Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein. Nat Genet 1995 9:141-145. 22. Tasinato A Boscoboinik D Bartoli GM Maroni P Azzi A. d-Alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations correlates with protein kinase C inhibition and is independent of its antioxidant properties. Proc Natl Acad Sci USA 1995 92:12190- 12194. 23. Boscoboinik D Ozer NK Moser U Azzi A. Tocopherols and 6-hydroxy-chro-

slide 289:

Molecular Basis of o-Tocopherol Action 239 man-2-carbonitrile derivatives inhibit vascular smooth muscle cell proliferation by a nonantioxidant mechanism. Arch Biochem Biophys 1995 318:241-246. 24. Boscoboinik DO Chatelain E Bartoli GM Stauble B Azzi A. Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-alpha• tocopherol. Biochim Biophys Acta 1994 1224:418-426. 25. Chatelain E Boscoboinik DO Bartoli GM Stauble B Azzi A. Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols. Biochim Biophys Acta 1993 1176:83-89. 26. Muller R Mumberg D Lucibello FC. Signals and genes in the control of cell• cycle progression. Biochim Biophys Acta 1993 1155:151-179. 27. Azzi A Boscoboinik D Hensey C. The protein kinase C family. Eur J Biochem 1992 208:547-557. 28. Newton AC. Protein kinase C: structure function and regulation. J Biol Chem 1995: 270:28495-28498. 29. Mellor H Parker PJ. The extended protein kinase C superfamily In Process Citation. Biochem J 1998: 332:281-292. 30. Korinek V Barker N Morin PJ van Wichen D de Weger R Kinzler KW. Yo• gelstein B Cleven H. Constitutive transcriptional activation by a beta• catenin-Tcf complex in APC-/- colon carcinoma. Science 1997 275: 1784-1787. 31. Morin PJ Sparks AB Korinek V Barker N Clevers H. Vogelstein B Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 1997 275:1787-1790. 32. Rubinfeld B Robbins P EI-Gamil M Albert I Porfiri E Polakis P. Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997 275: 1790-1792. 33. Matsushima H Shimohama S Tanaka S Taniguchi T Hagiwara M Hidaka H Kimura J. Platelet protein kinase C levels in Alzheimers disease. Neurobiol Aging 1994: 15:671-674. 34. Kinouchi T Sorimachi H Maruyama K Mizumo K Ohno S lshiura S Suzuki K. Conventional protein kinase C PKC-alpha and novel PKC epsilon but not delta increase the secretion of an N-terrninal fragment of Alzheimers disease amyloid precursor protein from PKC cDNA transfected 3Y I fibroblasts. FEBS Lett 1995 364:203-206. 35. Boyce JJ Shea TB. Phosphorylation events mediated by protein kinase C alpha and epsilon participate in regulation of tau steady-state levels and generation of certain "Alzheimer-like" phospho-epitopes. Int J Dev Neurosci 1997: 15:295- 307. 36. Govoni S Racchi M Bergamaschi S Trabucchi M Battaini F Bianchetti A Binetti AG. Defective protein kinase C alpha leads to impaired secretion of solu• ble beta-amyloid precursor protein from Alzheimers disease fibroblasts. Ann NY Acad Sci 1996: 777:332-337. 37. Haller H Lindschau C Quass P Distler A Luft FC. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha. Circ Res 1995 76:21-29.

slide 290:

240 Azzi et al. 38. Wang S Desai D Wright G Niles RM Wright GL. Effects of protein kinase C alpha overexpression on A7r5 smooth muscle cell proliferation and differentia• tion. Exp Cell Res I 997 236: I I 7- I 26. 39. Leng L Du B Consigli S McCaffrey TA. Translocation of protein kinase C• delta by PDGF in cultured vascular smooth muscle cells: inhibition by TGF-beta I. Artery 1996 22:140-154. 40. Schaffer SW Ballard C Mozaffari MS. Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart Mo Cell Biochem 1997 176:219-225. 41. Malhotra A Reich D Reich D Nakouzi A Sanghi V Geenen DL Buttrick PM. Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart which are prevented by angiotensin II receptor blockade. Circ Res 1997 81:1027-1033. 42. Ishii H Jirousek MR Koya D et al. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 1996 272:728- 731. 43. Chan AC Tran K. The uptake of R R Ralpha-tocopherol by human endothelial cells in culture. Lipids 1990 25: I 7-21. 44. Konneh MK Rutherford C Li SR Anggard EE Fems GA. Vitamin E inhibits the intimal response to balloon catheter injury in the carotid artery of the choles• terol-fed rat. Atherosclerosis I 995 I I 3:29-39. 45. Sirikci 0 Ozer NK Azzi A. Dietary cholesterol-induced changes of protein kinase C and the effect of vitamin E in rabbit aortic smooth muscle cells. Athero• sclerosis I 996 I 26:253-263. 46. Porreca E Ciccarelli R Di Febbo C Cuccurullo F. Protein kinase C pathway and proliferative responses of aged and young rat vascular smooth muscle cells. Atherosclerosis 1993 104:137-145. 47. Alexander DR Graves JD Lucas SC Cantrell DA Crumpton MJ. A method for measuring protein kinase C activity in permeabilized T lymphocytes by using peptide substrates. Evidence for multiple pathways of kinase activation. Biochem J 1990 268:303-308. 48. Azzi A Boscoboinik D Marilley D Ozer NK Stauble B Tasinato A. Vitamin E: a sensor and an information transducer of the cell oxidation state. Am J Clin Nutr 1995 62: I 337S- l 346S. 49. Martiny-Baron G Kazanietz MG Mischak H Blumberg PM Kochs G Hug H Marme D Schachtele C. Selective inhibition of protein kinase C isozyrnes by the indolocarbazole Go 6976. J Biol Chern I 993 268:9194-9 I 97. 50. Muller G Ayoub M Storz P Rennecke J Fabbro D Pfizenmaier K. PKC zeta is a molecular switch in signal transduction of TNf-alpha bifunctionally regu• lated by ceramide and arachidonic acid. EMBO J 1995 14:1961-1969.

slide 291:

16 Protein Kinase C Activation Development of Diabetic Vascular Complications and Role of Vitamin E in Preventing These Abnormalities To Cure Diabetes Naturally Click Here Sven-Erik Bursell Harvard Medical School Beetham Eye Institute Eye Research Boston Massachusetts George L. King Joslin Diabetes Center Harvard Medical School Boston Massachusetts Hyperglycemia induces multiple changes in vasculature or in neuronal cells in animal models of diabetes or in diabetic patients. The multifactorial nature of the changes is not surprising because the flux of glucose and its metabolites are known to affect many cellular pathways. The main challenge in this area has been to identify hyperglycemia-induced biochemical changes that can have a significant impact on vascular dysfunction and subsequent development of pathologies. Multiple theories have been proposed to explain the pathogene• sis of the various complications involving retina glomeruli peripheral nerves cardiovascular tissues wound healing and pregnancy. No one single theory however has emerged to account for all these changes. Extracellularly glucose can react nonenzymatically with the primary amines of proteins forming glycated compounds or oxidants 1 . These prod-

slide 292:

241

slide 293:

242 Bursell and King ucts can secondarily act on inflammatory cells or vascular cells directly via receptor- or nonreceptor-mediated processes to cause vascular dysfunction 23. Excessive glucose can also be transported intracellularly mainly by the glucose transporter GLUT- I and the resulting metabolism can cause changes in the redox potential increase sorbitol production via aldose reductase or alter signal transduction pathways such as the activation of diacylglycerol DAG and protein kinase C PKC pathway activation 4-10. It is probable that all hyperglycemia-induced intra- and extracellular changes and their ad• verse effects are being mediated through the alteration of some of these signal transduction pathways. The effect of hyperglycemia on signal transduction pathways has not been extensively studied except for the activation of the DAG-PKC pathway. This pathway is known to be important in vascular cells because it is associated with the regulation of vascular permeability contractility extrcellular matrix cell growth angiogenesis cytokine actions and leukocyte adhesions all of which are abnormal in diabetes 11 12. I. PROTEIN KINASE C PKC includes at least 11 isoforms a Pl p2 y 8 e 11 0 Aµ representing the major downstream targets for lipid second messengers or phorbol esters 11-13. The conventional PKC isoforms a Pl p2 y are Ca-t-dependent containing two cysteine-rich zinc finger-like motifs Cl region which are the binding sites of DAG or phorbol ester and a Ca2+ /phospholipid C2 re• gion. New PKCs 8 e 11 0 µ are DAG sensitive but Ca2 independent due to the absence of the C2 region. The two atypical PKCs /. and A are insensi• tive to DAG and lack one of the cysteine-rich motifs in the Cl region but they can be activated by phosphatidylserine. The source of DAG resulting in PKC activation can be derived from the hydrolysis of phosphatidylinositides Pl or from the metabolism of phos• phatidylcholine PC by phospholipase C PLC or D PLD. Recent data however have shown that each isoform can be regulated by more than one lipid second messenger 9 such as the activation of PKC / by PIP3 12-14. Multiple isoforms can be expressed in different cell types but despite exten• sive study the attribution of a specific function to a specific isoform cannot be consistently established suggesting that several isoforms can possibly me• diate a similar range of functions and that their actions may be cell specific 1215.

slide 294:

PKC Activation and the Effect of Vitamin E 243 II. MECHANISMS OF HYPERGLYCEMIA -INDUCED PKC ACTIVATION In association with the diabetic vascular complications increased total DAG content has been demonstrated in a variety of tissues including retina 16 aorta heart 17 and renal glomeruli 18 19 in both diabetic animal models and patients Table I. This has also been observed in classically termed "in• sulin-sensitive" tissues such as the liver and skeletal muscle 2021. In all vascular cells studied increasing glucose levels from 5 to 22 mM in the media elevated the cellular DAG contents 17. Increased DAG levels in response to elevated glucose may not occur immediately and can take as long as 3-5 days to reach a maximum after elevating glucose levels 1822. Xia et al. 22 also showed that increased DAG content was chronically maintained in the aorta of diabetic dogs even after 5 years of disease. In fact Inoguchi et al. 17 reported that euglycemic control by islet cell transplant after 3 weeks was not able to completely reverse the increases in DAG level or PKC activa• tion in the aorta of diabetic rats. These results clearly indicate that the activa• tion of the DAG-PKC pathway can be chronically sustained. Cellular DAG content can also be increased by agonist-stimulated hy• drolysis of PI or PC such as PLC or PLO 11-13. Because inositol phosphate products are not increased by hyperglycemia in aortic cells and glomerular Table 1 Summary of DAG Level and PKC Activity in Cultured Cells Exposed to High Glucose and Tissues Isolated from Diabetic Animals Diacylglycerol Protein k.inase C Cultured cells Retinal endothelial cells t r Aortic endothelial cells r r Aortic smooth muscle cells r r Renal rnesangial cells r r Pericytes r Tissues Retina diabetic rats and dogs r r Heart diabetic rats t i Aorta diabetic rats and dogs t t Renal glomeruli diabetic rats r t Brain diabetic rats ND Not changed Peripheral nerve r .J ND. not determined.

slide 295:

244 Bursell and King mesangial cells increases in PI hydrolysis are most likely not involved in diabetes 2223. Increases in DAG content could also arise from PC metabo• lism since Yasunare et al. 24 reported that PLO activity was increased by elevated glucose levels in aortic smooth muscle cells. They did not however quantitate the amount of total DAG. Most studies however have shown that the source of glucose-induced DAG increases were through the de novo syn• thesis pathway. In this case labeling studies using 6-3H- or U-14C-glucose demonstrated that elevated glucose levels increased the incorporation of la• beled glucose into the glycerol backbone of DAG in aortic endothelial cells 25 aortic smooth muscle cells 22 and glomeruli 18. These studies clearly established that the increased DAG content was partially derived from glyco• lytic intermediates 26-28. Palmitic acid and oleic acid are the predominate fatty acids incorporated into DAG through the de novo pathway and from the metabolism of PC which is consistent with the findings in vascular tissues from diabetic animals 25. In contrast DAG derived from PLC activation consists mainly of l-stearolyl 2-arachidonyl fatty acid which was not altered by glucose 2529. The activation of PKC by hyperglycemia may be tissue specific because it was noted in the retina 16 aorta heart 17 and glomeruli 818 but not clearly demonstrated in the brain 16 and peripheral nerves 30 Table 1 . Similar increases in DAG levels and PKC activation have also been shown in multiple types of cultured vascular cells in response to increased glucose levels Table I 8162231. Thus it is likely that the DAG-PKC pathway is activated by the hyperglycemic-diabetic state in all vascular cells. Among the various PKC isoforms in vascular cells PKC and o isofonns appear to be preferentially activated as shown by immunoblotting studies in aorta and heart of diabetic rats 17 and in cultured aortic smooth muscle cells exposed to high levels of glucose 32. However increases in other isoforms such as PKC a 2 and E in the retina 18 and PKC a l and o in the glomerular cells from diabetic rats have also been noted 3334. These results demon• strate that diabetes and hyperglycemia will activate the DAG-PKC pathways in many tissue types including vascular tissues and thus glucose and its me• tabolites can cause many cellular abnormalities. However for a hyperglyce• mia-induced change to be credible as a causal factor of diabetic complications it has to be shown to be able to be chronically altered difficult to reverse to cause similar vascular changes when activated without diabetes and to be able to prevent complications when it is inhibited. Thus far based on the evidence presented DAG-PKC pathway activation appears to fulfill the first two crite• ria. In the following sections data are presented to support a fulfillment of the final two criteria with respect to the DAG-PKC pathway.

slide 296:

PKC Activation and the Effect of Vitamin E 245 111. FUNCTIONAL ALTERATIONS IN VASCULAR CELLS MEDIATED BY DAG-PKC ACTIVATION Multiple cellular and functional abnormalities in the diabetic vascular tissues have been attributed to the activation of DAG-PKC pathways including vas• cular blood flow vascular permeability Na+-K+ ATPase and extracellular matrix components. A. Vascular Blood Flow Abnormalities in vascular blood flow and contractility have been found in many organs of diabetic animals or patients including the kidney retina pe• ripheral arteries and microvessels of peripheral nerves. In the retina of diabetic patients without clinical retinopathy 35-38 and animals with short durations of disease 39-43 retinal blood flows have been shown to be decreased. However retinal blood flow may be normal or increased with longer duration of retinopathy 37 3844 . Multiple lines of evidence have supported the hy• pothesis that the decreases in retinal blood flow are due to PKC activation. For example introduction of a PKC agonist such as a phorbol ester into the retina will decrease retinal blood flow 16. On the other hand decreases in retinal blood flow in diabetic rats have been reported to be normalized by PKC inhibitors 16 19. In nondiabetic animals the intravitreal injection of a DAG kinase inhibitor resulted in increased retinal DAG levels activation of PKC and a concomitant reduction in retinal blood flow 131 . DAG kinase metabolizes DAG to phosphatidic acid so DAG kinase inhibition will result in an increase in the available DAG pool. The results from this study showed that increased retinal DAG levels resulted in retinal blood flow decreases com• parable with those measured in the diabetic rats. In addition to the retina decreases in blood flow have also been reported in the peripheral nerves of diabetic animals which were normalized by PKC inhibition 4546 although some reports have noted increases in neuronal blood flow in diabetic rats 6. One of the potential mechanisms by which PKC activation could be causing vasoconstriction in the retina is by increasing the expression of endo• thelin-1 ET- I 47. We reported that the expression of ET-1 a potent vaso• constrictor plays a primary role in the regulation of retinal hemodynamics 48 and is increased in the retina of diabetic rats. Additionally intravitreous injection of an ET-A receptor antagonist BQ123 prevented the decrease in retinal blood flow in these diabetic rats 47. The decrease in blood flow to the retina could lead to local hypoxia. Hypoxia is known to be a potent inducer

slide 297:

246 Bursell and King of vascular endothelial growth factor VEGF causing increases in permeabil• ity and microaneurysm formation 4950. Abnormalities in hemodynamics have been clearly documented to pre• cede diabetic nephropathy 5152. Elevated renal glomerular filtration rate GFR and modest increases in renal blood flow are characteristic findings in insulin-dependent diabetes mellitus IDDM patients 5152 and experimental diabetic animals 53. Diabetic glomerular hyperfiltration is likely to be the result of hyperglycemia-induced decreases in arteriolar resistance especially at the level of afferent arterioles 5455 resulting in an elevation of glomeru• lar filtration pressure. Multiple mechanisms have been proposed to explain the increases in GFR and glomerular filtration pressure including an enhanced activity of angiotensin 56 and culturation in prostinoid productions 57-59. It is possible that the activation of DAG-PKC may also play a role in the enhancement of angiotensin actions because angiotensin mediates some of its activity by the activation of the DAG-PKC pathway 57. In addition in• creases in vasodilatory prostanoids such as prostaglandin E2 PGE2 and pras• tafandin 12 could also be involved in causing glomerular hyperfiltration in dia• betes 5859. The enhanced production of PGE2 induced by diabetes and hyperglycemia could be the result of sequential activation of PKC and cyto• solic phospholipase A2 cPLA2 a key regulator of arachidonic acid synthesis 60-63. In the microvessels increases in nitric oxide NO activities a potent vasodilator may also enhance glomerular filtration 64. Urinary excretions of N02/N03 stable metabolites of NO have been reported to be increased in diabetes of short duration 64-66 possibly due to enhanced expression of inducible NO synthase iNOS gene and increased production of NO in mesan• gial cells 67. In addition both increases in iNOS gene expression and NO production can be mimicked by PKC agonist and inhibited by PKC inhibitors when induced by hyperglycemia 67 suggesting that NO production might be increased in diabetes through PKC-induced iNOS overexpression. In addition Graier et al. 68 suggested that NO production was enhanced by the elevation of glucose levels possibly by the increased flux of Ca2+ and its activation of eNOS. However Craven et al. 69 reported that the production of glomerular NO and its second messenger cGMP in diabetic rats in response to choliner• gic agents were decreased and that PKC inhibitors restored the glomerular cGMP production. Several authors also reported that elevated levels of glucose decreased NOS expression in vascular smooth muscle cells and that these effects of glucose were reversed by PKC inhibitors 7071 . Thus PKC can regulate renal hemodynamics by increasing or decreasing NO production dependent on the cell type and tissue location.

slide 298:

PKC Activation and the Effect of Vitam in E 247 In the macrovessels increases in contractility found in diabetes are due to a delay in the relaxation response after contraction induced by cholinergic agents 72- 75. These abnormal responses can also be prevented by PKC inhibitors 76 suggesting that PKC activation plays a general role in causing abnormal peripheral hemodynamics in diabetes. B. Vascular Permeability and Neovascularization Increased vascular permeability is another characteristic systemic vascular ab• normality in diabetic animals in which increased permeability to albumin can occur as early as after 4-6 weeks of diabetes 77 suggesting endothelial cell dysfunctions. PKC activation can directly increase the permeability of albumin and other macromolecules through barriers formed by endothelial cells 7879 and skin chamber granulation tissues 80 probably by phosphorylating cytoskeletal proteins forming the intracellular junctions 8182. Interestingly phorbol ester-induced increases in endothelial permeability may be regulated by PKC p I activation 83 which is consistent with the preferential activation of PKC p isoforms in diabetes. PKC activation could also regulate vascular permeability and neovascu• larization via the expression of growth factors such as the VEGF/vascular permeability factor VPF which is increased in ocular fluids from diabetic patients and has been implicated in the neovascularization process of prolifera• tive retinopathy 49. We reported that both the mitogenic and the permeabil• ity-inducing actions of VEGF/VPF are due in part to the activation of the PKC P isoform via tyrosine phosphorylation of PLC 84. Further in vivo studies in the rat have shown that VEGF-associated increases in retinal vascu• lar permeability are mediated through the PKC pathway 85. Inhibition by the PKC P isoform selective inhibitor L Y33353 l resulted in the decrease of both endothelial cell proliferation and angiogenesis 84 and permeability in• creases induced by VEGF 85. In addition Williams et al. 50 showed that the expression of YEGF was increased in aortic smooth muscle cells by elevat• ing glucose concentration and was inhibited by PKC inhibitors. In the kidney the expression of transforming growth factor-B TGF• P has been shown to be increased in the glomeruli of diabetic patients and experimental animals. Similar increases of TGF-P have also been reported in cultured mesangial cells exposed to high glucose levels 9. Because TGF-P can directly cause the overexpression of extracellular matrix PKC inhibitors have been shown both to inhibit TGF-P expression by hyperglycemia and to prevent the mesangial expansion observed in diabetic nephropathy 7 9 11 .

slide 299:

248 Burselland King C. Na+-K+ ATPase Na+ -K + ATPase an integral component of the sodium pump is involved in the maintenance of cellular integrity and functions such as contractility growth and differentiation 5. It is well established that Na+ -K + ATPase activity is generally decreased in the vascular and neuronal tissues of diabetic patients and experimental animals 586-88. However studies on the mecha• nisms by which hyperglycemia inhibited Na ·1-K + AIPase activity have pro• vided some conflicting results regarding the role of PKC. Phorbol esters activators of PKC have been shown to prevent the inhib• itory effect of hyperglycemia on Na+ -K + ATPase 5 which suggested that PKC activity might be decreased in diabetic conditions. Recently however we showed that elevated glucose level 20 mM will increase PKC and cPLA2 activities leading to increases of arachadonic acid release and PGE2 produc• tion resulting in decreases in Na·1-K• ATPase activity. Inhibitors of PKC or PLA2 prevented glucose-induced reduction in Na•-K+ ATPase activities in aortic smooth muscle cells and mesangial cells 61 . The apparent paradoxical effects of phorbol ester and hyperglycemia are probably due to both the quanti• tative and the qualitative differences of PKC stimulation induced by these stimuli. Phorbol ester which is not a physiological activator can increase the activity of many PKC isoforms and overall PKC activity by 5-10 times whereas hyperglycemia only increases PKC by twofold a physiological rele• vant change 61 that appears to affect only a few isoforms. Thus the results derived from the studies using phorbol esters are difficult to interpret with respect to their physiological significance. D. Extracellular Matrix Components The thickening of the capillary basement membrane is one of the early struc• tural abnormalities observed in almost all the tissues including the vascular system in diabetes 89. Because the basement membrane can affect numerous functions such as structural support vascular permeability cell adhesion pro• liferation differentiation and gene expressions alterations in its components may cause vascular dysfunction 90. Histologically increases in type IV and VI collagen fibronectin and laminin and decreases in proteoglycans are observed in the mesangium of diabetic patients 9192. These observations can be replicated in mesangial cells incubated in media of increasing glucose levels 5-20 mM. These mes• angium changes could be prevented by general PKC inhibitors 93-98. As described above the increased expression of TGF-P has been implicated in the development of mesangial expansion and basement membrane thickening in diabetes 99-104. Ziyadeh et al. 105 106 reported that neutralizing TGF-

slide 300:

PKC Activation and the Effect of Vitamin E 249 j3 antibodies significantly reduced collagen synthesis and gene expression of type IV collagen and fibronectin in the renal cortex of diabetic rats and in cultured mesangial cells exposed to high glucose levels. Because PKC activa• tion can increase the production of extracellular matrix and TGF-j3 expression it is not surprising that several reports have shown that PKC inhibitors can also prevent hyperglycemia- or diabetes-induced increases in extracellular ma• trix and TGF-13 in mesangial cells or renal glomeruli 32. E. Selective PKCjl lsoform Inhibition Numerous studies have used PKC inhibitors such as staurosporine H- 7 and GF109203X to characterize the role of PKC activation in diabetic vascular complications. Long-term studies involving PKC inhibitors however have not been possible due to their toxicity which is associated with their nonspe• cificity with respect to inhibition of other kinases 19 107. Because analyses of retina kidney and cardiovascular tissues from diabetic rats showed that the PKCj3 isoforms were preferentially activated 17 1932 a specific inhibi• tor for the PKCj3 isoforms could potentially be more effective and less toxic than the general isoform nonspecific PKC inhibitors. Recently we reported that increases in albuminuria and abnormal retinal and renal hemodynamics in diabetic rats can be ameliorated by an orally avail• able PKCj3 isofonn selective inhibitor LY33353 I. These physiological changes are concomitant with the inhibition of diabetes-induced PKC activa• tion in retina and renal glomeruli 19. L Y33353 I prevented the overexpres• sion of TGF-j3 a 1 IV collagen and fibronectin in renal glomeruli of diabetic rats 33. These results suggested that activation of PKCj3 isoforms are in• volved in the development of some of the early abnormalities of diabetic vas• cular complications. PKC inhibitors could also mediate their effect by the inhibition of angiotensin actions. Angiotensin action appears to be increased because angiotensin-converting enzyme inhibitors have been shown to delay the progression of nephropathy I 07. However long-term studies are needed to clarify the usefulness of L Y33353 I to prevent the chronic pathological changes of diabetic vascular complications. IV. VITAMIN E AND PKC INHIBITION Oxidative stress has been postulated as an underlying cause of diabetic vascu• lar complications I 08-112. Antioxidants such as vitamin E have been the subject of considerable interest with respect to their potential ability to amelio• rate diabetic complications. There has also been considerable interest in the

slide 301:

250 Bursell and King use of vitamin E as an antioxidant agent for potential beneficial effects in coronary disease and cancers. Results from large multicenter clinical trials are now becoming available. A study on coronary heart disease in women 113 and in men 114 showed that increased vitamin E intake was associated with a significant risk reduction for coronary heart disease. Additionally a recently published study involving male smokers in Finland 115 showed a 32 de• crease in the incidence of prostate cancer in subjects taking 50 mg vitamin E/day. Interestingly in this study there was a 23 increase in prostate cancer in those subjects randomized to -carotene. Clinical studies aimed at charac• terizing the effect of vitamin E in the eye have focused primarily on the poten• tial benefit of vitamin E in age-related macular degeneration 116 I I 7 retini• tis pigmentosa I I 8 and retinopathy of prematurity 119. The results from these studies are suggestive that vitamin E specifically and other antioxidants in general may be beneficial in treating macular degeneration and retinopathy of prematurity. There have been however no clinical studies aimed at investi• gating the effect of vitamin E in diabetes. In the rat retina vitamin E levels were fivefold higher than in other tissues such as the aorta 120. Vitamin E supplementation further increased these retinal vitamin E levels. Other investigators have shown that vitamin E is present in primate and human retinas 121-123 that the regional retinal distribution of vitamin E suggests an antioxidant protective effect against age• related macula degeneration and that the level of vitamin E in the retina corre• lates with serum vitamin E levels 121. Vitamin E in addition to its antioxidant potential has the other interest• ing property of being effective in inhibiting the activation of the DAG-PKC pathway in vascular tissues and cultured vascular cells exposed to high glucose levels 32 120. When retinal vascular endothelial cells exposed to high glu• cose were treated with vitamin E d-a-tocopherol DAG decreased and PKC activation was normalized 32120. We reported that vitamin E can inhibit PKC activation probably by decreasing DAG levels 32 120 because the direct addition of vitamin E to purified PKC a or isoforms in vitro did not have any inhibitory effect 120. These results are consistent with other studies demonstrating that d-a-tocopherol will inhibit PKC activation 124-126. Boscoboinik et al. 124 first demonstrated in 1991 that PKC activation was inhibited by d-a-tocopherol in a manner unrelated to d-a-tocopherols antioxi• dant action 124-127. They also showed that the magnitude of the inhibition was related to the level of PKC activation 128 with little effect of d-o• tocopherol if cellular PKC was not activated. Recently the activation of DAG kinase has been suggested to be one potential site of action for vitamin E to inhibit PKC. Results indicate an indi• rect effect through activation of DAG kinase and increased metabolic break-

slide 302:

PKC Activation and the Effect of Vitamin E 251 down of DAG to phosphatidic acid that resulted in decreased DAG levels and decreased PKC activation 129. Koya et al. 130 confirmed these results in the kidney and showed that glomerular dysfunction in diabetic rats could be prevented by d-a-tocopherol treatment through PKC inhibition most likely mediated through increased DAG kinase activity. In vivo studies in the diabetic rat have shown that the decreased retinal blood flow is related to elevation of retinal DAG levels inhibition of DAG kinase 131 and the activation of PKC 1632 particularly the isoform of PKC 19 131 . The results from these studies showed that the effects of in• creased DAG levels and PKC activation on retinal hemodynamics in nondia• betic rats can mimic the hemodynamic changes measured in untreated diabetic rats. In diabetic rats vitamin E treatment through regular intraperitoneal in• jections prevented the increases in both DAG levels and the activation of PKC in the retina aorta heart and renal glomeruli 120 131. Functionally vitamin E treatment prevented the abnormal hemodynamics in retina and kidney of diabetic rats in parallel with the inhibition of DAG-PKC activation 120 131 . In addition increased albuminuria was prevented by vitamin E treatment in diabetic rats 131 . Normalization of the physiological parameters studied in these diabetic rats was achieved despite chronically maintained elevated blood glucose levels. Thus it is possible that some of the PKC activation induced by diabetes could also be the result of excessive oxidants which are known to activate PKC and can be produced by hyperglycemia leading to the devel• opment of vascular dysfunction in the early stages of diabetes 132. In diabetic patients with no or minimal diabetic retinopathy retinal blood flow was reduced to an extent comparable with that measured in diabetic rats 3537. Studies have shown that the reduction in retinal blood flow in these patients is associated with the level of glycemic control 35. These clinical results combined with prior animal studies provide the support for performing clinical studies aimed at evaluating whether vitamin E treatment is also effective in normalizing retinal blood flow and renal function in patients with IDDM. Additionally multicenter clinical trials will need to be initiated to answer the question of whether high doses of vitamin E can prevent the development of microvascular complications in diabetes. V. SUMMARY The results presented above are consistent with the activation of the DAG• PKC signal transduction pathway in diabetes. The initiating factors are chiefly metabolic with hyperglycemia as the main triggering element. Other metabolic

slide 303:

252 Bursell and King changes such as those associated with free fatty acids are also potentially in• volved. The finding that the secondary metabolic products of glucose such as glycation products and oxidants can also increase DAG-PKC suggest that the activation of DAG-PKC could be a common downstream mechanism by which multiple byproducts of glucose are exerting their adverse effects. It is not surprising that changes in the DAG-PKC pathway can play a role in dia• betic microvascular complications as this signal transduction pathway is known to regulate many vascular actions and functions as described above 11 12. It is also likely that hyperglycemia and diabetes may affect other signal transduction pathways besides the DAG-PKC pathway because a num• ber of these other pathways can also regulate vascular functions. Hyperglycemia or diabetes has also been associated with the activation of more than one PKC isoform. Again this is not surprising because many isoforms are DAG sensitive and each cell usually contains several PKC iso• forms 11 12. However it is surprising that the results of immunoblotting and the use of PKC isoform inhibitor appear to suggest that PKC isoforms a.re predominantly activated in all vascular tissues and may be responsible for many of the vascular dysfunctions. The correlation between the activation of DAG-PKC and diabetic vas• cular and neurological complications are substantial in rodent models of diabe• tes 7 8 17-1931 however limited data are available to indicate that DAG• PKC levels are increased in the vasculature of diabetic patients. This is primar• ily due to the difficulty of obtaining fresh human vascular or neurological tissues for the measurement of DAG-PKC levels. Thus further studies are needed to confirm whether DAG-PKC activation plays a role in the develop• ment of diabetic complications. First the activation of the DAG-PKC path• way needs to be chronically inhibited in a long-term animal model of diabetes to demonstrate which of the various retinal and renal pathologies can be pre• vented. Long-term experiments to chronically inhibit PKC can be accom• plished through the use of specific PKC isoform inhibitors or by characterizing the pathologic changes in transgenic mice strains lacking a specific PKC iso• form. These experimental approaches are now possible because a specific and relatively nontoxic oral inhibitor of PKC isoforms is now available and can be used to test which of the vascular dysfunctions are due to PKC isoform activation 19. Second most of the reported findings to date have been performed in animal tissues and not in human vascular tissues. Thus there may be differ• ences between human and animal vascular tissue responses in relation to glu• cose metabolism and PKC isoform expression. A PKC isofonn inhibitor will only be useful in diabetic patients if the same profile of PKC isoforms are

slide 304:

PKC Activation and the Effect of Vitamin E 253 activated or expressed in diabetic patients as in the diabetic rodent models. In addition the secondary markers of PKC activation need to be identified because they can be used to monitor the effectiveness of PKC inhibition when treated with intensive glycemic control or with PKC inhibitors. Progress has been made to identify some of these potential secondary parameters of vascu• lar pathologies such as the levels of VEGF changes in retinal hemodynamics and endothelial cell function 40495061. The most important requirement for determining the role of activation of the DAG-PKC pathway in the vascular complication of diabetic patients has to be clinical trials using specific PKC isoform inhibitors. These trials are now in progress specifically with the orally available PKCP inhibitor. The need for clinical trials is vital as multiple agents have been shown to be capable of reversing vascular abnormalities induced by hyperglycemia in rodent mod• els of diabetes. None of these agents however has been shown to be effective in clinical trials 133134 clearly indicating the difficulties in extrapolating results to humans from those obtained using rodent models for diabetic com• plications. An additional potential problem with any therapeutic PKC inhibitor used clinically is the issue of toxicity because PKC activation is involved in so many vital functions of the cell. This is especially true in the clinical arena as these agents can be used by patients over long periods of time. Thus a large body of evidence has suggested that the activation of the DAG-PKC pathway by hyperglycemia and diabetes plays a role in the devel• opment of some vascular dysfunctions and neurovascular changes noted in diabetes. However definitive studies as described above are ongoing and should determine clearly the role of DAG-PKC in the development of the various complications of diabetic patients. REFERENCES I. Brownlee M Cerami A Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988 318:1315-1321. 2. Vlassara H. Advanced glycation end-products and atherosclerosis. Ann Med 1996 28:419-426. 3. Schmidt AM Hori 0 Cao R Yan SD Brett J Wautier JL Ogawa S Kuwabara K Matsumoto M Stern D. RAGE: a novel cellular receptor for advanced glyca• tion end products. Diabetes 1996 45:S77-S80. 4. Kaiser N Sasson S Feener EP Boukobza-Vardi N Higashi S Moller DE Davidheiser S Przybylski RJ King GL. Differential regulation of glucose

slide 305:

254 Bursell and King transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes 1993 42:80-89. 5. Greene D Lattimer SA Sima AAF. Sorbitol phosphoinositides and sodium• potassium-ATPase in the pathogenesis of diabetic complications. N Engl J Med 1987: 316:599-606. 6. Williamson JR Chang K Frangos M Hasan KS Ido Y Kawamura T Nyen• gaard JR Van den Enden M Kilo C Tilton RG. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993 42:801-813. 7. King GL Ishii H Koya D. Diabetic vascular dysfunctions: a model of excessive activation of protein kinase C. Kidney Int 1997 52:S77-S85. 8. Derubertis FR Craven PA. Activation of protein kinase C in glomerular cells in diabetes. Mechanisms and potential link to the pathogenesis of diabetic glo• merulopathy. Diabetes 1994 43: 1-8. 9. Sharma K Ziyadeh FN. Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-ji as a key modulator. Diabetes 1995 44: 1139- 1146. I 0. Baynes JW Thorpe SR. The role of oxidative stress in diabetic complications. CmT Opin Endocrinol 1996 3 :277-284. 11. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activa• tion of protein kinase C. Science 1992 258:607-614. 12. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular re• sponses. FASEB J 1995 9:484-496. 13. Liscovitch M Cantley LC. Lipid second messengers. Cell 1994: 77:329-334. 14. Nakanishi H Brewer KA Exton JH. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 345-trisphosphate. J Biol Chem 1993 268: 13-16. 15. Chang EY Szallasi Z. Acs P Raizada V Wolfe PC Fewtrell C Blumberg PM Rivera J. Functional effects of overexpression of protein kinase C-alpha - beta -delta -epsilon and -eta in the mast cell line RBL-2H3. J Irnmunol 1997 159:2624-2632. 16. Shiba T Inoguchi T Sportsman JR Heath W Bursell S King GL. Correlation of diacylglycerol and protein kinase C activity in rat retina to retinal circulation. Am J Physiol 1993 265:E783-E793. 17. Inoguchi T Battan R Handler E Sportsman JR Heath W King GL. Preferen• tial elevation of protein kinase C isoform j3JI and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility of glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 1992 89: 11059-11063. 18. Craven PA Davidson CM DeRubertis FR. Increase in diacylglycerol mass in isolated glomeruli by glucose from de novo synthesis of glycerolipids. Diabetes 1990 39:667-674. 19. Ishii H Jirousek MR Koya D Takagi C Xia P. Clermont A Bursell S-E Kem TS Ballas LM Heath WF Stramm LE Feener EP King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC j3 inhibitor. Science 1996 272:728- 731. 20. Considine RV Nyce MR Allen LE Morales LM Triester S Serrano J Colberg

slide 306:

PKC Activation and the Effect of Vitamin E 255 J Lanza-Jacoby S Caro JF. Protein kinase C is increased in the liver of humans and rats with non-insulin-dependent diabetes mellitus: an alteration not due to hyperglycemia. J Clin Invest I 995 95:2938-2944. 21. Saha AK Kurowski TG Colca JR Ruderman NB. Lipid abnormalities in tis• sues of the KKAy mouse: effects of pioglitazone on malonyl-CoA and diacyl• glycerol. Am J Physiol 1994 267:E95-EIOI. 22. Xia P lnoguchi T Kern TS. Engerman RL Oates PJ King GL. Characteriza• tion of the mechanism for the chronic activation of diacylglycerol-protein ki• nase C pathway in diabetes and hypergalactosemia. Diabetes 1994 43: I I 22- 1129. 23. Craven PA DeRubertis FR. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. J Clin Invest I 989 83: 1667- I 675. 24. Yasanare K Kahno M Kano H Yokokawa K Horio T Yoshikawa J. Possible involvement of phospholipase and protein kinase C in vascular growth induced by elevated glucose concentration. Hypertension I 996 28: I 59- I 68. 25. lnoguchi T Pu X Kunisaki M Higashi S Feener EP King GL. Insulins effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vas• cular tissues. Am J Physiol I 994 267:E369-379. 26. Berne C. The metabolism of lipids in mouse pancreatic islets. The biosynthesis of triacylglycerol and phospholipids. Biochem J 1975 152:667-673. 27. Dunlop ME Larkins RG. Pancreatic islets synthesize phospholipids de novo from glucose via acyl-dihydroxyacetone phosphate. Biochem Biophys Res Commun 1985 132:467-473. 28. Brindley DN Sturton RG. Phosphatidate metabolism and its relation to triacyl• glycerol biosynthesis. In: Hawthorne JN Ansel GB eds. Phospholipids. New York: Elsevier 1982:179-207. 29. Holub BJ Kuksis A. Metabolism of molecular species of diacylglycerol phos• pholipids. Adv Lipid Res 1986 16:1-125. 30. Ido Y McHowat J Chang KC Arrogori-Martelli E Orfaliam Z Kilo C Corr PB Williamson JR. Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L carritine. Diabetes I 994 43: I 469-1477. 31. Ayo SH. Radnik R Garoni JA Troyer DA Kreisberg JI. High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures. Am J Physiol 199 I 26I:F571-577. 32. Kunisaki M Bursell S-E Umeda F Nawata H King GL. Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabe• tes 1994 43: 1372- I 377. 33. Koya D Jirousek MR Lin Y-W Ishii H Kubok.i K King GL. Characteristics of protein kinase C isoform activation on the gene expression of transforming growth factorB extracellular matrix components and prostanoids in the glomer• uli of diabetic rats. J Clin Invest 1997 I 00: 115-126. 34. Kikkawa R Haneda M Uzu T Koya D Sugimoto T Shigeta Y. Translocation of protein kinase a and in rat glomerular mesangial cells cultured under high glucose conditions. Diabetologia 1994 37:838-841.

slide 307:

256 Bursell and King 35. Bursell S-E Clermont AC Kinsley BT Simonson DC Aiello LM Wolpert HA. Retinal blood flow changes in patients with insulin-dependent diabetes mellitus and no diabetic retinopathy. lnvest Ophthalmol Vis Sci 1996 37:886- 897. 36. Feke GT Buzney SM Ogasawara H Fujio N Goger DG Spack NP Gabbay KH. Retinal circulatory abnormalities in type I diabetes. Invest Ophthalmol Vis Sci 1994 35:2968-2975. 37. Clermont AC Aiello LP Mori F Aiello LM Bursell SE. Vascular endothelial growth factor and severity of nonproliferative diabetic retinopathy mediate reti• nal hemodynamics in vivo: a potential role for vascular endothelial growth fac• tor in the progression of nonproliferative diabetic retinopathy. Am J Ophthal• mol 1997 124:433-446. 38. Konno S Feke GT Yoshida A Fujio N Goger DG Buzney SM. Retinal blood flow changes in type I diabetes: a long term follow-up study. Invest Ophthamol Vis Sci 1996 37: 1140-1148. 39. Small KW Stefansson E Hatchell D. Retinal blood flow in normal and diabetic dogs. Invest Ophthalmol Vis Sci 1987 28:672-675. 40. Clermont AC Brittis M Shiba T McGovern T King GL Bursell S-E. Normal• ization of retinal blood flow in diabetic rats with primary intervention using insulin pumps. Invest Ophthalmol Vis Sci 1994 35:981-990. 41. Miyamoto K Ogura Y Nishiwaki H Matsuda N Honda Y Kato S Ishida H Seino Y. Evaluation of retinal microcirculatory alterations in the Goto-Kakizaki rat. Invest Ophthalmol Vis Sci 1996 37:898-905. 42. Takagi C King GL Clermont AC Cummins DR Takagi H Bursell SE. Rever• sal of abnormal retinal hemodynamics in diabetic rats by acarbose an alpha• glucosidase inhibitor. Curr Eye Res 1995 14:741- 749. 43. Higashi S Clermont AC Dhir V Bursell SE. Reversibility of retinal flow ab• normalities is disease-duration dependent in diabetic rats. Diabetes 1998 47: 653-659. 44. Kohner EM. Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy. Diabetes 1995 44:603-607. 45. Tesfayes S Malik R Ward JD. Vascular factors in diabetic neuropathy. Diabe• tologia 1994 37:847-851. 46. Cameron NE Cotter MA Lai K Hohman TC. Effect of protein kinase C inhibi• tion on nerve function blood flow and Na +K + ATPase defects in diabetic rats. Diabetes 1997 46suppl 1:31A-0121. 47. Takagi C Bursell S-E Lin Y-W Takagi H Duh E Jiang Z Clermont AC King GL. Regulation of retinal hemodynamics in diabetic rats by increased expression and action of endothelin-1. Invest Ophthalmol Vis Sci 1996 37: 2504-2518. 48. Takagi C King GL Takagi H Lin Y-W Clermont AC Bursell SE. Endothe• lin-1 action via endothelin receptors is a primary mechanism modulating retinal circulatory response to hyperoxia. Invest Ophthalmol Vis Sci 1996 37: 2099-2109.

slide 308:

PKC Activation and the Effect of Vitamin E 257 49. Aiello LP Avery RL Arrigg PG Keyt BA Jampel HD Shah ST Pasquale LR Thieme H Iwamoto MA Park JE Nguyen HY Aiello LM Ferrara N King GL. Vascular endothelial growth factor in ocular fluids of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994 331: 1480- 1487. 50. Williams 8 Gallachen 8 Patel H Orme C. Glucose-induced protein kinase C activation regulates vascular permeability factor mRNA expression and peptide production vascular smooth muscle cells in vitro. Diabetes 1997 46: 1497- 1503. 51. Ditzel J Schwartz M. Abnormally increased glomerular filtration rates in short• term insulin treated diabetic subjects. Diabetes 1967 16:264-267. 52. Christiansen JS Gammelgaard J Frandsen M Parving HH. Increased kidney size. glomerular filtration rate and renal plasma flow in short-term insulin• dependent diabetes. Diabetologia 1981 20:451-456. 53. Hostetter TH. Troy JL Brenner BM. Glomerular hemodynamics in experimen• tal diabetes mellitus. Kidney Int 1981 19:410-415. 54. Viberti GC. Early functional and morphological changes in diabetic nephropa• thy. Clin Nephrol 1979 12:47-53. 55. ODonnell MP Kusiske BL Keane WF. Glomerular hemodynamic structural alterations in experimental diabeties mellitus. FASEB J 1988 2:2339- 2347. 56. Anderson S Jung FF Ingelfinger JR. Renal renin-angiotensin system in diabe• tes: functional immunohistochemical and molecular biologica correlations. Am J Physiol 1993 265:F477-F486. 57. Ruan X. Arendshorst WJ. Role of protein kinase C in angiotensin 11-induced renal vasoconstriction in genetically hypertensive rats. Am J Physiol 1996 270: F945-F952. 58. Craven PA Caines MA DeRubertis FR. Sequential alterations in glomerular prostaglandin and thromboxane synthesis in diabetic rats: relationship to the hypertiltration of early diabetes. Metabolism 1987 36:95-103. 59. Perico N Benigni A Gabanelli M Piccinelli A Rog M Riva CD Remuzzi G. Atrial natriuretic peptide and prostacyclin synergistically mediate hyperfil• tration and hyperperfusion of diabetic rats. Diabetes 1992 41 :533-538. 60. Williams 8 Schrier RW. Glucose-induced protein kinase C activity regulates arachidonic acid release and eicosanoid production by cultured glomerular mes• angial cells. J Clin Invest 1993 92:2889-2896. 61. Xia P Kramer RM King GL. Identification of the mechanism for the inhibition of Na " K + -adenosine triphosphatasc by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A: J Clin Invest 1995 96:733- 740. 62. Haneda M Arakis I Togaro M Sugimoto T Isono M Kikkawa R. Mitogen activated protein kinase C is activated in glomeruli of diabetic rats and glomeru• lar mesangial cells cultured under high glucose conditions. Diabetes 1997 46: 847-853.

slide 309:

258 Bursell and King 63. Lin L-L Wartmann M Lin A Y Knopf JL Seth A Davis RJ. cPLA2 is phos• phorylated and activated by MAP kinase. Cell 1993 72:269-278. 64. Bank N Aynedjian HS. Role of EDRF nitric oxide in diabetic renal hyperfil• tration. Kidney Int 1993 43: 1306-1312. 65. Tolins JP Shultz PJ Raij L Brown DM Mauer SM. Abnormal renal hernody• namic response to reduced renal perfusion pressure in diabetic rats: role of NO. Am J Physiol 1993 265:F886-F895. 66. Komers R Allen TJ Cooper ME. Role of endothelium-derived nitric oxide in the pathogenesis of the renal hemodynamic changes of experimental diabetes. Diabetes 1994 43:1190-1197. 67. Sharma K Danoff TM DePiero A Ziyadeh FN. Enhanced expression of induc• ible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: possible mediation via protein kinase C. Bio• chem Biophys Res Commun 1995 207:80-88. 68. Graier WF Simecek S Kukovetz WR Kostner GM. High D-glucose-induced changes in endothelial Ca21 /EDRF signaling are due to generation of superox• ide anions. Diabetes 1996 45:1386-1395. 69. Craven PA Studer RK DeRubertis FR. Impaired nitric oxide-dependent cyclic guanosine monophosphate generation in glomeruli from diabetic rats. J Clin Invest I 994 93:311-320. 70. Muniyappa R Srinivas PR Ram JL Walsh MF Sowers JR. Calcium and pro• tein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells. Hypertension 1998 31 I Pt 2:289- 295. 71. Nishio E Watanabe Y. Glucose-induced down-regulation of NO production and inducible NOS expression in cultured rat aortic vascular smooth muscle cells: role of protein kinase C. Biochem Biophys Res Commun 1996 229:857- 863. 72. Kamala K Miyata N Kasuya Y. Involvement of endothelial cells in relaxation and contraction responses of the aorta to isoproterenol in native and streptozo• tocin-induced diabetic rats. J Pharmacol Exp Ther 1989 249:890-894. 73. Mayhan WG. Impairment of endothelium-dependent dilatation cerebral arteri• oles during diabetes mellitus. Am J Physiol 1989 256:H621-H625. 74. Tesfamariam B Jakubowski JA Cohen RA. Contraction of diabetic rabbit a011a caused by endothelium-derived PGH2-TxA2• Am J Physiol 1989 257:Hl327- Hl333. 75. McVeigh GE Brennan GM Johnston GD McDermott BJ McGrath LT Henry WR Andrews JW. Impaired endothelium-dependent and independent vasodila• tion in patients with Type 2 non-insulin-dependent diabetes mellitus. Diabeto• logia 1992 35:771-776. 76. Tesfamariam B Brown ML Cohen RA. Elevated glucose impairs endothelium• dependent relaxation by activating protein kinase C. J Clin Invest 1991 87: 1643-1648. 77. Williamson JR Chang K Tilton RG Prater C Jeffrey JR Weigel C Sherman

slide 310:

PKC Activation and the Effect of Vitamin E 259 WR Eades DM Kilo C. Increased vascular permeability in spontaneously dia• betic BB/W rats and in rats with mild versus severe streptozotocin-induced diabetes. Diabetes 1987 36:813-821. 78. Oliver JA. Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions. J Cell Physiol 1990 145:536-542. 79. Lynch JJ Ferro TJ Blumenstock FA Brockenauer AM Malik AM. Increased endothelial albumin permeability mediated by protein kinase C activation. J Clin Invest 1990 85: 1991-1998. 80. Wolf BA Williamson JR Easom RA Chang K Sherman WR Turk J. Diacyl• glycerol accumulation and microvascular abnormalities induced by elevated glucose levels. J Clin Invest 1991 87:31-38. 81. Werth DK Niedel JE Pastan I. Vinculin a cytoskeletal substrate of protein kinase C. 1 Biol Chem 1983 258:11423-11426. 82. Stasek JE Patterson CE Garcia JGN. Protein kinase C phosphorylates caldes• mon- and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. 1 Cell Physiol 1992 153:62- 75. 83. Nagpala PG Malik AB Vuong PT Lum H. Protein kinase C p1 overexpression augments phorbol ester-induced increase in endothelial permeability. J Cell Physiol 1996 166:249-255. 84. Xia P Aiello LP Ishii H Jiang Z Park DJ Robinson GS Takagi H Newsome WP Jirousek MR King GL. Characterization of vascular endothelial growth factors effect on the activation of protein kinase C its isoforrns and endothelial cell growth. J Clin Invest 1996 98:2018-2026. 85. Aiello LP Bursell SE Clermont A Duh E Ishii H Takagi C Mori F Ciulla TA Ways K Jirousek M Smith LE King GL. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective P-isoform-selective inhibitor. Diabetes 1997 46: 1473-1480. 86. King GL Shiba T Oliver J Inoguchi T Bursell S-E. Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus. Annu Rev Med 1994 45: 179-188. 87. Winegrad AI. Does a common mechanism induce the diverse complications of diabetes Diabetes 1987 36:396-406. 88. MacGregor LC Matschinsky FM. Altered retinal metabolism in diabetes II: measurement of sodium-potassium ATPase and total sodium and potassium in individual retinal layers. J Biol Chem 1986 261 :4052-4058. 89. Williamson JR Kilo C. Extracellular matrix changes in diabetes mellitus. In: Scarpelli DG Migahi G eds. Comparative pathobiology of major age-related diseases. New York: Liss 1984:269-288. 90. Lu TT Yan LG Madri JA. Integrin engagement mediates tyrosine dephosphor• ylation on platelet-endothelial cell adhesion molecule I. Proc Natl Acad Sci USA 1996 93: 11808-11813. 91. Scheinman JL. Fish AJ Matas AJ Michael AF. The immunohistopathology of

slide 311:

260 Bursell and King glomerular antigens. II. The glomerular basement membrane actomyosin and fibroblast surface antigens in normal diseased and transplanted human kidneys. Am J Pathol 1978 90:71-88. 92. Bruneval P Foidart JM Nochy D Camilleri JP Bariety J. Glomerular matrix proteins in nodular glomerulosclerosis in association with light chain deposition disease and diabetes mellitus. Hum Pathol 1985 16:477-484. 93. Studer RK Craven PA DeRubertis FR. Role for protein kinase C in the media• tion of increased fibronectin accumulation by mesangial cells grown in high• glucose medium. Diabetes 1993 42: 118-126. 94. Ayo SH Radnik RA Garoni J Glass II WF Kreisberg JI. High glucose causes an increase in extracellular matrix proteins in cultured mesangial cells. Am J Pathol 1990 136:1339-1348. 95. Ayo SH Radnik RA Glass II WF Garoni JA Rampt ER Appling DR Kreis• berg JI. Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Am J Physiol 1991 260:Fl85-Fl91. 96. Haneda M Kikkawa R Horide N Togawa M Koya D Kajiwara N Ooshima A Shigeta Y. Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 1991 34: 198-200. 97. Pugliese G Pricci F Pugliese F Mene P Lenti L Andreani D Galli G Casini A Bianchi S Rotella CM. Mario UD. Mechanisms of glucose-enhanced extra• cellular matrix accumulation in rat glomerular mesangial cells. Diabetes 1994 43:478-490. 98. Furno P. Kuncio GS Ziyadeh FN. PKC and high glucose stimulate collagen cx:1 IV transcriptional activity in a reporter mesangial cell line. Am J Physiol 1994 267:F632-F638. 99. MacKay K Striker LJ Stauffer JW Doi T Agodoa LY Striker GE. Trans• forming growth factor p. Murine glomerular receptors and responses of isolated glomerular cells. J Clin Invest 1989 83:1160-1167. 100. Suzuki S Ebihara I Tomino Y. Koide H. Transcriptional activation of matrix genes by transforming growth factor beta I in mesangial cells. Exp Nephrol 1993: I :229-237. IO I. Nakamura T Miller D Ruoslahti E Border WA. Production of extracellular matrix by glomerular epithelial cells regulated by transforming growth factor• p . Kidney Int 1992 41:1213-1221. 102. Yamamoto T Nakamura T Noble NA Ruoslahti E Border WA. Expression of transforming growth factor p is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci USA 1993 90: 1814-1818. 103. Sharma K Ziyadeh FN. Renal hypertrophy is associated with upregulation of TGF-P I gene expression in diabetic BB rat and NOD mouse. Am J Physiol 1994 267:FI094-Fl 101. 104. Pankewycz OG Guan J-X Bolton WK Gomez A Benedict JF. Renal TGF• p regulation in spontaneously diabetic NOD mice with correlations in mesan• gial cells. Kidney Int 1994 46:748- 758. 105. Ziyadeh FN Sharma K Ericksen M Wolf G. Stimulation of collagen gene

slide 312:

PKC Activation and the Effect of Vitamin E 261 expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-B. J Clin Invest 1994 93:536-542. 106. Sharma K Jin Y Guo J Ziyadeh FN. Neutralization of TGF- by anti-TGF• antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 1996 45:522- 530. 107. Lewis EJ Hunsicker LG Bain RP Rohde RD. The effect of angiotensin con• verting enzyme inhibitor on diabetic nephropathy. N Engl J Med 1993 329: 1456-1462. 108. Baynes JW Thorpe SR. The role of oxidative stress in diabetic complications. Curr Opin Endocrinol 1996 3:277-284. 109. Giugliano D Cerellio A Paolisso G. Diabetes mellitus hypertension and car• diovascular disease: which role for oxidative stress. Metabolism 1995 44:363- 368. I IO. Giugliano D Cerellio A Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996 19:257-265. l l I. Asahina T Kashiwagi A Yoshihiko N et al. Impaired activation of glucose oxidation and NADH supply in human endothelial cells exposed to H02 in high glucose medium. Diabetes 1995 44:520-526. 112. Chappey 0 Dosquet C Wautier MP Wautier JL. Advanced glycation end products oxidant stress and vascular lesions. Eur J Clin Invest 1997 27:97- 108. 113. Stampfer MJ Hennekens CH Manson JE Colditz GA Rosner B Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993 328:1444-1449. 114. Rimm EB Stampfer MJ Ascherio A Giovannucci E Colditz GA Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993 328:1450-1456. 115. Heinonen OP Albanes D Virtamo J et al. Prostate cancer and supplementation with u-tocopherol and -carotene: incidence and mortality in a controlled trial. J Natl Cancer Inst 1998 90:440-446. 116. Mares-Perlman JA Brady WE Klein R et al. Serum antioxidants and age• related macular degeneration in a population-based case-control study. Arch Ophthalmol 1995 113:1518-1523. 117. Snodderly OM. Evidence for protection against age-related macular degenera• tion by carotenoids and antioxidant vitamins. Am J Clin Nutr 1995 62: 1448S- 1461S. 118. Berson EL Rosner B Sandberg MA et al. A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa. Arch Ophthalmol 1993 111:761-772. 119. Johnson L Quinn GE Abbasi S Gerdes J Bowen FW. Bhutani V. Severe retinopathy of prematurity in infants with birth weights less than 1250 grams: incidence and outcome of treatment with pharmacologic serum levels of vita-

slide 313:

262 Bursell and King min E in addition to cryotherapy from 1985 to 1991. J Pediatr 1995 127:632- 639. 120. Kunisaki M Bursell S-E Clermont AC Ishii H Ballas LM Jirousek MR Umeda F Nawata H King GL. Vitamin E prevents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am J Phys• iol 1995 269:E239-E246. 121. Crabtree DV Snodderly DM Adler AJ. Retinyl palmitate in macaque retina• retinal pigment epithelium-choroid: distribution and correlation with age and vitamin E. Exp Eye Res 1997 64:455-463. 122. Crabtree DV Adler AJ Snodderly DM. Radial distribution of tocopherols in rhesus monkey retina and retinal pigment epithelium-choroid. Invest Ophthal• mol Vis Sci 1996 37:61-76. 123. Friedrichson T Kalbach HL Buck P van Kuijk FJ. Vitamin E in macular and peripheral tissues of the human eye. Curr Eye Res 1995 14:693- 701. 124. Boscoboinik D Szewczyk A Hensey C Azzi A. Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C. J Biol Chem 1991 266:6188- 6194. 125. Boscoboinik DO Chatelain E Bartoli G-M Stauble B. Azzi A. Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-a.• tocopherol. Biochim Biophys Acta 1994 1224:418-426. 126. Ozer NK Palozza P Boscoboinik D Azzi A. d-a.-Tocopherol inhibits low den• sity lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells. FEBS Lett 1993 322:307-310. 127. Boscoboinik D Ozer NK Moser U Azzi A. Tocopherols and 6-hydroxy-chro• man-2carbonitrile derivatives inhibit vascular smooth muscle cell proliferation by a nonantioxidant mechanism. Arch Biochem Biophys 1995 318:241- 246. 128. Tasinato A Boscoboinik D Bartoli GM Maroni P Azzi A. d-a.-Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations correlates with protein kinase C inhibition and is independent of its antioxidant properties. Proc Natl Acad Sci USA 1995 92: 12190- 12194. 129. Tran K Proulx PR Chan AC. Vitamin E suppresses diacylglycerol DAG level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity. Biochim Biophys Acta 1994 1212:193-202. 130. Koya D Lee 1-K Ishii H Kanoh H King GL. Prevention of glomerular dys• functions in diabetic rats by treatment of d-o-tocopherol. J Am Soc Nephrol 1997 8:426-35. 131. Bursell S-E Takagi C Clermont AC et al. Specific retinal DAG and PKC- isoform modulation mimics abnormal retinal hemodynamics in diabetic rats. Invest Ophthalmol Vis Sci 1997 38:2711-2720. 132. Konishi H Tanaka M Takemura Y Matsuzaki H Ono Y Kikkawa U Nishi• zuka Y. Activation of protein kinase C by tyrosine phosphorylation in response to H202. Proc Natl Acad Sci USA 1997 94:11233-11237.

slide 314:

PKC Activation and the Effect of Vitamin E 263 133. Po11e D Jr Schwartz MW. Diabetes complications: why is glucose potentially toxic Science 1996 272:699- 700. 134. Nicolucci A Carinci F Cavaliere D Scorpiglione N Belfiglio M Labbrozzi D Mari E Benedetti MM Tognoni G Liberati A. A meta-analysis of trials on aldose reductase inhibitors in diabetic peripheral neuropathy. The Italian Study Group. The St. Vincent Declaration. Diabetes Med 1996 13:1017- 1026.

slide 315:

This Page Intentionally Left Blank

slide 316:

17 Oxidative Stress and Pancreatic 3-Cell Destruction in Insulin • Dependent Diabetes Mellitus To Get Best Natural Diabetes Treatment Click Here Mizuo Hotta Eiji Yamato and Jun-ichi Miyazaki Osaka University Medical School Osaka Japan Insulin-dependent diabetes mellitus IDDM is considered to be an autoim• mune disease l 2. Recent reports suggest that reactive oxygen species ROS participate in the development of IDDM 34 . Thioredoxin TRX is a small l 2 kDa reduction/oxidation redox protein 56 and has protective effects on cells against oxidative stress by scavenging ROS 5- 7 by repairing DNA and proteins damaged by ROS 5-8 and by blocking apoptosis induced by ROS 69. Nonobese diabetic NOD mice are well known as an excellent ani• mal model for human IDDM l 0-12. To elucidate the roles of oxidative stress in autoimmune diabetes we generated NOD transgenic mice overex• pressing TRX exclusively in pancreatic -cells. Spontaneous diabetes was prevented or delayed in the NOD transgenic mice. The results indicate that ROS in pancreatic -cells may play an essential role in the development of IDDM. Here we describe the proposed mechanisms of the -cell destruction by ROS in IDDM and the protective effects of the TRX system against -cell destruction.

slide 317:

265

slide 318:

266 Hotta et al. I. PATHOGENESIS OF IDDM PATIENTS AND NOD MICE IDDM is caused by autoimmune destruction of pancreatic P-cells 12. Be• cause P-cells are almost completely destroyed at clinical onset of this disease the affected individuals require daily injection of insulin to prevent diabetic ketoacidosis and to sustain their Jives. NOD mice spontaneously develop autoimmune diabetes I 0-12 after the infiltration of inflammatory cells into pancreatic islets termed "insulins" and are known as an excellent animal model for studying human IDDM 10- 12. Infiltrating cells are composed of T and B lymphocytes macrophages Ml and natural killer NK cells. The P cells are thought to be destroyed by these immunocytes directly or via cytotoxic cytokines such as interleukin• IL-I tumor necrosis factor-aTNF-a and interferon- 34 l 3 . II. OXIDATIVE STRESS AND IDDM Recent studies have indicated that ROS such as nitric oxide NO superoxide anion radical Oi and hydrogen peroxide H202 are generated by Mt in islets or induced in P-cells by cytotoxic cytokines secreted from immunocytes 34 Fig. 1 . lt has been shown that the expression of inducible nitric oxide Cytokines IL·1 IFN·y TNF·a Streptozotocin Alloxan Pancreatic cell ROS NO 02· H202 etc. p cell death Figure 1 A proposed mechanism of pancreatic -cell destruction by oxidative stress in autoimmune diabetes and drug-induced diabetes.

slide 319:

Oxidative Stress and Pancreatic p-Cell Destruction 267 synthase iNOS is augmented in P-cells of NOD mice 14. It has also been suggested that NO was induced by iNOS in the P-cells of human IDDM pa• tients 15. These data indicate that oxidative stress may be one of the effector mechanisms of P-cell destruction by infiltrating inflammatory cells in autoim• mune diabetes. It has previously been suggested that pancreatic P-cells are especially vulnerable to oxidative stress. In fact P-cells are selectively destroyed by ROS-generating agents streptozotocin STZ and alloxan 16-18. Such sus• ceptibility to ROS is probably due to low levels of key enzymes scavenging ROS such as superoxide dismutase SOD catalase and glutathione peroxi• dase 19-22. Overexpression of Drosophila Cu/Zn SOD in p cells has been shown to confer resistance to alloxan-induced diabetes 23. Antioxidative agents such as nicotinamide 2425 and vitamin E 26 have been shown to have protective effects against diabetes of NOD mice. Recently it was re• ported that transgenic mice overexpressing iNOS in P cells develop insulin• dependent diabetes without insulitis 27. These results suggest that ROS and the antioxidative systems in P-cells play pivotal roles in the destruction of P• cells in the development of autoimmune diabetes. Ill. OXIDATIVE STESS AND APOPTOSIS Recent histochemical analysis suggested that apoptosis is the mechanism of P-cell destruction in autoimmune diabetes 2829. The Fas/Fas ligand Fasl. system is profoundly related to apoptosis 30 Fig. 2. Transgenic mice over• expressing FasL in 3-cells were reported to be highly susceptible to autoim• mune diabetes 31 whereas Fas deficient lpr/lpr mice were resistant to auto• immune diabetes 3132. Fas/Fasl system was also suggested to mediate P-cell destruction in human IDDM patients 15. NF-KB has been implicated in apoptosis although the functions of NF• KB are complicated and there is controversy about the functions of NF-KB in apoptosis 3334. It has recently been reported that NF-KB is induced by IL- I in rat islets and RINm5F cells 35. Because NF-KB is known to activate iNOS 3334 these results indicate that excessive NO may be generated by IL- I 36 via activation of NF-KB 3334 resulting in P-cell destruction possibly due to apoptosis 37. IV. TRX AND ANTIOXIDATIVE EFFECTS TRX is a small reduction/oxidation redox protein present in both prokaryotic and eukaryotic cells 56. In human this protein was found as an adult T-

slide 320:

----lf-1----4 ..:i i....--4z 1----4 LL atic NFKB t iNOS t ROS NO 02-. H202 etc. 268 Hotta et al. IL-1 IFN-y TNF-a Fasl Pancre p cell p cell death/apoptosis Figure 2 A proposed mechanism of pancreatic -cell death/apoptosis by cytotoxic cytokines in autoimmune diabetes. cell leukemia-derived factor 6. TRX is induced by various types of stress such as viral infection ischemic insult ultraviolet light x-ray irradiation and H202 56. Recently TRX has been shown to have protective effects on cells against oxidative stress. TRX itself can function as an ROS scavenger 67. TRX originally studied as a cofactor of ribonucleotide reductase in DNA synthesis has been revealed to be a substrate of the antioxidative and antiapop• totic enzyme TRX peroxidase 38. Recent studies have indicated that TRX plays essential roles in the repair of DNA and proteins damaged by ROS 5- 839-43. Augmented expression of TRX is often seen in neoplastic cells 5644- 46 and appears to protect neoplastic cells against the cytotoxicity of ROS• generating antineoplastic agents such as cis-diaminedichloroplatinum II adriamycin etoposide and mitomycin C 694546. Recombinant TRX pre• vents cell death induced by ROS 6. TRX also protects cells against apoptosis induced by TNF and Fas-agonistic antibody 647. A number of in vitro stud-

slide 321:

Oxidative Stress and Pancreatic p-Cell Destruction 269 ies have suggested that the TRX system plays an important role in the protec• tion of cells against the cytotoxicity of oxidative stress 6944-48. V. TAX AS A REGULATOR OF INTRACELLULAR SIGNALING AGAINST OXIDATIVE STRESS Recent reports revealed that TRX also functions as a regulator of intracellular signalings 6. NF-KB 64950 and Ref- l 6843 which have TRX binding sites are regulated by TRX. Ref-I regulates activator factor AP-1 6843. NF-KB and AP-I have been implicated in apoptosis 33S I although the pre• cise functions and mechanisms are complicated and still unknown. It has been reported that overexpression of TRX inactivates NF-KB and activates AP- I 49. Inactivation of NF-KB by TRX overexpression may reduce excessive NO production under oxidative stress. Very recently mammalian TRX has been shown to function as a direct inhibitor of apoptosis signal-regulating kinase ASK-1 52. Das et al. 53 suggested that TRX can induce antioxidative proteins such as MnSOD. These results indicate that TRX acts as a regulator of intracellular signalings against oxidative stress. VI. ENDOGENOUS TRX EXPRESSION IN PANCREATIC 13-CELLS In our study using immunoblot analysis we found the expression of endoge• nous TRX to be much lower in islet cells than in pancreatic exocrine cells. Based on immunohistochemical analysis Hansson et al. 54 also suggested that TRX expression of islet cells was lower compared with pancreatic exo• crine cells. Their result was consistent with our study. This attenuation of TRX expression in -cells may be one of the reasons why -cells are vulnerable to oxidative stress. VII. PREVENTIVE EFFECTS OF TRX OVEREXPRESSION AGAINST AUTOIMMUNE DIABETES IN VIVO To directly assess the roles of oxidative stress in -cell destruction in autoim• mune diabetes we generated NOD transgenic mice overexpressing TRX ex• clusively in -cells. The incidence of diabetes in NOD transgenic mice was

slide 322:

270 Hatta et al. Thioredoxin transgenic mice ----i-- C57BU6J mice NOD I NOD x 86 F1 transgenic mice Streptozotocin STZ treatment 250 mg/kg Measurements a. Blood glucose levels b. Insulin contents of pancreas Figure 3 The generation of NOD X 86 Fl transgenic mice and the evaluation of pancreatic -cell damage after streptozotocin treatment. remarkably reduced compared with their negative littermates. Although the incidence of diabetes was reduced the severity of insulitis before overt diabe• tes was not significantly different between NOD transgenic mice and their negative littermates. To study the protective effects of TRX against ROS• generating agents we produced NOD X C57BL/6J 86 Fl transgenic mice and injected the ROS-generating agent STZ into them Fig. 3. The elevation of blood glucose levels in NOD X B6 Fl transgenic mice was reduced and the reduction of insulin contents was suppressed compared with their negative littermates. These results suggest that ROS play pivotal roles in -cell destruc• tion in autoimmune diabetes and in STZ-induced diabetes. VIII. SUMMARY TRX overexpression has preventive effects on both autoimmune diabetes and ROS-generating agent-induced diabetes as described. Our data suggest that the pancreatic -cell-targeted control of redox systems utilizing TRX and/or the antioxidative therapies targeted to -cells are anticipated to prevent or delay the overt diabetes even after the development of insulitis. REFERENCES I. Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Dia• betes 1965 14:619-633.

slide 323:

Oxidative Stress and Pancreatic 13-Cell Destruction 271 2. Foulis AK Clark A. Pathology of the pancreas in diabetes mellitus. In: Kahn CR Weir GC eds. Joslins diabetes mellitus 13th ed. Philadelphia: Lea Feb• iger 1994:265-281. 3. Mandrup-Poulsen T. The role of interleukin- I in the pathogenesis of IDDM. Diabetologia 1996 39: 1005-1029. 4. Eizirik DL Flodstrom M Karlsen AE et al. The harmony of the spheres: induc• ible nitric oxide synthase and related genes in pancreatic beta cells. Diabetologia 1996 39:875-890. 5. Holmgren A. Thioredoxin. Annu Rev Biochem 1985 54:237-271. 6. Nakamura H Nakamura K Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997 15:351-369. 7. Mitsui A Hirakawa T Yodoi J. Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin. Biochem Biophys Res Commun 1992 186:1220-1225. 8. Hirota K Matsui M Iwata S et al. AP- l transcriptional activity is regulated by a direct association between thioredoxin and Ref-l. Proc Natl Acad Sci USA 1997 94:3633-3638. 9. Baker A Payne CM Briehl MM et al. Thioredoxin a gene found overexpressed in human cancer inhibits apoptosis in vitro and in vivo. Cancer Res 1997 15: 5162-5167. 10. Makino S Kunimoto K Muraoka Y et al. Breeding of a non-obese diabetic strain of mice. Jikken Dobutsu 1980 29: 1-13. l l. Kikutani H Makino S. The murine autoimmune diabetes model: NOD and re• lated strains. Adv Immunol 1992 51 :285-322. 12. Andre I Gonzalez A Wang 8 et al. Checkpoints in the progression of autoim• mune disease: lessons from diabetes models. Proc Natl Acad Sci USA 1996 93: 2260-2263. 13. Pujol-Borrell R Todd I Doshi M et al. HLA class II induction in human islet cells by interferon gamma plus tumour necrosis factor or lymphotoxin. Nature 1987 326:304-306. 14. Rabinovitch A Suarez-Pinzon WL Sorensen 0 et al. Inducible nitric oxide syn• thase iNOS in pancreatic islets of nonobese diabetic mice: identification of iNOS-expressing cells and relationships to cytokines expressed in the islets. En• docrinology 1996 137:2093-2099. 15. Stassi G Maria RD Trucco G et al. Nitric oxide primes pancreatic beta cells for Fas-mediated destruction in insulin-dependent diabetes mellitus. J Exp Med 1997 186:1193-1200. 16. Grankvist K Marklund S Sehlin J et al. Superoxide dismutase catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 1979 182: 17-25. 17. Malaisse WJ Malaisse-Lagae F Sener A et al. Determinants of the selective toxicity of alloxan to the pancreatic beta cell. Proc Natl Acad Sci USA 1982 79:927-930. 18. Takasu N Komiya I Asawa T et al. Streptozocin- and alloxan-induced HP2

slide 324:

272 Hotta et al. generation and DNA fragmentation in pancreatic islets. H02 as mediator for DNA fragmentation. Diabetes 1991 40:1141-1145. 19. Grankvist K Marklund SL Taljedal IB. CuZn-superoxide dismutase Mn-super• oxide dismutase catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse. Biochem J 1981 199:393-398. 20. Gandy SE 3d Galbraith RA Crouch RK et al. Superoxide dismutase in human islets of Langerhans. N Engl J Med 1981 304:1547-1548. 21. Cornelius JG Luttge BG Peck AB. Antioxidant enzyme activities in IDD-prone and IDD-resistant mice: a comparative study. Free Radie Biol Med 1993 14: 409-420. 22. Lenzen S Drinkgern J Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radie Biol Med 1996 20:463-466. 23. Kubisch HM Wang J Bray TM et al. Targeted overexpression of Cu/Zn super• oxide dismutase protects pancreatic beta-cells against oxidative stress. Diabetes 1997 46:1563-1566. 24. Yamada K Nonaka K Hanafusa T et al. Preventive and therapeutic effects of large-dose nicotinamide injections on diabetes associated with insulitis. Diabetes 1982 31 :749- 753. 25. Elliott RB Pilcher CC Stewart A et al. The use of nicotinamide in the preven• tion of type I diabetes. Ann NY Acad Sci 1993 696:333-341. 26. Beales PE Williams AJ Albertini MC et al. Vitamin E delays diabetes onset in the non-obese diabetic mouse. Horm Metab Res 1994 26:450-452. 27. Takamura T Kato I Kimura N et al. Transgenic mice overexpressing type 2 nitric-oxide synthase in pancreatic beta cells develop insulin-dependent diabetes without insulitis. J Biol Chem 1998 273:2493-2496. 28. Kurrer MO Pakala SV Hanson HL et al. Beta cell apoptosis in T cell-mediated autoimmune diabetes. Proc Natl Acad Sci USA 1997 94:213-218. 29. OBrien BA Harmon BV Cameron DP et al. Apoptosis is the mode of beta cell death responsible for the development of IDDM in the nonobese diabetic NOD mouse. Diabetes 1997 46:750- 757. 30. Nagata S Golstein P. The Fas death factor. Science 1995 267:1449-1456. 31. Chervonsky AV Wang Y Wong FS et al. The role of Fas in autoimmune diabe• tes. Cell 1997 89:17-24. 32. ltoh N Imagawa A Hanafusa T et al. Requirement of Fas for the development of autoimmune diabetes in nonobese diabetic mice. J Exp Med 1997 186:613-618. 33. Sonenshein GE. Rel/NF-kappa B transcription factors and the control of apoptosis. Semin Cancer Biol 1997 8:113-119. 34. Sibenlist U Franzoso G Brown K. Structure regulation and function of NFKB. Annu Rev Cell Biol 1994 10:405-455. 35. Kwon G Corbett JA Hauser S et al. Evidence for involvement of the proteo• some complex 26S and NFKB in IL-I -induced nitric oxide and prostaglandin production by rat islets and RINm5F cells. Diabetes 1998 47:583-591. 36. Corbett JA Wang JL Sweetland MA et al. Interleukin-I beta induces the forma-

slide 325:

Oxidative Stress and Pancreatic 13-Cell Destruction 273 tion of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evi• dence for the beta-cell as a source and site of action of nitric oxide. J Clin Invest 1992 90:2384-2391. 37. Kaneto H Fujii J Seo HG et al. Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells. Diabetes 1995 44:733- 738. 38. Zhang P Liu B Kang SW et al. Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2. J Biol Chem 1997 272: 30615-30618. 39. Laurent TC Moore EC Reichard P. Enzymatic synthesis of deoxyribonucleo• tides. IV. Isolation and characterzation of thioredoxin the hydrogen donor from Escherichia coli B. J Biol Chem 1964 239:3436-3444. 40. Huber HE Russel M Model P et al. Interaction of mutant thioredoxins of Esche• richia coli with the gene 5 protein of phage T7: the redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J Biol Chem 1986 261: 15006-15012. 41. Steitz TA. A mechanism for all polymerases. Nature 1998 391:231-232. 42. Ramana CV Boldogh I Izumi T et al. Activation of apurinic/apyrimidinic endo• nuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals. Proc Natl Acad Sci USA 1998 95:5061-5066. 43. Qin J Clore GM Kennedy WP et al. The solution structure of human thiore• doxin complexed with its target from Ref- I reveals peptide chain reversal. Struc• ture 1996 4:613-620. 44. Berggren M Gallegos A Gasdaska JR et al. Thioredoxin and thioredoxin reduc• tase gene expression in human tumors and cell lines and the effects of serum stimulation and hypoxia. Anticancer Res 1996 I 6:3459-3466. 45. Sasada T Iwata S Sato N et al. Redox control of resistance to cis diammine• dichloroplatinum II CDDP: protective effect of human thioredoxin against CDDP-induced cytotoxicity. J Clin Invest 1996 97:2268-2276. 46. Yokomizo A Ono M Nanri H et al. Cellular levels of thioredoxin associated with drug sensitivity to cisplatin mitomycin C doxorubicin and etoposide. Can• cer Res 1995 55:4293-4296. 47. Matsuda M Masutani H Nakamura H et al. Protective activity of adult T cell leukemia-derived factor ADF against tumor necrosis factor-dependent cytotox• icity on U937 cells. J lmmunol 1991 147:3837-3841. 48. Gallegos A Berggren M Gasdaska JR et al. Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent sele• nium. Cancer Res 1997 57:4965-4970. 49. Schenk H Klein M Erdbrugger W et al. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP- I. Proc Natl Acad Sci USA 1994: 91:1672-1676. 50. Qin J Clore GM Kennedy WM et al. Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the tran• scription factor NF kappa 8. Structure 1995: 3:289-297.

slide 326:

274 Hotta et al. 51. Karin M Liu Zg Zandi E. AP-I function and regulation. Curr Opin Cell Biol 1997 9:240-246. 52. Saitoh M Nishitoh H Fujii M et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase ASK-I. EMBO J 1998 17:2596-2606. 53. Das KC Lewis-Molock Y White CW. Elevation of manganese superoxide dis• mutase gene expression by thioredoxin. Am J Respir Cell Mol Biol 1997 17: 713-726. 54. Hansson HA Holmgren A Rozell B et al. Immunohistochemical localization of thioredoxin and thioredoxin reductase in mouse exocrine and endocrine pan• creas. Cell Tissue Res 1986 245: 189-195.

slide 327:

18 InterrelationshipBetween Oxidative Stress and Insulin Resistance To Get Rid Of Diabetes Permanently Click Here Karen Yaworsky Romel Somwar and Amira Klip The Hospital for Sick Children and Universityof Toronto Toronto Ontario Canada I. INSULIN RESISTANCE: A KEY FACTOR IN TYPE 2 DIABETES Insulin is the predominant hormone responsible for the maintenance of glucose homeostasis through its regulation of metabolic activites in muscle liver and adipose tissue. Insulin causes an increase in glucose uptake into peripheral tissues specifically muscle and fat cells conversely in the liver the hormone decreases gluconeogenesis thereby reducing hepatic glucose output. Insulin is also responsible for the promotion of protein synthesis. These effects result from both rapid and long-term metabolic actions of the hormone . One hundred million people worldwide suffer from type 2 diabetes 2 yet despite intense research the primary lesions responsible for type 2 diabe• tes remains unknown. The genetic susceptibility of this disease fails to follow simple Mendelian inheritance but is of a polygenic nature with superimposed environmental influences 3. An aggregation of small genetic effects rather than the effect of one single gene gene-to-gene and gene-to-environment in• teractions are thought to contribute to the development of this disease yet most genes involved in the origin of type 2 diabetes remain unknown R. Heggele personal communication. The environmental factors that favor the

slide 328:

development of this disease in genetically predispositioned individuals include high-fat diets low levels of physical activity and increasing age 3. 275

slide 329:

276 Yaworsky et al. Type 2 diabetes is characterized by resistance to the insulin stimulation of glucose uptake in skeletal muscle and adipose tissue by impaired insulin• dependent inhibition of hepatic glucose production and by dysregulated insu• lin secretion 3. Both the insulin resistance and insulin secretory defects ap• pear to be the result of genetic and environmental factors associated with the disease. It is largely acknowledged that insulin resistance is a primary factor responsible for glucose intolerance in the prediabetic state. Initially to com• pensate for the insulin resistance insulin secretion increases to maintain nor• mal glycemic levels. However when the insulin secretory capacity fails to adequately compensate for the impaired insulin action hyperglycemia ensues. This in turn further exacerbates the primary insulin resistance through the effects of high glucose collectively known as glucose toxicity and through the increased circulation of fatty acids and triglycerides 4. Hence insulin resistance has primary and secondary causes in type 2 diabetes Fig. 1 . A. Molecular Basis of Insulin Action and Insulin Resistance in Peripheral Tissues To understand the molecular basis of insulin resistance whether primary or secondary in muscle and fat cells it is imperative to gain knowledge of the normal mechanisms of insulin action. In these tissues insulin stimulates glu• cose uptake by rapidly mobilizing preexisting glucose transporters primarily the GLUT4 isofonn from an intracellular storage organelle or vesicle to the plasma membrane 56. This is achieved by a series of signals elicited from the receptor which are detected in an unknown fashion by the intracellular organelle. The latter is then free to find and interact with docking sites on the plasma membrane 7 that will ultimately enable fusion of the two membranes to provide functional glucose transporters. Detailed knowledge has emerged on the signals emanating from the receptor that are essential for GLUT4 trans• location and the nature of the proteins engaged in vesicle docking and fusion with the plasma membrane. The signaling events involved in the insulin-mediated GLUT4 transloca• tion include autophosphorylation of the insulin receptor tyrosine phosphoryla• tion of docking proteins known as insulin receptor substrates IRS 1-4 their subsequent binding to the enzyme phosphatidylinositol 3-kinase Pl 3-kinase and the resultant activation of PI 3-kinase to produce phosphorylated phospho• inositides. How these signals then translate into translocation of the GLUT4 vesicle is the subject of vigorous study. A serine/threonine kinase identified

slide 330:

111 p p 11 1 p p 1111 I I PP aa PI3K Oxidative Stress and Insulin Resistance 277 Insulin Receptor 11tllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll\llllllllllllllllll11 i 1 I : ./ . G L UT 4 y. I 11\ • ii 111 1111111 ...J\..1\1\.... ··\ ....../· r\........fL.. 1111 II IIIJlflIIIIIIIJlll jTTTT\"" ..\ f""III IIIIIIIIII II II litll 11 Figure 1 Proposed model for the development of insulin resistance and type 2 diabe• tes. It is proposed that elevations in insulin levels as a result of genetic defects and excess carbohydrate and fat intake lead to increased levels of nonesterified fatty acids NEFAand hyperlipidemia. These elevated levels of lipids result in impaired -cell function and induce insulin resistance through changes in glucose metabolism in skele• tal muscle and in the liver. These changes in metabolism in insulin responsive tissues lead to hyperglycemia and the diabetic state ensues. as protein kinase B also referred to as Akt has been shown to be activated by these phosphoinositides in vitro 89 and in response to insulin in vivo 10 and has been proposed to be a mediator of GLUT4 translocation l l 12 Fig. 2. Among the proteins involved in vesicle docking and fusion with the target plasma membrane are the vesicular proteins of the synaptobrevin family and the plasma membrane proteins syntaxin4 and SNAP23 13-17. These proteins are isoforms offunctional equivalents that participate in synaptic vesi• cle docking and fusion with the presynaptic plasma membrane of neurons. ln contrast to this knowledge little is known about how the glucose transporter• containing vesicles sense the insulin signal or travel to the plasma membrane. It follows that the diminished response to insulin of glucose uptake into muscle and fat could result from any of the following possibilities: defects in

slide 331:

:it.. I + 278 Yaworsky et al. Genetic Defect Excesscarbohydrate Initial Hyperinsulinemia ¥"and fat intake \ Genetic Defect Increasedllpogenesis"and growth of adipose tissue Hyperlipldemiaand elevated NEFA Genetic Defect .¥ Impairedglucose metabolism Impairedglucose-induced High hepaticglucose production lnsulln release Ins ulin R esist r an g ce ly:m P : -cell Dysfunction NIDDM Figure 2 Schematic diagram of the glucose transporter translocation hypothesis. Insulin-responsive tissues specifically adipose tissue and skeletal muscle contain intra• cellular stores of glucose transporter proteins GLUT. The binding of insulin and the subsequent increase in tyrosine kinase activity of the insulin receptor initiates a signal• ing cascade which results in the tyrosine phosphorylation of insulin receptor substrates IRS l-n their binding to the enzyme phosphatidylinositol 3-kinase Pl 3-kinase and the resultant activation of PI 3-kinase to produce phosphorylated phosphoinositides. This signaling cascade leads to the mobilization and insertion of stored glucose trans• porters into the plasma membrane allowing for increased glucose influx into the cell in response to insulin. insulin signals defects in detecting/transducing the signal a reduction in the total amount of glucose transporters and/or inability of the transporters to properly dock with and incorporate into the plasma membrane. Of these de• fects in insulin signaling and glucose transporter levels have been amply ex• plored and emerging evidence is being provided for defects in glucose trans• porter translocation. No studies have examined the mechanism of glucose transporter interaction with the plasma membrane in either humans or animals with diabetes. Finally because the mechanism whereby the intracellular or•

slide 332:

ganelle detects the insulin signal is largely unknown the possibility that this step is defective remains unexplored. A brief account of the status of glucose transporter levels GLUT4 translocation and defects in the insulin signaling pathway in diabetes is provided below. It is important to realize that for the

slide 333:

Oxidative Stress and Insulin Resistance 279 most part these studies do not distinguish whether the defects found relate to the primary or secondary insulin resistance. 1. GLUT4 Expression The levels of expression of the GLUT4 glucose transporter have been analyzed in a variety of animal models of type 2 diabetes and in tissue from individuals with type 2 diabetes for review see 18 19. GLUT4 protein content is mark• edly diminished in adipose tissues of human and most animal models. It has been found that GLUT4 expression in adipocytes decreases as diabetes devel• ops in older Zucker rats 18 19 and adipose cells taken from humans with type 2 diabetes also show a reduction in GLUT4 content 20. However this change in GLUT4 levels is restricted to adipose tissue and is not seen in skele• tal muscle of these animal models of type 2 diabetes as normal expression of GLUT4 is observed in muscle of db/db mice and Zucker rats 21-24. Muscle biopsies taken from individuals with type 2 diabetes also show normal skeletal muscle GLUT4 content 25. However a small number of studies have exam• ined the amount of GLUT4 protein on the plasma membrane of muscle from diabetic animals and it was found to be abnormal 18 19. This may suggest that sorting of the transporter is a key factor in muscle whereas net synthesis of the transporter is more pertinent in fat. Whether these changes cause the diabetic state or ensue from hyperglycemia hyperinsulinemia and/or hypertri• glyceridemia remains to be established. In a study attempting to shed light on this question brown adipose tissue was ablated in transgenic mice resulting in a decrease in the total GLUT4 protein in adipocytes. This led to the develop• ment of diabetes suggesting that a reduction in the level of GLUT4 could be causative in this disease 26. However genetic knockout of the GLUT4 gene in muscle and fat did not create a phenotype of diabetes although glucose intolerance was generated 27. 2. GLUT4 Translocation In skeletal muscle of humans with type 2 diabetes the plasma membrane does not show a reduction and in fact may show a small increase in the amount of GLUT4 transporters. Yet the stimulation of glucose uptake by insulin is totally blunted. Zierath et al. 28 showed that the membranes of these muscles dis• play a diminished gain in glucose transporters in response to an insulin clamp. A similar observation was also made in two animal models of diabetes 2429. Defective GLUT4 translocation is also seen in fat cells from these animals 30 and in fat cells from humans with type 2 diabetes 20. As a result several explanations have been put forward to account for this reduced translocation

slide 334:

280 Vaworsky et al. of GLUT4 to the cell surface in skeletal muscle and adipocytes.These include topics that have been briefly mentioned such as impaired translocation ma• chinery and an inability of the transporters to functionally incorporate into the plasma membrane in addition to the next topic to be discussed an alteration in the signaling emerging from the insulin receptor. 3. Insulin Signaling In animal models of type 2 diabetes and in humans with type 2 diabetes there is considerable evidence for defects in the early stages of insulin action 31- 33. There is an approximately 50 decrease in insulin receptor phosphoryla• tion and an 80 decrease in IRS- I phosphorylation in liver and skeletal mus• cle of oh/oh mice 34. This was associated with a more than 90 decrease in insulin-stimulated PI 3-kinase activity associated with IRS- I and no detectable stimulation of total Pl 3-kinase activity. In addition insulin-stimulated Akt kinase activity in skeletal muscle of the lean diabetic Goto-Kakizaki rat was reduced by 68 35. Skeletal muscle isolated from individuals with type 2 diabetes also show defects at the level of the insulin receptor tyrosine kinase activity IRS-I expression and phosphorylation and IRS-I-associated PI 3- kinase activity 36. A reduction in IRS- I expression by 70 and IRS-1- associated PI 3-kinase activity has also been reported in adipose cells isolated from individuals with type 2 diabetes 20. Thus in type 2 diabetes there are defects at four early steps of insulin action. Whether there are also defects distal to the initial signaling events that contribute to impaired translocation of GLUT4 remains to be determined. In addition to alterations in the level of expression or activation of the signaling molecules in type 2 diabetes the isoform selectivity of signaling also changes. In adipose cells isolated from humans with type 2 diabetes IRS- 2 becomes the main docking protein for PI 3-kinase and Grb2 in response to insulin 20. This is not surprising because expression of IRS-2 increases and this protein predominates as the main insulin receptor substrate in mice lacking lRS-1 3738. Importantly mice genetically manipulated to lack IRS-I do not develop diabetes 38 whereas mice Jacking IRS-2 do 39. In summary changes in the levels of glucose transporter expression defects in the insulin signaling pathway and alterations in pattern of signaling molecules may all contribute to either or both primary and secondary insulin resistance in type 2 diabetes. B. Factors That May Trigger Insulin Resistance It has been suggested that circulating and metabolic factors could play an important role in the etiology of insulin resistance. This is supported by the

slide 335:

Oxidative Stress and Insulin Resistance 281 observation that insulin resistance of in vitro muscle preparations can be re• versed by incubation in solutions of normal insulin and glucose levels 40. Circulating factors such as tumor necrosis factor-a TNF-a and free fatty acids FFA and intracellular metabolites such as glucosamine can induce an insulin-resistant state in vitro and may contribute to the development of insulin resistance in vivo. J. Tumor Necrosis Factor-a The level of expression of adipose tissue TNF-a a multifunctional cytokine rises as a consequence of obesity. It is also closely correlated with circulating insulin levels which serve as an index of insulin resistance 41 and is ex• pressed in increased levels in skeletal muscle of individuals with insulin resis• tance 42. Mice homozygous for a targeted null mutation in the TNF-a gene were significantly less insulin resistant compared with normal obese mice 43 suggestive of a causative role of TNF-a in obesity-related insulin resistance. The mechanism by which TNF-a exerts an impairment of insulin action re• mains incompletely understood. In adipocytes chronic TNF-a exposure in• duced serine phosphorylation of IRS- I inhibiting the insulin receptor tyrosine kinase 44. However acute exposure of TNF-a in rat hepatoma cells induced serine phosphorylation of IRS- I without any inhibitory effect on the insulin receptor tyrosine kinase yet IRS- I tyrosine phosphorylation and association with the p85 regulatory subunit of Pl 3-kinase was blunted 45. Greater con• fusion on the TNF-a mechanism of action was seen in 3T3-Ll adipocytes whereby TNF-a increased IRS- I tyrosine phosphorylation and association with p85 46. Further studies are required to elucidate the mechanism by which TNF-a acts as a mediator directly or indirectly of obesity-related insu• lin resistance. 2. Free Fatty Acids It has been demonstrated that increased levels of circulating FFA correlate with peripheral insulin resistance in humans 4748. Animal models ofhyper• lipidemia lend support to a correlation between increased FFA and insulin resistance 4950. In addition infusion of intralipid in vivo was shown to inhibit insulin-stimulated glucose uptake 4851 and numerous studies also demonstrate that elevated plasma FFA levels decrease insulin-stimulated glu• cose uptake in skeletal muscle 485152. However the underlying mecha• nism of how FFA induce insulin resistance remains unknown. Elevated plasma FFA levels have been suggested to have an inhibitory effect on glucose oxida• tion via the classic glucose-fatty acid cycle 53 and have been linked to impaired glycogen synthesis 54. It has also been suggested that the decrease

slide 336:

282 Yaworsky et al. in insulin-stimulated glucose uptake in skeletal muscle mediated by elevated FFA occurs at early steps in glucose utilization specifically at the level of glucose transport and/or glucose phosphorylation 5155. In support of a mechanism whereby elevated FFA induces insulin resistance through inhibi• tion of glucose transport/phosphorylation it was recently reported that eleva• tions in FFA induce insulin resistance in vivo via inhibition of components of the insulin signaling cascade specifically inhibition of IRS- I-associated PI 3-kinase activity 56. Overall these results suggest that elevated FFA may contribute to insulin resistance by action at several cellular processes that con• trol glucose uptake. 3. Glucosamine An intracellular mechanism that has received much attention lately as a possi• ble generator of insulin resistance is increased flux of glucose through the hexosamine pathway 57. This pathway utilizes intracellular glucose and the amino acid glutamine to produce glucosamine which is a precursor of UDP• N-acetylglucosamine UDP-GlcNAc and UDP-N-acetylgalactosamine UDP• GalNAc used in protein glycosylation. How glycosylation leads to insulin resistance is not known but increasing evidence supports the concept that a rise in N-acetylglucosamine is linked to insulin resistance. The first observa• tions were made in primary cultures of rat adipocytes in which it was observed that increased glucose flux through the hexosamine biosynthetic pathway is the mechanism by which prolonged exposure to high levels of glucose and insulin resulted in impaired glucose transport 5859. Induction of insulin resistance via increased glucosamine flux can be reproduced by administration of glucosamine in vitro and in vivo and is associated with increased accumula• tions of UDP-GlcNAc and UDP-GalNAc 6061 . These serve as an index of the amount of carbon flux through the pathway. Impaired insulin-mediated glucose disposal induced by elevated glucosamine levels is associated with decreased muscle GLUT4 translocation to the sarcolemmal fraction in re• sponse to insulin 62. Furthermore overexpression of glucosamine fructose amido transferase the rate-limiting enzyme for the hexosamine biosynthetic pathway resulted in decreased GLUT4 translocation to the plasma membrane in response to insulin 63. Increased glucose flux though the hexosamine biosynthetic pathway could therefore be responsible for inducing insulin resis• tance through the downregulation of the glucose transport system. In contrast to the evidence presented whereby circulating factors such as TNF-cx FFA and the metabolite glucosamine may be involved in the induc• tion of insulin resistance there is no formal evidence to support or discard a

slide 337:

Oxidative Stress and Insulin Resistance 283 role of oxidative stress in the origin of insulin resistance. The folllowing sec• tion examines the possibility that a link might exist between them. 11. OXIDATIVE STRESS IN RELATION TO DIABETES AND INSULIN RESISTANCE A. Methods of Detection of Oxidative Stress Oxidative stress is defined as the oxidative damage inflicted by an excess of reactive oxygen species ROS on a cell or organ 64. As the natural balance between toxic oxidants and protective antioxidant defenses is altered oxida• tive stress results. Such conditions include an increase in free radical concen• tration or a decrease in the antioxidant capacity-or oxidant scavenging abil• ity-of the cell 65. Free radicals are highly reactive atoms or molecules containing one or more unpaired electrons 64. These toxic metabolites such as peroxides su• peroxides and hydroxyl radicals can be generated through both essential and nonessential oxidation-reduction reactions of the cytosol and mitochondria 66. Free radical-mediated oxidative damage has been observed in a variety of pathological conditions including type 2 diabetes. However difficulties arise in measuring oxygen radicals due to the highly reactive nature of these species because they rapidly react with various substrates including them• selves and have short lifespans 10-6 10-9 s in aqueous systems 65. None• theless measurements of free radicals via direct and indirect measures al• though complicated allow for an approximate assessment of oxidative stress. These measurements include spin trapping to directly measure the levels of free radicals 65 direct measurement of superoxide radicals in plasma 64 and identification of products of the oxidation of polyunsaturated fatty acids PUFA by lipid hydroperoxide assays 65. In addition the plasma GSH/ GSSG ratio reduced glutathione/oxidized glutathione serves as an indirect measurement of free radical reactions and as an indicator of the oxidative stress that may occur under physiological and pathological conditions 65. Several of these methods have been used to measure oxidative stress in diabetes 67 as discussed elsewhere in this book. A few examples follow. Animal models have been used to address the possible in vivo relation• ship between oxidative stress and diabetes. Accelerated accumulation of ad• vanced glycation end products AGEs was measured as the amount of fluo• rescent protein adducts with lipoperoxidative aldehydes malondialdehyde MDA and 4-hydroxynonenal HNE in rat skin collagen of diabetic BB rats

slide 338:

284 Yaworsky et al. 68. The diabetic rats had significantly higher levels of autoantibody against albumin modified by the lipoperoxidative aldehydes MDA and HNE in addi• tion to reactive oxygen species ROS 69. This suggests an increased genera• tion of oxygen free radicals and lipoperoxidative aldehydes in these diabetic rats. Also the duration of diabetes correlated significantly with the develop• ment of antibodies directed against MDA and HNE. The presence of antibod• ies against oxidatively modified proteins in spontaneously diabetic rats pro• vides indirect evidence of the occurrence of oxidative modification of proteins in vivo 69. In addition decreased plasma concentrations of ascorbic acid AA have been reported in individuals with diabetes 70. In a recent study lens and renal AA levels were partially restored when normal glycemia was approached by insulin treatment in streptozotocin-diabetic BB rats 71. These in vivo studies provide evidence for the coexistence of oxidative stress associ• ated with the increased level of oxidative damage in diabetes. Studies of humans with type 2 diabetes also reveal the coexistence of oxidative stress with the disease. Serum has antioxidant activity against transi• tion metal ion-catalyzed reactions a result of the ferrous iron oxidizing fer• roxidase activity of caeruloplasmin and of the iron-free fraction of transferrin 72. Increased levels of transferrin ferritin ferroxidase activity of caeru• loplasmin and iron-binding capacity were described in the serum of 67 sub• jects with type 2 diabetes 73. This observation has been interpreted to repre• sent a protective response to the increased level of oxidative stress in subjects with diabetes. Free radical-mediated oxidative stress has been implicated in the patho• genesis of complications associated with type 2 diabetes. This wide field is not discussed here because it is treated amply elsewhere in this book. The following paragraphs address the origin of oxidative stress in diabetes and its possible link to insulin resistance. B. Origin of Oxidative Stress in Type 2 Diabetes Suspected causative agents of the increased level of oxidative stress associated with type 2 diabetes are hyperglycemia hyperinsulinemia and an alteration of serum antioxidant activity Fig. 3. 1. Hyperglycemia Hyperglycemia has been strongly implicated in the development of diabetic complications an effect also known as glucose toxicity. The mechanisms of glucose toxicity are the subject of extensive investigation and include glucose

slide 339:

Glu In Sy Oxidative Stress and Insulin Resistance 285 cose Oxidation . Increased oxygen radical production Glycation . Protein Oxidation creased activity . Depletion of NADPH of Polyol Pathway stores HIGH INSULIN mpathetic Nervous . System Overdrive Elevated NEFA . concentrations Catecholamine-induced free radical production Decreased GSH levels Increased free radical production Figure 3 How oxidative stress may arise in diabetes. The characteristic high levels of glucose ancl insulin observed in type 2 diabetes may contribute to oxidative stress through the production of free radicals protein oxidation and by depletion of intracel• lular reductants and antioxidants. This increased level of oxidative stress may be attrib• uted to the increased levels of glucose oxidation glycation increased activity of the polyol pathway sympathetic nervous system overdrive and elevated nonesterified free fatty acid NEFA concentrations resulting from high levels of glucose and insulin. oxidation glycation increased levels of polyols and elevation of the hexos• amine biosynthetic pathway. Most of these actions can contribute to oxidative stress. Glucose Enolization. Glucose enolizes in vitro reducing molecular oxygen catalyzed by transition metals to yield oxidizing intermediates and o-ketoaldehydes 74 Fig. 4. Hydroxyl radicals superoxide anions and hy• drogen peroxide are the reduced oxygen products formed in this glucose oxida• tion reaction all of which are capable of protein damage through cross-linking fragmentation and lipid oxidation 6475. Glycation and AGEs. The protein reactive n-ketoaldehydes which are formed via protein glycation are in turn protein reactive and are thus responsible for the formation of AGEs and protein cross-linking 74 Fig. 4. AGEs have been implicated in the generation of ROS activation of the transcription factor NF-KB and in modulation of endothelial physiology 66. Sorbitol and Fructose Levels. Intracellularly elevated glucose levels can promote glucose reduction into sorbitol and fructose due to increased free glucose and an elevation in aldose-reductase and sorbitol-dehydrogenase ac• tivities 76. The rise in aldose-reductase activity diminishes NADPH cellular

slide 340:

I I I I I I I I I I I --JLR O OHH OHOH H U CH20H H OH H H Protein -NH o-Glucose " -o."_ll Hydroxyaldehyde Enedlol H OHH OHOH Protein -N-1 cHoH H OH H H Schiffs Base o•o /- Metaln+ /f Metaln-1+ H N-CH i O H OH OH II CHOH RH Enediol Radical Anion I .: i Protein OH H H Amadori Product/Adduct i / 0 0 R_JJ_JLH o.Ketoal:ehydes Protein -NH Protein- NH + o.Ketoaldehydes t Protein OHO H H Ketoaminomethylol Protein Crosslinking AGE formation on + Protein -NH t Hydroxyl Radical-Mediated

slide 341:

Protein Damage Figure 4 Glucose oxidation and glycation: glucose-induced protein damage and in• creased oxidative stress. Glucose via glycative and oxidative processes can result in protein damage and increased levels of oxidative stress as a result of the increased production of advanced glycation end products AGEs and hydroxyl radicals. These processes can therefore be attributed to type 2 diabetes due to the increased glucose levels hyperglycemia associated with this disease. From Hunt JV Dean RT Wolff SP. Hydroxyl radical production and autoxidative glycosylation: glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochern J 1998 256:205-212. Copyright 1988 by The Biochemical Soci• ety and Portland Press.

slide 342:

Oxidative Stress and Insulin Resistance 287 levels thereby lowering the reducing power of the cell. lt has been proposed that this leads to inhibition of the activity of NADPH-requiring enzymes in• cluding nitric oxide synthase and glutathione reductase 64. Vasoconstriction and tissue injury can result from the diminished nitric oxide synthase activity 77 and the low glutathione reductase activity could result in an increased cellular susceptibility to free radical damage 78 and oxidative stress. 2. Hyperinsulinemia Hyperinsulinemia a hallmark of insulin resistance has recently been invoked as a plausible causative agent in the development of oxidative stress-induced diabetic complications. Experimental evidence suggests a relationship be• tween hyperinsulinemia and increased free radical production 79. In human fat cells increased accumulation of hydrogen peroxidase occurs upon expo• sure to elevated insulin levels 79. It has been hypothesized that the increase in free radical production associated with elevated levels of insulin may result from heightened sympathetic nervous system activity and from elevated FFA concentrations 64. High insulin leads to an overdrive of the sympathetic nervous system that results in increased catecholamine release 8081. Cate• cholamines have been associated with an augmented production of free radi• cals in diabetic animals through a rise in metabolic rate and autooxidation 82. In vitro and in vivo studies suggest that the hyperinsulinemia-induced rise in fasting FFA concentrations may be associated with oxidative stress. An in vitro study with cultured endothelial cells showed an association between increased fatty acid levels in the medium with both an increase in oxidative stress and a decrease in initial glutathione levels 83. Further studies in hu• mans have supported a relationship between increased FFA levels and free radical-associated oxidative stress the latter was inferred from the lower GSH/ GSSG ratio in the plasma of subjects with diabetes compared with controls 64. 3. Natural Antioxidant Activities A brief summary of certain properties of selected antioxidants is addressed in the following section. Antioxidants are scavengers of free radicals that consti• tute an important component of the cellular defense mechanism against oxida• tive stress. As a result reduction or changes in the activity of these compounds result in deleterious effects to the cell due to the development of free radical• mediated oxidative stress. In previous and in following sections evidence is provided to discuss the status of antioxidants in diabetes.

slide 343:

288 Yaworsky et al. Vitamin E tocopherol is the predominant lipophilic antioxidant and is localized to the plasma membrane 66. It is one of the most important chain• breaking antioxidants responsible for the prevention of the propagation of free radical-induced reactions 84. Interestingly vitamin E has been shown to decrease the covalent linking of glucose to serum proteins in vitro and to inhibit the glycation of serum proteins in vivo 85. Glutathione is one of the most important water-soluble antioxidants. It is a tripeptide and the reduced form is the major hydrophilic intracellular reductant responsible for protection and repair against oxidant damage 66. Ascorbate vitamin C a key aqueous phase antioxidant is involved in vitamin E reduction 86 thereby promoting many of the antioxidant prop• erties of vitamin E including the ability to prevent the propagation of free radical-induced chain reactions. u-Lipoic acid is the most potent endogenous antioxidant and is also a natural cofactor of mitochondrial dehydrogenase complexes. The redox poten• tial of the dihydrolipoate DHLA/a-lipoic acid couple is -0.32 mV 87. This strong reductant is responsible for the regeneration of reduced glutathione and vitamin E. It can also prevent lipid peroxidation possibly through its ability to scavenge superoxide and hydroxyl radicals and is involved in the inhibition of the activation of NF-KB by directly preventing its translocation from the cytoplasm to the nucleus 66. The ability of o-Iipoic acid to protect against oxidative stress is an important feature that might be applied to coun• teract the oxidative stress-mediated diabetic complications. This approach is discussed elsewhere in this book. C. Can Oxidative Stress Cause Insulin Resistance The question of precedence between hyperinsulinemia and oxidative stress into the subsequent development of diabetes remains unanswered 64. The studies mentioned above suggest that at least the secondary hyperinsulinemia could plausibly precede or cause increased free radical production and the resulting oxidative stress. Further studies are necessary to address whether oxidative stress as a result of hyperinsulinemia-mediated increased free radi• cal production could precede insulin resistance and lead to the onset of diabe• tes. To date there is no evidence that primary insulin resistance is linked to oxidative effects. The following sections analyze two emerging lines of study in this direction. 1. Oxidative Stress Antioxidants. and Insulin Resistance In Vivo There is little evidence for a role of antioxidant therapy in the prevention of insulin resistance and type 2 diabetes. Serum of individuals with type 2 diabe-

slide 344:

Oxidative Stress and Insulin Resistance 289 tes have a lower vitamin E level and a higher GSH/GSSG ratio compared with control subjects 88. Individuals with type 2 diabetes who received vitamin E supplementation had improved metabolic control as indicated by the signifi• cant drop in circulating levels of glycosylated hemoglobin 89. In a small randomized trial with humans with or without type 2 diabetes vitamin E sup• plementation reduced oxidative stress and improved insulin action 6488 verifying an important role for antioxidant therapy in diabetes. This study involved the use of a euglycemic hyperinsulinemic clamp to study the effects of vitamin E supplementation on insulin sensitivity 90. The results showed a significant gain in insulin-mediated nonoxidative glucose disposal after sup• plementation of 900 mg vitamin E 90. In addition the vitamin E supplemen• tation significantly increased plasma vitamin E levels and significantly reduced GSH/GSSG ratios in all subjects 90. It was proposed that the mechanism by which vitamin E improves insulin responsiveness in individuals with and without diabetes was related to its role as an antioxidant 88. The hypothesis was put forward that increased lipid peroxidation as a result of increased free radical-induced oxidative stress or a reduction in the antioxidant capacity of the cell could cause changes in the fluidity of the membrane this in turn would purportedly lower glucose uptake 91 . The ability of an antioxidant to quench free radicals and reduce lipid peroxidation could presumably provide protection from changes to membrane fluidity and restore normal glucose transporter function. The first prospective population study undertaken to address the role of free radical stress and antioxidants in relation to the incidence of diabetes examined whether low vitamin E concentrations are a risk factor for the inci• dence of type 2 diabetes 92. The authors computed the levels of plasma vitamin E and the incidence of developing diabetes over a 4-year period in 944 men aged 42-60 who were determined not to have diabetes at baseline examination 92. Type 2 diabetes was defined by either a fasting blood glu• cose concentration of ".6.7 mM a blood glucose concentration ". 10.0 mM 2 h after a glucose load or by a clinical diagnosis of diabetes with either dietary oral or insulin treatments. Forty-five men developed diabetes over the 4-year follow-up period 92. However these 45 men also had a raised baseline body mass index elevated blood glucose and serum fructosamine concentrations a higher ratio of saturated fatty acids to the sum of monounsa• turated and polyunsaturated fatty acids and a higher serum triglyceride con• centration 92. From the multivariate logistic regression model used in this study it was found that the baseline body mass index was the strongest pre• dictor of diabetes. Other factors with significant associations to an excess risk of diabetes included low plasma vitamin E concentrations a high ratio of saturated to other fatty acids in serum and a high socioeconomic status 92.

slide 345:

290 Yaworsky et al. From this multivariate logistic regression model a 3.9-fold risk of developing diabetes was associated with a low lipid standardized plasma vitamin E con• centration below median 92. In addition another model was used whereby lipid standardized vitamin E concentrations were replaced by unstandardized vitamin E concentrations when other risk factors such as serum low-density• lipoprotein cholesterol and triglyceride concentrations were taken as the strongest predictors of diabetes. From this model a decrease of I µmol/L of uncategorized vitamin E concentration was associated with an increment of 22 in the risk of developing diabetes 92. Hence a significant relationship was proposed to exist between low vitamin E concentrations and an increased risk of diabetes. Clinical trials are needed to support the effect of antioxidants in the prevention of diabetes. It has been suggested that these trials should address the need for a "free radical initiative" 93 to understand how free radicals could affect the intrinsic mechanisms of diabetes 65. Also trials are necessary to confirm a role of vitamin E or other antioxidants in the preven• tion of type 2 diabetes 92. 2. Oxidative Stress and Insulin Resistance In Vitro In vitro insulin resistance can be induced by prolonged insulin treatment exposure of cells to high glucose concentrations or by the preexposure of cells to glucosamine Fig. 5. Insulin resistance at the level of glucose transport can result from various signaling defects as previously mentioned including alterations in insulin receptor function depletion of the GLUT4 transporter pool and alterations in the postreceptor signaling pathway 94. Prolonged insulin treatment of 3T3-L I adipocytes induced an insulin• resistant state as a result of changes in the insulin signal transduction cascade 95. Treatment of 3T3-Ll adipocytes with 500 nM insulin for 24 h increased basal glucose transport and led to an insulin-resistant state for this transport 59697. This treatment did not modify the insulin receptor or levels of GLUT4 however it prevented GLUT4 translocation in response to acute insu• lin stimulation 95. Prolonged insulin treatment also induced a decrease in the level of IRS- I expression and phosphorylation and a reduced ability of insulin to stimulate Pl 3-kinase and MAP kinase 98. Thus insulin resistance induced by prolonged insulin treatment of 3T3-Ll adipocytes is associated with multiple signaling defects including defects at the level of IRS- I PI 3- kinase MAP kinase and impaired GLUT4 translocation 98. These defects are similar to those observed in the hyperinsulinemic states of obese humans and rodents 98 validating this insulin-resistant model as an effective ap• proach to study alterations of the insulin signaling pathway and insulin resis• tance.

slide 346:

Oxidative Stress and Insulin Resistance 291 I In Vitro Models of InsulinResistance I On cells in culture or Isolated tissue Glucose Oxidase Measure Insulin action on glucose uptake Figure 5 In vitro models of insulin resistance provide an effective tool to study alterations in the insulin signaling pathway and insulin resistance. Treatment of cells in culture or tissue with high levels of glucose insulin glucosamine or glucose oxidase gnerate systems of insulin resistance thus enabling further studies of insulin action in the insulin-resistant state. Hyperglycemia has been linked to a worsening of insulin resistance 99- 10 I attributed to a disruption of normal cellular metabolism and of insulin• induced glucose disposal 102. In vitro adipocytes exposed to high concentra• tions of glucose develop impaired insulin signaling reduced insulin respon• siveness and diminished recruitment of glucose transporters to the plasma membranes in response to insulin 58 I 03. These alterations may be a result of the adverse metabolic consequences of the hyperglycemia. Furthermore it was discovered that an additional factor glucosamine was necessary for the glucose-induced desensitization of the insulin-stimulated glucose transport system 104. Glucosamine was also found to be more potent than glucose in the ability to induce insulin resistance and to decrease insulin responsiveness 104. It has been hypothesized from these findings that hexo• samine metabolism may be the pathway by which cells sense and respond to ambient glucose levels and when glucose flux is excessive downregulation of glucose transport occurs and insulin resistance results I 02 I 04 . Thus these models may lead to a better understanding of the mechanisms involved in the alterations of glucose metabolism seen in the insulin-resistant state. To assess the role of oxidant stress and insulin resistance in vitro Rudich et al. 105 used 3T3-LI adipocytes preexposed to an enzymatic system capa• ble of generating ROS. Exposure of the 3T3-LI adipocytes to 25 mU/mL

slide 347:

292 Yaworsky et al. glucose oxidase for 18 h resulted in steady production of H202 with a concom• itant threefold increase in basal 2-deoxyglucose uptake activity and a reduction in insulin-dependent 2-deoxyglucose uptake 105. The increase in basal trans• port as a result of increased oxidative stress was associated with an increase in GLUTI mRNA and protein level 105. A reduction in GLUT4 protein and mRNA content was also observed and this may account for a portion of the reduced insulin-stimulated glucose transport in the glucose oxidase-treated ad• ipocytes I 05. Basal lipogenesis was also enhanced by this treatment whereas acute insulin stimulation of glucose oxidase-treated adipocytes significantly reduced lipogenesis activity 105. A further alteration of insulin-stimulated metabolism was also observed in these adipocytes as exposure to glucose oxi• dase lowered both the basal and insulin-stimulated glycogen synthase a activ• ity 105. Recently Rudich et al. 106 observed that GLUT4 translocation was selectively impaired in glucose oxidase-treated 3T3-LI adipocytes as there was impaired redistribution of PI 3-kinase to the LDM fraction of cells upon insulin stimulation. This suggests that oxidative stress could impair the insulin-mediated PI 3-kinase cellular redistribution which results in the im• paired GLUT4 translocation. D. o-Llpolc Acid: Stimulation of Glucose Uptake via Components of the Insulin Signaling Pathway As previously mentioned a-lipoic acid is a naturally occurring cofactor of oxidative metabolism which is found as lipoamide covalently bound to a lysyl residue in five eukaryotic proteins including mitochondrial dehydrogenase complexes 107. A natural antioxidant lipoic acid has been used for the treat• ment of diabetic neuropathy l 08 and ischemia-reperfusion injury 109 and has been indicated to improve glucose metabolism 110. In vitro and in vivo studies have demonstrated that exogenously supplied a-lipoic acid is taken up and reduced to DHLA by NADH- or NADPH-dependent enzymes in a variety of cells and tissues 111 112. Furthermore a-lipoic acid has the ability to decrease the NADH/NAD+ ratios elevated as a result of sorbitol oxidation to fructose under hyperglycemic conditions by the consumption of N ADH 113 . Further antioxidant properties attributed to a-lipoic acid include its ability to directly scavenge ROS and to recycle thioredoxin glutathione vitamin E and vitamin C 114. However it is not known whether these potent antioxidant prop• erties of c-Iipoic acid contribute to its ability to improve glucose utilization. o-Lipoic acid has been shown in vitro to stimulate glucose utilization in isolated rat diaphragms 115 to enhance insulin-stimulated glucose metab• olism in insulin-resistant skeletal muscle of obese Zucker rats 116 and to

slide 348:

Oxidative Stress and Insulin Resistance 293 stimulate glucose transport activity in skeletal muscle isolated from both lean and obese Zucker rats 117. In streptozotocin-diabetic rats chronic o-lipoic acid treatment reduced blood glucose concentrations by enhancement of mus• cle GLUT4 content and increased muscle glucose utilization 118. In addi• tion acute and repeated parenteral administration of n-lipoic acid improved insulin-stimulated glucose disposal in individuals with type 2 diabetes 110119 strengthening its therapeutic value as an antidiabetic agent. Estrada et al. 120 established the ability of u-lipoic acid to stimulate glucose uptake into the insulin-responsive L6 skeletal muscle cells and 3T3-LI adipocytes in culture. The naturally occurring R+ isoform of lipoic acid was shown to have a significantly greater effect on the stimulation of glucose uptake in L6 cells in comparison with the S- isoform or the racemic mixture 120. In addition R+ lipoic acid had a positive effect on both basal and insulin• stimulated glucose uptake but did not improve the sensitivity of glucose uptake to submaximal concentrations of insulin 120. It was suggested that this in• crease in glucose uptake could not be entirely attributed to the antioxidant abilities of this agent alone. The increase in glucose uptake was mediated by a rapid translocation of the GLUTl and GLUT4 glucose transporter isoforms from the internal membrane fraction to the plasma membrane of L6 myotubes 120. We have recently shown that o-lipoic acid similarly stimulates the trans• location of GLUT and GLUT4 from the internal membrane fractions to the plasma membrane in 3T3-Ll adipocytes K. Yaworsky unpublished data. To account for the mechanism by which o-Iipoic acid could stimulate this in• crease in glucose uptake via rapid glucose transporter translocation an inhibi• tor of Pl 3-kinase wortmannin was used. As stated earlier PI 3-kinase activity is essential for the propagation of the insulin signal responsible for the media• tion of insulin-stimulated glucose uptake as a result of the translocation of glucose transporters 12 l . Wortmannin significantly lowered the c-Iipoic acid stimulated increase in glucose uptake in L6 myotubes suggesting the involve• ment of PI 3-kinase in lipoic acids mechanism of action 120. More recent evidence has shown that n-lipoic acid directly stimulates IRS- I immunopre• cipitated Pl 3-kinase activity in L6 myotubes and this increase in PI 3-kinase activity is wortmannin sensitive R. Somwar K. Yaworsky and A. Klip un• published data. These data suggest that ce-lipoicacid engages components of the insulin signaling pathway in its ability to stimulate glucose uptake in L6 myotubes. This differs from other stimuli of glucose uptake such as the exer• cise and/or hypoxia pathway that do not use PI 3-kinase in their ability to stimulate glucose uptake although they require glucose transporter transloca• tion 122133. The unique action of o-lipoic acid to increase glucose uptake

slide 349:

aa 294 Yaworsky et al. Insulin a-Lipoic Acid Receptor 11111111111111111111111111111111 111111111111111111111111111111111111111111111111111111111 111 p p 1111 11 p p \ 1 II I I pp Pl3K ----.. ./ . GLUT4 GLUTVI I 1 ·. Vesicle ./ Vesicle / Jl- c - V - . 11 .L j_ ii \ T T i"1 I 11 111111 . " J V \J V \J V \.. " . I l I I I II I I I I I I I I I I I I I r\ ... .J LJL..... 11 11111111111111111111111 lttt\ """\ w+t\J"""\.f""" 11 Figure 6 Schematic diagram of the proposed n-lipoic acid signaling mechanism. a• Lipoic acid uses components of the insulin signaling pathway in its ability to stimulate glucose uptake via the rapid translocation of GLUT and GLUT4 to the plasma mem• brane. In L6 myotubes it has been shown that lipoic acid activates phosphatidylinositol 3-kinase PI3K and Akt in a wortmannin-sensitive fashion. These results indicate that lipoic acid uses components of the insulin signal transduction cascade in its ability to increase glucose uptake into insulin-responsive cells in culture. via an insulin-sensitive pathway was further exemplified by studies whereby o-lipoic acid increased Akt activity in L6 myotubes R. Somwar K. Yawor• sky and A. Klip unpublished data Fig. 6. These actions of o-lipoic acid are distinctive from other currently used antidiabetic agents and highlight a-lipoic acid as an attractive therapeutic strategy for the treatment of insulin resistance in type 2 diabetes. REFERENCES I. Heesom K Harbeck M Kahn C Denton R. Insulin action on metabolism. Dia• betologia 1997 40:83-89. 2. Groop LC Tuomi T. Non-insulin dependent diabetes mellitus-a collision be• tween thrifty genes and an affluent society. Ann Med 1997 29:37-53.

slide 350:

Oxidative Stress and Insulin Resistance 295 3. Kahn BB. Type 2 diabetes: when insulin secretion fails to compensate for insu• lin resistance. Cell 1998 92:593-596. 4. Day C Grove J Daly A Stewart M Avery P Walker M. Tumour necrosis factor-alpha gene promoter polymorphism and decreased insulin resistance. Di• abetologia 1998 41 :430-434. 5. Cushman S Wardzala L. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem 1980 255:4758-4762. 6. Suzuki K Kono T. Evidence that insulin causes translocation of glucose trans• port activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci USA 1980 77:2542-2545. 7. Rea S James D. Moving GLUT4 the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes 1997 46: 1667-1677. 8. Franke TF Yang SI Chan TO et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 1995 81 :727-736. 9. Franke TF Kaplan DR Cantley LC Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-34-bisphosphate see com• ments. Science 1997 275:665-668. 10. Kohn AD Kovacina KS Roth RA. Insulin stimulates the kinase activity of RAC-PK a pleckstrin homology domain containing ser/thr kinase. EMBO 1995 14:4288-4295. 11. Kohn AD Summers SA Birnbaum MJ Roth RA. Expression of a constitu• tively active Akt Ser/Thr kinase in 3T3-L I adipocytes stimulates glucose up• take and glucose transporter 4 trans location. J Biol Chem 1996: 271: I 372- 1378. 12. Cong L Chen H Li Y et al. Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 1997 I I : 1881- 1890. 13. Sollner T Whiteheart S Brunner M et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 1993 362:318-324. 14. Volchuk A Ewart HS et al. Syntaxin 4 in 3T3-LI adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol Biol Cell 1996 7: I 075-1082. 15. Sudhof T De Camilli P. Niemann H Jahn R. Membrane fusion machinery: insights from synaptic proteins. Cell 1993 75: 1-4. 16. Rothman J. Mechanisms of intracellular protein transport. Nature 1994 372: 55-63. 17. Wong P Daneman N Volchuk A et al. Tissue distribution of SNAP-23 and its subcellular localization in 3T3-LI cells. Biochem Biophys Res Commun 1997 230:64-68. 18. Klip A Tsakiridis T Marette A Ortiz PA. Regulation of expression of glu• cose transporters by glucose-a review of studies in vivo and in cell cultures. FASEB J 1994 8:43-53.

slide 351:

296 Vaworsky et al. 19. Tsakiridis T Marette A Klip A. Glucose transporters in skeletal muscle of animal models of diabetes. In: Shafrir E ed. Lessons from Animal Models of Diabetes 1994: 141-159. 20. Rondinone CM Wang L-M Lonnroth P Wesslau C Pierce JH Smith U. Insu• lin receptor substrate IRS I is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin• dependent diabetes mellitus. Proc Natl Acad Sci USA 1997: 94:4171-4175. 21. Friedman JE Sherman WM Reed MJ Elton CW Dohm GL. Exercise training increases glucose transporter protein GLUT-4 in skeletal muscle of obese Zucker fa/fa rats. FEBS Lett 1990 268: 13-16. 22. Koranyi L James D. Mueckler M Permutt MA. Glucose transporter levels in spontaneously obese db/db insulin-resistant mice. J Clin Invest 1990:85:962- 967. 23. Kahn BB. Rossetti L Lodish HF Charron MJ. Decreased in vivo glucose up• take but normal expression of GLUT and GLUT4 in skeletal muscle of dia• betic rats. J Clin Invest 1991 87:2197-2206. 24. King PA Horton ED Hirshman MF Horton ES. Insulin resistance in obese Zucker rat fa/fa skeletal muscle is associated with a failure of glucose trans• porter translocation. J Clin Invest 1992 90: 1568-1575. 25. Pedersen 0 Bak JF Andersen PH et al. Evidence against altered expression of GLUT or GLUT4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 1990 39: 865-870. 26. Hamann A Benecke H. et al. Characterization of insulin resistance and NIDDM in transgenic mice with reduced brown fat. Diabetes 1995 44:1266-1273. 27. Katz E Stenbit A Hatton K DePinho R Chan-on M. Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 1995 377:151-155. 28. Zierath JR He L Guma A Odegoard Wahlstrom E Klip A Wallberg-Henriks• son H. Insulin action on glucose transport and plasma membrane GLUT4 con• tent in skeletal muscle from patients with NIDDM. Diabetologia 1996 39: 1180-1189. 29. Gibbs EM Stock JL McCoid SC et al. Glycemic improvement in diabetic dbl db mice by overexpression of the human insulin-regulatable glucose transporter GLUT4. J Clin Invest 1995 95:1512-1518. 30. Kahn 8. Glucose transport: pivotal step in insulin action. Diabetes 1996 45: 1644-1654. 31. Kahn CR. Insulin action diabetogenes and the cause of type II diabetes. Diabe• tes 1994 43:1066-1084. 32. Saad MJA Araki E Miralpeix M Rothenberg PL White MF Kahn CR. Regu• lation of insulin receptor substrate- I in liver and muscle of animal models of insulin resistance. J Clin Invest 1992 90:1839-1849. 33. Le Marchand-Brustel Y Gremeaux T Ballotti R Yan Obberghen E. Insulin receptor tyrosine kinase is defective in skeletal muscle of insulin-resistant obese mice. Nature 1985 315:676-678.

slide 352:

Oxidative Stress and Insulin Resistance 297 34. Folli F Saad MJA Backer JM Kahn CR. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes-mellitus. J Clin Invest 1993: 92: 1787-1794. 35. Krook A Kawano Y Song XM et al. Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki rats. Diabetes 1997 46:2110-2114. 36. Bjomholm M Kawano Y Lehtihet M Zierath JR. Insulin receptor substrate• \ phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation. Diabetes 1997 46:524- 527. 37. Tamemoto H Kadowaki T Tobe K et al. Insulin resistance and growth retarda• tion in mice lacking insulin receptor substrate-I. Nature 1994 372: 182- 186. 38. Araki E Lipes MA Patti M-E et al. Alternative pathway of insulin signaling in mice with targeted disruption of the IRS- I gene. Nature 1994 372: 186-190. 39. Withers DJ Gutierrez JS Towery H et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 1998: 391 :900-904. 40. Zierath J Galuska D Nolte A Thome A Kristensen J. Wallberg-Henriksson H. Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM-in vitro reversal of muscular insulin resistance. Diabe• tologia 1994 37:270-277. 41. Hotamisligil G Amer P Caro J Atkinson R Spiegelman B. Increased adipose tissue expression of tumour necrosis factor alpha in human obesity and insulin resistance. J Clin Invest 1995 95:2409-2415. 42. Saghizadeh M Ong J Garvey W Henry R Kem P. The expression of TNF alpha by human muscle. J Clin Invest 1996 97: I l l l -1116. 43. Uysal K Weisbrock S Marino M Hotamisligil G. Protection from obesity• induced insulin resistance in mice lacking TNF-alpha function. Nature 1997 389:610-614. 44. Hotamisligil G Peraldi P Budavari A Ellis R White M Spiegelman B. RS• I-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha and obesity-induced insulin resistance. Science 1996 271 :665-668. 45. Kanety H Hemi R Papa M Karasik A. Sphingomyelinase and ceramide sup• press insulin-induced tyrosine phosphorylation of the insulin receptor substrate• . J Biol Chem 1996 271:9895-9897. 46. Guo D Donner D. Tumour necrosis factor promotes phosphorylation and bind• ing of insulin receptor substrate-I to phosphatidylinositol 3-kinase in 3T3-Ll adipocytes. J Biol Chem 1996 271:615-618. 47. Falholt K Jensen I Lindkaer Jensen S et al. Carbohydrate and lipid metabolism of skeletal muscle in type 2 diabetic patients. Diabetic Med 1988 5:27-31. 48. Kelley D Mokan M Simoneau J-A Mandarino L. Interaction between glucose and free fatty acid metabolism in human skeletal muscle. J Clin Invest 1993 92:91-98. 49. Shillabeer G Chamoun C Hatch G Lau DCW. Exogenous triacylglycerol in-

slide 353:

298 Yaworsky et al. hibits insulin-stimulated glucose transport in L6 muscle cells in vitro. Biochem Biophys Res Commun 1995 207:768- 774. 50. Zarjevski N Doyle P Jeanrenaud B. Muscle insulin resistance may not be a primary etiological factor in the genetically obese fa/fa rat. Endocrinology 1992 130:1564-1570. 51. Boden G Jadali F White J et al. Effects of fat on insulin-stimulated carbohy• drate metabolism in normal men. J Clin Invest 1991 88:960-966. 52. Vaag A Handberg A Skott P Richter E Beck-Nielson H. Glucose-fatty acid cycle operates in humans at the levels of both whole body and skeletal muscle during low and high physiological plasma insulin concentrations. Eur J Endo• crinol 1994 130:70-79. 53. Randle P Garland P Hales C Newsholme E. The glucose-fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963: 785- 789. 54. Shagro E Woldegiorgis G Ruoho A DiRusso C. Fatty acyl-CoA esters as regulators of cell metabolism. Prostaglandins Leukot Essent Fatty Acids 1995 52: 163-166. 55. Hargreaves M Kiens B Richter E. Effect of increased plasma free fatty acid concentrations on muscle metabolism in exercising men. J Appl Physiol 1991 70:194-201. 56. Marcucci M Griffin M Estrada P Barucci N Cline G Shulman G. Elevations in free fatty acids induce insulin resistance via inhibition of IRS- I -associated PI 3-kinase activity in vivo. Diabetes 1998 47suppl I :A284. 57. ODoherty R Stein D Foley J. Insulin resistance. Diabetologia 1997 40:810- 815. 58. Garvey W Olefsky J Matthaei S Marshall S. Glucose and insulin coregulated the glucose transport system in primary cultured adipocytes. J Biol Chem 1987 262: 189-197. 59. Marshall S Bacote V Traxinger R. Discovery of a metabolic pathway mediat• ing glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem 1991 266:4706-4712. 60. Rossetti L Hawkins M Chen W Gindi J Barzilai N. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995 96: 132-140. 61. Hawkins M Angelov I Liu R Barzilai N Rossetti L. The tissue concentration of UDP-N-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake. J Biol Chem 1997 272:4889-4895. 62. Baron A Zhu J-S Zhu J-H Weldon H Maianu L Garvey W. Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity. J Clin Invest 1995 96:2792- 2801. 63. Chen H Ing B Robinson K Feagin A Buse M Quon M. Effects of overexpres• sion of glutamine: fructose-6-phosphate amidotransferase GFAT and glucos-

slide 354:

Oxidative Stress and Insulin Resistance 299 amine treatment on translocation of GLUT4 in rat adipose cells. Mol Cell Endo• crinol 1997 135:67-77. 64. Paolisso G Giugliano D. Oxidative stress and insulin action: is there a relation• ship Diabetoiogia 1996 39:357-363. 65. Corninacini L Garbin U Cascio V. The need for a "free radical initiative." Diabetologia 1996 39:364-366. 66. Hofmann M. Bierhaus A Ziegler R Wahl P Nawroth P Tritschler H. Lipoate effects on atherogenesis. In: Fuchs J Packer L Zimmer G eds. Lipoic Acid in Health and Disease. New York: Marcel Dekker 1997. 67. Cross C Halliwell B Borish E. Oxygen radicals and human disease. Ann Intern Med 1987: 107:526-545. 68. Odetti P Traverso N Cosso L Noberasco G Pronzato M Marinari U. Good glycemic control reduces oxidation and glycation end-products in collagen of diabetic rats. Diabetologia 1996 39:1440-1447. 69. Traverso N Menini S Cosso L et al. Immunological evidence for increased oxidative stress in diabetic rats. Diabetologia 1998 41:365-370. 70. Paolisso G DAmore A Balbi V et al. Plasma vitamin C affects glucose ho• meostasis in healthy subjects and in non-insulin-dependent diabetics. Am J Physiol 1994 266:E26 l-E268. 71. Lindsay R Jamieson N Walker S McGuigan C Smith W. Baird J. Tissue ascorbic acid and polyol pathway metabolism in experimental diabetes. Diabe• tologia 1998 41 :516-523. 72. Stocks J. Gutteridge J Sharp R Dorrnandy T. The inhibition of lipid autoxida• tion by human serum and its relationship to serum proteins and tocopherol. Clin Sci Mol Med 1974 47:223-233. 73. Jones A Winkles J Jennings P Florkowski C Lunec J Barnett A. Serum antioxidant activity in diabetes mellitus. Diabetes Res 1988 7:89-92. 74. Wolff S Jiang Z Hunt J. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Radie Biol Med 1991 J0:339-352. 75. Brownlee M. Cerami A Vlassara H. Advanced glycosylation end products in tissue and biochemical basis of diabetic complication. N Engl J Med 1988 318: 1315-1322. 76. Hohman T Beg M. Diabetic complication: progress in the development of treat• ments. Exp Opin Invest Drugs 1994 3:1041-1049. 77. Lowenstein C Dinerman J Snyder S. Nitric oxide: a physiologic messenger. Ann Intern Med 1994 120:227-237. 78. Chari S Noth N Rothi A. Glutathione and its redox system in diabetic polymor• phonuclear leukocytes. Am J Med Sci 1984 297:14-15. 79. Krieger-Brauer H Kather K. Human fat cells possess a plasma membrane bound H202 generating system that is activated by insulin via a mechanism bypassing the receptor kinase. J Clin Invest 1992 89:1006-1013. 80. DeFronzo R Ferrannini E. Insulin resistance: a multifaceted syndrome respon• sible for NIDDM obesity hypertension and atherosclerotic cardiovascular dis• ease. Diabetes Care 1991 14:173-194.

slide 355:

300 Yaworsky et al. 81. Rowe J Young J Minaker K Stevens A Pallotta J Landsberg L. Effect of insulin and glucose infusion on sympathetic nervous system activity in normal men. Diabetes 1981 30:219-225. 82. Singal P Beamish R Dhalla N. Potential oxidative pathways of catecholamines in the formation of lipid peroxides and genesis of heart disease. Adv Exp Med Biol 1983 161:391-401. 83. Henning B Enoch C Chow C. Protection by vitamin E against endothelial cell injury by linoleic acid hydroperoxides. Nutr Res 1983 7: 1253-1259. 84. Young I Tomey J Trimble E. The effect of ascorbate supplementation on oxi• dative stress in the streptozotocin diabetic rat. Free Radie Biol Med 1992 I 3: 41-46. 85. Ceriello A. Quatraro A Giugliano D. New insights on non-enzymatic glycosyl• ation may lead to therapeutic approaches for the prevention of diabetic compli• cations. Diabet Med 1992 9:297-299. 86. Sies H. Strategies of antioxidant defense. Eur 1 Biochem 1993 215:213-219. 87. Jocelyn P. The standard redox potential of cysteine-cystine from the thioldisul• phide exchange reaction with glutathione and lipoic acid. Eur J Biochem 1967 2:327-331. 88. Caballero B. Vitamin E improves the action of insulin. Nutr Rev 1993 5 l: 339-340. 89. Cerierro A Giugliano D Quatraro A Donzella C Dipalo G Lefebvre P. Vita• min E reduction of protein glycosylation in diabetics: new prospect for preven• tion of diabetic complications. Diabetes Care 1991 14:68- 72. 90. Paolisso G D Amore A Giugliano D Ceriello A. Vericchio M DOnofrio F. Pharmacologic doses of vitamin E improve insulin action in healthy subjects and noninsulin-dependent diabetic patients. Am 1 Clin Nutr 1993 57:650- 656. 91. Whitesell R Reyen D Meth A Pelletier D Aburnrad N. Activation energy of slowest step in the glucose carrier cycle: correlation with membrane lipid fluid• ity. Biochemistry 1989 28:5818-5825. 92. Salonen J Nyyssonen K Tuomainen T et al. Increased risk of non-insulin dependent diabetes mellitus at low plasma vitamin E concentrations: a four year follow up study in men. Br Med 1 1995 311: 1124-1127. 93. Meshnick S. Oxidant stress distressed. Redox Rep 1995 1:77-78. 94. Kahn B. Facilitative glucose transporters: regulatory mechanisms and dysregu• lation in diabetes. J Clin Invest 1992 89: 1367-1374. 95. Kozka I Clark A Holman G. Chronic treatment with insulin selectively down regulates cell-surface GLUT4 glucose transporters in 3T3-Ll adipocytes. J Biol Chem 1991 266:11726-11731. 96. Tordjman K Leingang K James D Mueckler M. Differential regulation of two distinct glucose transporter species expressed in 3T3-L I adipocytes: effect of chronic insulin and tolbutamide treatment. Proc Natl Acad Sci USA 1989 86: 7761-7765.

slide 356:

Oxidative Stress and Insulin Resistance 301 97. Rosen 0 Simith C Fung C Rubins C. Development of hormone receptors and hormone responsiveness in vitro. Effect of prolonged insulin treatment on hex• ose uptake in 3T3-LI adipocytes. J Biol Chem 1978 253:7579- 7583. 98. Ricort J-M Tanti J-F Van Obberghen E Le Marchand-Brustel Y. Alterations in insulin signaling pathway induced by prolonged insulin treatment of 3T3- LI adipocytes. Diabetologia 1995 38: 1148-1156. 99. Yki-Jarvinen H Helve E Koivisto V. Hyperglycemia decreases glucose uptake in type I diabetes. Diabetes 1987 36:892-896. 100. Unger R Grundy S. Hyperglycemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance. Diabetologia 1985 28: I I 9- 121. IO I. DelPrato S Sheehan P Leonetti F Simonson D. Effect of chronic physiologic hyperglycemia on insulin secretion and glucose metabolism Abstr. Diabetes 1986 35suppl I: l 96A. 102. McClain DA Crook ED. Hexosamines and insulin resistance. Diabetes 1996 45: 1003-1009. 103. Garvey W Huecksteadt T Birnbaum M. Pretranslational suppression of an insulin-responsive glucose transporter in rats with diabetes mellitus. Science 1989 245:60-63. 104. Marshall S Bacote V Traxinger R. Discovery of a metabolic pathway mediat• ing glucose-induced desensitization of the glucose transport system. J Biol Chem 1991 266:4706-4712. 105. Rudich A Kozlovsky N Potashnik R Bashan N. Oxidant stress reduces insulin responsiveness in 3T3-LI adipocytes. Am J Physiol 1997 272:E935-E940. 106. Rudich A Kozlovsky N Potashnik R Tirosh A Pessler D Bashan N. Regula• tion of glucose transporters gene expression and protein function in oxidative stress induced insulin resistance. VII International Symposium on Insulin Re• ceptor Insulin Action: Molecular and Clinical Aspects Jerusalem Israel 1998. 107. Packer L Roy S Sen C. Alpha-lipoic acid: a metabolic antioxidant and poten• tial redox modulator of transcription. Adv Pharmacol 1996 38:79-10 I. 108. Ziegler D Hanefeid M Ruhnau K et al. Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidant alpha-lipoic acid. Diabetologia 1996 38: 1425-1433. 109. Panigrahi M Sadguna Y Shivakumar B et al. Alpha lipoic acid protects against reperfusion injury following cerebral ischemia in rats. Brain Res 1996 717: 79-101. I I 0. Jacob S Henriksen E Schiemann A et al. Enhancement of glucose disposal in patients with type 2 diabetes by alpha-lipoic acid. Drug Res 1995 45:872- 874. 111. Handleman G Han D Tritschler H Packer L. Alpha-lipoic acid reduction by mammalian cells to the dithiol form and release into the culture medium. Bio• chem Pharmacol 1994 47: 1725-1730.

slide 357:

302 Yaworsky et al. 112. Hararnaki N Han D Handelman G Tritschler H Packer L. Cytosolic and mito• chondrial systems for NADH- and NADPH-dependent reduction of alpha-lipoic acid. Free Radie Biol Med 1997 22:535-542. 113. Roy S. Sen C Tritschler H Packer L. Modulation of cellular reducing equiva• lent homeostasis by alpha lipoic acid. Biochem Pharmacol 1997 53:393-399. 114. Packer L Witt E Tritschler H. Alpha-lipoic acid as a biological antioxidant. Free Radie Biol Med 1995 19:227-250. 115. Haugaard N Haugaard E. Stimulation of glucose utilization by thioctic acid in rat diaphragm incubated in vitro. Biochim Biophys Acta 1970 222:583-586. 116. Jacob S Streeper R Fogt D et al. The antioxidant alpha lipoic acid enhances insulin-stimulated glucose metabolism in insulin-resistant rat skeletal muscle. Diabetes 1996 45:1024-1029. 117. Henriksen E. Jacob S Streeper R Fogt D Hokama J Tritschler H. Stimulation by alpha lipoic acid of glucose transport activity in skeletal muscle of lean and obese Zucker rats. Life Sci 1997 61:805-812. 118. Khamaisi M Potashnik R Tirosh A et al. Lipoic acid reduces glycaemia and increases muscle GLUT4 content in streptozotocin-diabetic rats. Metabolism 1997 46:763- 768. 119. Jacob S Henriksen E Tritschler H Augustin H Dietze G. Improvement of insulin-stimulated glucose disposal in type 2 diabetes after repeated parenteral administration of thioctic acid. Exp Clin Endocrinol Diabetes 1996 I 04:284- 288. 120. Estrada D Ewart H Tsakiridis T et al. Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioctic acid. Diabetes 1996 45: I 798- 1804. 121. Cheatham B Vlahos C Cheatham L Wang L Blenis J Kahn C. Phosphatidyl• inositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase SNA synthesis and glucose transporter translocation. Mol Cell Biol 1994 14: 4902-4911. 122. Lee A Hansen P Holloszy J. Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle. FEBS Lett 1995 361 :51-54. 123. Yeh J Gulve E. Rameh L Birnbaum M. The effects of wortmannin on rat skeletal muscle: dissociation of signaling pathways for insulin- and contraction• activated hexose transport. J Biol Chem 1995 270:2107-2111.

slide 358:

19 Oxidative Stress and Antioxidant Treatment: Effects on Muscle Glucose Transport in Animal Models of Type 1 and Type 2 Diabetes To Kill Diabetes Forever Click Here Erik J. Henriksen University of Arizona Tucson Arizona By definition diabetes mellitus is a group of pathophysiological conditions of varying etiologies that has as a common denominator-thederangement of blood glucose regulation i.e. hyperglycemia. Two major forms of diabetes mellitus exist: type I a less common form in which there is an absolute defi• ciency of circulating insulin due to destruction of the P-cells of the pancreas and type 2 the most common form characterized primarily by a decreased ability of insulin to stimulate skeletal muscle glucose transport and metabo• lism. Although there is increasing information that oxidative stress character• ized by the localized production of free radicals and other reactive oxygen species may be associated with metabolic abnormalities present in both type l and type 2 diabetes and several studies have been published recently support• ing the effectiveness of antioxidant interventions in improving the defective metabolic state characteristic of diabetes the relationship between oxidative stress and insulin resistance remains controversial 12.

slide 359:

The purpose of this chapter is to briefly review the regulation of skeletal muscle glucose transport by insulin under normal conditions and the underly• ing defects in this regulation present in type I and type 2 diabetes and the available information regarding the role of oxidative stress in diabetes and the 303

slide 360:

304 Henriksen utility of antioxidant interventions with a focus on the water-soluble antioxi• dant lipoic acid 3 in ameliorating these metabolic abnormalities associated with type 1 and type 2 diabetes. In this context I discuss primarily evidence from animal model studies because Chapter 20 specifically addresses clinical investigations involving diabetes and antioxidant interventions. I. REGULATION OF MUSCLE GLUCOSE TRANSPORT Skeletal muscle is the major tissue responsible for the peripheral disposal of glucose in the face of a glucose or insulin challenge or during exercise 45. Skeletal muscle glucose transport activity is acutely regulated by insulin through the activation of a series of intracellular proteins including insulin receptor autophosphorylation and tyrosine kinase activation tyrosine phos• phorylation of insulin receptor substrate- I IRS-I and activation of phos• photidylinositol 3-kinase Pl 3-kinase ultimately resulting in the translo• cation of a glucose transporter protein isoform the GLUT4 protein to the sarcolemmal membrane where glucose transport takes place via a facilitative diffusion process for a review see 6. Recent evidence from rodent studies indicates that the amount of GLUT4 protein incorporated into the sarcolemmal membrane correlates closely with the degree of insulin-stimulated glucose transport 78 and supports the idea that GLUT4 translocation represents the major mechanism for insulin stimulation of glucose transport in skeletal muscle. Skeletal muscle glucose transport is also stimulated by an insulin-inde• pendent process that is activated by contractions 9-11 via a GLUT4 translo• cation mechanism 7 12. Evidence that these insulin-dependent and insulin• independent pathways for stimulation of glucose transport are mediated by different mechanisms came initially from studies demonstrating that the maxi• mal effects of the two pathways are completely additive 11 13-15. More• over the additive effect of insulin and contractions in combination is due to an additivity of the effects of these stimuli on GLUT4 translocation into the sarcolemma 7. More recently studies by Goodyear et al. 16 have provided a molecular basis for these two distinct pathways for activation of glucose transport in skeletal muscle. Although insulin increases tyrosine phosphoryla• tion of the insulin receptor and IRS- I and activates IRS- I-associated PI 3- kinase muscle contraction alone has no effect on these factors indicating that distinct intracellular pathways exist in muscle for activation of GLUT4 translocation and glucose transport by insulin and contractions 16.

slide 361:

Oxidative Stress and Antioxidant Treatment 305 II. INSULIN RESISTANCE IN DIABETES Insulin resistance is defined as a reduced ability of insuin to activate specific insulin-dependent biological processes in cells of target organs. In poorly con• trolled type l diabetes insulin resistance is thought to be a secondary effect of the dyslipidemic state elevated free fatty acids and the prolonged hyper• glycemic state. In type 2 diabetes insulin resistance of skeletal muscle glucose disposal is generally considered to be a primary factor in the etiology of this disease. In this latter state the skeletal muscle insulin resistance is often ac• companied by a variety of other metabolic abnormalities including obesity dyslipidemia hypertension and atherosclerosis 17-19 a condition referred to variously as "syndrome X" 18 19 or the "insulin resistance syndrome" 17. The link among these disorders has been attributed to hyperinsulinemia a consequence of the insulin resistance 17. Indeed the increased cardiovas• cular mortality associated with this condition has been directly attributed by some leading investigators to the hyperinsulinemia itself 20-22. Interventions that improve insulin action on skeletal muscle glucose me• tabolism in insulin-resistant individuals are therefore expected to decrease conversion rates to overt diabetes and to reduce cardiovascular mortality in diabetic populations. Therefore an understanding of the pathophysiologyun• derlying this insulin resistance and the search for optimal interventions for improving insulin action on skeletal muscle are of substantial interest. Ill. ANIMAL MODELS OF TYPE 1 AND TYPE 2 DIABETES A. Type 1 Diabetes The most widely used animal model of type l diabetes is the streptozotocin• induced diabetic rat. Streptozotocin is a compound that causes hypersecretion of insulin from the pancreatic -cells resulting in their eventual dysfunction and leading to a hypoinsulinemic state 23. The streptozotocin-diabeticrat is characterized by marked postprandial hyperglycemia and by an elevation in free fatty acids without ketoacidosis 23. Skeletal muscle from the streptozo• tocin-diabetic rat is markedly insulin resistant for stimulation of glucose trans• port 2425 and expresses a significantly reduced protein expression of the GLUT4 glucose transporter isoform 2425. B. Type 2 Diabetes Although numerous rodent models of type 2 diabetes exist the focus here is on the obese Zucker fa/fa rat. The obese Zucker rat is an animal model of

slide 362:

306 Henriksen severe skeletal muscle insulin resistance also characterized by marked hyper• insulinemia 26 glucose intolerance 2728 dyslipidemia 26 moderate hypertension 29 and central adiposity 30. It is therefore an excellent model with which to study the underlying pathophysiology and potential interven• tions in the insulin-resistance syndrome. Studies have identified at least one cellular locus for the insulin resistance of glucose transport in this animal model. Insulin-stimulated GLUT4 protein translocation 831 and glucose transport activity 83233 are substantially impaired in isolated skeletal mus• cle from these obese animals. Anai et al. 34 have very recently shown that in skeletal muscle from obese Zucker rats there are significant defects in crucial aspects of the insulin signaling cascade. Compared with age-matched lean Zucker rats in hindlimb muscle from the obese Zucker rats there is a 60 smaller IRS- I protein level and insulin-stimulated IRS- I phosphorylation is only 72 of control values despite elevated basal levels. The amount of the regulatory subunit of PI 3- kinase detected using a p85a. antibody associated with the tyrosine-phos• phorylated IRS-I in the insulin-stimulated state is only 29 of control. Finally IRS-I-associated PI 3-kinase activity in muscle immunoprecipitates from the obese animals is 54 of the level observed in lean animals 34. These findings likely represent the molecular basis for the skeletal muscle insulin resistance present in the obese Zucker rat. IV. OXIDATIVE STESS INSULIN RESISTANCE AND ANTIOXIDANT TREATMENT IN DIABETES A. Streptozotocin-Diabetic Rat There is ample evidence that markers of oxidative stress are increased in the most widely accepted rodent model of type l diabetes the streptozotocin• diabetic rat. For example plasma and liver lipid peroxides as measured by the thiobarbituric acid reactive substances assay are elevated in the streptozo• tocin-diabetic rat 35. In addition recent evidence indicates that in this model of type I diabetes sciatic nerve levels of reduced glutathione GSH are lower and the ratio of oxidized to reduced glutathione GSSG/GSH is elevated com• pared with tissue from normoglycemic control animals 36. Chronic treatment with the antioxidant lipoic acid brings about a nearly complete normalization of the GSH and GSSG/GSH profiles in sciatic nerve from the streptozotocin• diabetic rats and also significantly improves nerve blood flow and conduction velocity 36.

slide 363:

Oxidative Stress and Antioxidant Treatment 307 Plasma glucose is markedly elevated and insulin action on skeletal mus• cle glucose transport activity is substantially reduced in the streptozotocin• diabetic rat possibly as a result of reduced muscle GLUT4 protein levels 25. Acutely lipoic acid can cause a marked lowering of plasma glucose in these diabetic animals 25. Chronically a f O-daytreatment period of these diabetic animals with lipoic acid also results in a significant lowering of plasma glucose levels and causes profound increases in both skeletal muscle GLUT4 protein levels and insulin-stimulated glucose transport activity 25. Collectively these results provide evidence that the beneficial metabolic effects of lipoic acid in this severely hyperglycemic diabetic animal model may be associated with an improvement in the oxidant/antioxidant status of the animal. B. Obese Zucker Rat Much less information regarding the oxidant/antioxidant status is presently available for the obese Zucker rat. It should be stressed that this animal model displays only mild fasting hyperglycemia with more severe abnormalities ob• served when the animal is presented with a glucose load 273738. Neverthe• less Nourooz-Zadeh 39 reported that the isoprostane 8-epi-PGF2. a marker of oxidative stress is elevated in the plasma of the diabetic Zucker rat com• pared with lean controls. Interestingly these elevated levels of oxidative stress are significantly reduced with antioxidant treatment such as a-tocopherol 39. These results concerning oxidative stress in the diabetic Zucker rat are consistent with observations of human type 2 diabetes. During a euglycemic hyperinsulinemic clamp a significant inverse relationship has been observed between insulin action on nonoxidative glucose disposal and plasma superox• ide ion and a significant positive relationship has been seen between insulin action on nonoxidative glucose disposal and plasma GSH/GSSG ratio in type 2 diabetic patients 40. Patients with impaired glucose tolerance a prediabetic state or overt type 2 diabetes have significantly reduced erythrocyte levels of the antioxidant enzymes catalase and superoxide dismutase and diminished plasma GSH 41 . Decreased serum vitamin E content a marker of impaired oxidant/antioxidant status was recently reported to be associated with in• creased risk of developing type 2 diabetes in a Finnish population 42 and type 2 diabetic patients themselves display significantly reduced plasma vita• min E levels 43. Finally plasma hydroperoxides another marker of oxidative stress are higher in subjects with type 2 diabetes compared with healthy con• trol subjects and are significantly inversely con-elatedwith the degree of meta• bolic control 43.

slide 364:

308 Henriksen The effectivenessof antioxidant interventions particularly chronic treat• ment with lipoic acid in ameliorating the metabolic abnormalities present in the obese Zucker rat has been demonstrated in a series of studies from our laboratory. The results of these studies are summarized below. In these studies the obese Zucker rats were treated intraperitoneally with a racemic mixture 50 R- and 50 S-enantiomers of lipoic acid for 10-12 days and were investigated after an overnight fast food restricted to 4 g at 5 P.M. of the previous evening. As shown in Figure 1 the obese Zucker rat displays only mild hyperglycemia and this slight elevation in plasma glucose is completely reversed with chronic lipoic acid treatment 30 mg/kg 4445. More striking is the marked hyperinsulinemia and dyslipidemia of the obese Zucker rat com• pared with the lean Zucker rat 4445. Chronic lipoic acid treatment leads to significant reductions in both plasma insulin -20 and free fatty acids -15 Fig. l . It should be noted that these alterations due to the racemic mixture of lipoic acid are entirely due to the R-enantiomer as treatment with the S-enantiomer actually exacerbates the hyperinsulinemia and has no sig• nificant effect in lowering plasma free fatty acids 45. More recently we have shown that glucose tolerance after a 1-g/kg oral glucose feeding is improved by lipoic acid in a dose-dependent fashion Fig. Plasma Glucose mM Plasma Insulin nM Plasma FFA mM 8.0 6.0 4.0 2.0 0 • Lean Control D Obese Control • Obese Lipoic Acid Figure 1 Effect of chronic treatment of obese Zucker rats with lipoate on plasma glucose insulin and free fatty acids. Values are means ::: SE. p 0.05 vs. obese vehicle-treated control p 0.05 vs. lean control. From Ref. 44.

slide 365:

D Obese V • Obese 1 • Obese 3 Oxidative Stress and Antioxidant Treatment 309 j Glucose Response I 250 ie 200 - 150 100 ehicle 0 mg/kg Lipoic Acid 0 mg/kg Lipoic Acid 0 Insulin Response 300 200 100 0 15 30 45 60 Time min Figure 2 Effect of chronic treatment of obese Zucker rats with lipoate on glucose and insulin responses to a 1-g/kg oral glucose tolerance test. Values are means :: SE. p 0.05 vs. obese vehicle-treated control.

slide 366:

310 Henriksen 2 with a significantly smaller area under the curve AUC of the glucose response in a group of obese animals treated for 10 days with 30 mg/kg lipoic acid compared with control Fig. 3 left. Moreover this improved glucose response was seen in the face of a reduced insulin response during the test Fig. 2 and a smaller insulin AUC Fig. 3 middle. The glucose-insulin index the product of the glucose and insulin AUCs and an indirect index of in vivo insulin action was significantly lower in the 30 mg/kg lipoic acid-treated obese group compared with the obese control group implying that peripheral insulin action was enhanced by lipoic acid. Consistent with this finding was our observation that insulin-mediated glucose transport activity in both fast glycolytic muscle m. epitrochlearis Fig. 4 and slow oxidative muscle m. soleus Fig. 5 was improved in the 30 mg/kg lipoic acid-treated obese group compared with the obese control group. To determine the functional relevance of this improvement of insulin• mediated glucose transport we assessed the correlation between insulin-medi• ated glucose transport activity in either the epitrochlearis or the soleus and the glucose-insulin index in obese animals treated with either vehicle IO mg/ kg lipoic acid or 30 mg/kg lipoic acid Fig. 6. The correlation coefficients between the glucose-insulin index and insulin action on glucose transport in the epitrochlearis r -0.598 p 0.05 and in the soleus r -0.654 p 0.05 were statistically significant indicating that the improved insulin action on muscle glucose transport was at least in part responsible for the improvement in whole-body glucose tolerance observed after lipoic acid treat• ment. Because the whole homogenate level of GLUT4 protein in skeletal mus• cle from lipoic acid-treated obese Zucker rats is not significantly elevated com• pared with obese controls 44 this would imply that lipoic acid enhances the ability of insulin to activate translocation of intracellular GLUT4 protein into the sarcolemmal membrane a process that is defective in obese Zucker rats 8.31 . This hypothesis however remains to be tested experimentally. V. PERSPECTIVES: ANTIOXIDANTS AND INSULIN RESISTANCE A growing body of knowledge supports a role of oxidative stress in the compli• cations associated with the hyperglycemic state of diabetes. Moreover in• creasing evidence though still fairly limited at this point indicates that oxida• tive stress may be associated with the skeletal muscle insulin resistance inherent to both type I and type 2 diabetes. Therefore interventions that can

slide 367:

- en - - .. C - - Index units X 106 0 C ii ID . - . 15000 Glucose AOC mg/di X min 16000 InsulinAUC µU/ml X min Glucose-Insulin D D Ill Ill I» :: 200 a. 14000 10000 6000 :: 160 o· ii I» :: 120 -t D I» 3 D :: 40 Obese Vehicle Control Obese Obese 10mg/kg 30 mefq ALA ALA 2000 Obese Vehicle Control Obese Obese 10 mg/kg 30 mg/kg ALA ALA 0 Obese Obese Obese Vehicle lOmg/kg 30mg/kg Control ALA ALA

slide 368:

80 w Figure 3 Areas under the curve AUC for the glucose and insulin responses to an oral glucose tolerance test in control and chronic lipoate-treated obese Zucker rats. The glucose-insulin index represents the product of the glucose AUC and the insulin AUC. Values are means ::: SE. p 0.05 vs. obese vehicle-treated control. ....

slide 369:

312 300 200 100 0 Obese ZuckerEpitrochlearis IB 4 w 0 0 10 30 cx-LipoicAcid mg/kg/day Henriksen Figure 4 Effect of chronic treatment of obese Zucker rats with lipoate on in vitro insulin-stimulated glucose transport activity in the isolated epitrochlearis muscle. D Basal 2-deoxyglucose uptake •. increase in 2-deoxyglucose uptake due to insulin 2 mU/mL. This increase is shown in the box for each bar. Values are means :::: SE. p 0.05 vs. 0 mg/kg lipoate. ameliorate the oxidant/antioxidant imbalance in this condition will be helpful in improving peripheral insulin action on glucose transport and metabolism in skeletal muscle. Indeed several animal model and clinical investigations support the beneficial effects of antioxidants particularly lipoic acid in the diabetic state. Ample evidence now exists in the literature indicating that one locus of action of lipoic acid in improving metabolic control in animal models of insu• lin resistance is at the level of the skeletal muscle itself. Chronic treatment of the streptozotocin-diabetic rat a model of type l diabetes leads to a reduction in blood glucose that is associated with an increase in muscle GLUT4 protein expression and insulin-stimulated muscle glucose transport. Likewise chronic treatment of obese Zucker rats an animal model of the insulin resistance syn• drome with lipoic acid enhances whole body glucose tolerance and is associ• ated with significant improvements in insulin action on skeletal muscle glu-

slide 370:

Oxidative Stress and Antioxidant Treatment 313 800 600 400 200 I Obese Zucker Soleus j 0 0 10 30 o.-Lipoic Acid mg/kg/day Figure 5 Effect of chronic treatment of obese Zucker rats with lipoate on in vitro insulin-stimulated glucose transport activity in the isolated soleus muscle strips D basal 2-deoxyglucose uptake •. increase in 2-deoxyglucose uptake due to insulin 2 mU/mL. This increase is shown in the box for each bar. Values are means :::: SE. p 0.05 vs. 0 mg/kg lipoate. cose transport and metabolism and with reductions in plasma insulin and free fatty acids. There are however areas where our knowledge of lipoic acid action on metabolism is incomplete. For example we still need more information on the underlying molecular mechanisms responsible for the lipoic acid-induced improvement in insulin action. Some limited evidence indicates that there may be some interaction between lipoic acid and the insulin signaling cascade in the L6 muscle cell line 46 and in isolated muscle from the Zucker rat 47 however a more complete characterization of this interaction in skeletal mus• cle is necessary. In addition the relationship between lipoic acid action on skeletal muscle metabolism and its effects on cell oxidant/antioxidant status need to be more thoroughly investigated. It is clear that although investiga• tions of the metabolic actions of lipoic acid have yielded much important information over the last few years there is still much more work to be done

slide 371:

Epitrochlearis Soleus ---- •.. r ·0.598 P0.06 r ·0.654 P0.05 ·- • • - ..... o d • • .J •• . -.p O r1 ·---- ·o 0 .-. 0 0 0 . 100 200 300 0 200 300 314 Henrikse n I . - . -- i . . .. . . --- . Glucose-Insulin Index units X 106 Glucose-Insulin Index units X 106 Figure 6 Correlations between the glucose-insulin index and skeletal muscle insulin• mediated glucose transport activity in epitrochlearis left or soleus right muscles from obese Zucker rats treated chronically with lipoate. in the future to further our understanding of this important antioxidant com• pound. ACKNOWLEDGMENTS I would like to thank Dr. Stephan Jacob and Dr. Hans Tritschler for their intellectual contributions to the lipoic acid studies conducted in my laboratory and AST A Medica AWD GmbH for continued financial support of these studies. REFERENCES I. Paolisso G Giugliano D. Oxidative stress and insulin action: is there a relation• ship Diabetologia 1996 39:357-364. 2. Cominacini L Garbin U Cascio L. The need for a free radical initiative. Diabeto• logia 1996 39:364-366. 3. Packer L Witt EH Tritschler HJ. Alpha-lipoic acid as a biological antioxidant. Free Radie Biol Med 1995 19:227-250.

slide 372:

Oxidative Stress and Antioxidant Treatment 315 4. DeFronzo RA Ferrannini E Hendler R Felig P Wahren J. Regulation of splanchnic and peripheral glucose uptake by insulin and hyperglycemia in man. Diabetes 1983 32:32-45. 5. Baron AD Brechtel G Wallace P Edelman SY. Rates and tissue sites of non• insulin- and insulin-mediated glucose uptake in humans. Am J Physiol 1988 255:E769-E774. 6. Cheatham B Kahn CR. Insulin action and the insulin signaling network. Endo• crine Rev 1995 16:117-142. 7. Gao J Ren J Gulve EA Holloszy JO. Additive effect of contractions and insulin on GLUT-4 translocation into the sarcolemma. J Appl Physiol 1994 77:1587- 1601. 8. Etgen GJ Wilson CM Jensen J Cushman SW Ivy JL. Glucose transport and cell surface GLUT-4 protein in skeletal muscle of the obese Zucker rat. Am J Physiol 1996 271 :E294-E30 I. 9. Holloszy JO Narahara HT. Studies of tissue permeability. X. Changes in perme• ability to 3-methylglucose associated with contraction of frog muscle. J Biol Chem 1965 240:3493-3500. JO. Garetto LP Richter EA Goodman MN Ruderman NB. Enhanced muscle glu• cose metabolism after exercise in the rat: the two phases. Am J Physiol 1984 246:E47 I-E475. 11. Nesher R Karl IE Kipnis DM. Dissociation of effects of insulin and contraction on glucose transport in rat epitrochlearis muscle. Am J Physiol 1985 249:C226- C232. 12. Goodyear LJ Hirshman MF Horton ES. Exercise-induced translocation of skele• tal muscle glucose transporters. Am J Physiol 1991 261:E795-E799. 13. Richter EA Garetto LP Goodman MN Ruderman NB. Enhanced muscle glu• cose metabolism after exercise: modulation by local factors. Am J Physiol 1984 246:E476-E482. 14. Wallberg-Henriksson H Constable SH Young DA Holloszy JO. Glucose trans• port into rat skeletal muscle: interaction between exercise and insulin. J Appl Physiol 1988 65:909-913. 15. Henriksen EJ Bourey RE Rodnick KJ Koranyi L Permutt MA Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol 1990 259:E593-E598. 16. Goodyear LJ Giorgino F Balon TW Condorelli G Smith RJ. Effects of contrac• tile activity on tyrosine phosphoproteins and PI 3-kinase activity in rat skeletal muscle. Am J Physiol 1995 268:E987-E995. 17. DeFronzo RA Fcrrannini E. Insulin resistance. A multifaceted syndrome respon• sible for NIDDM obesity hypertension dyslipidernia and atherosclerotic car• diovascular disease. Diabetes Care 1991 14: 173-194. 18. Reaven GM. Role of insulin resistance in human disease. Diabetes 1988 37: 1595-1607. 19. Reaven GM. Role of insulin resistance in human disease syndrome X: an ex• panded definition. Annu Rev Med 1993 44: 121-131.

slide 373:

316 Henriksen 20. Ferrannini E. Haffner SM Mitchell BD Stern MP. Hyperinsulinaemia: the key feature of a cardiovascular and metabolic syndrome. Diabetologia 1991: 34:416- 422. 21. Reaven GM. The fourth Musketeer-fromAlexander Dumas to Claude Bernard. Diabetologia 1995 38:3-13. 22. King GL. The role of hyperglycaemia and hyperinsulinaemiain causing vascular dysfunction in diabetes. Ann Med 1996: 28:427-432. 23. Asayama K Nakane T Uchida N Hayashibe H Dobashi K Nakazawa S. Serum antioxidant status in streptozotocin induced diabetic rats. Horm Metab Res 1994: 26:313-315. 24. Kainulainen H Breiner M Schurmann A Marttinen A Vi1jo A Joost HG. In vivo glucose uptake and glucose transporter proteins GLUT and GLUT4 in heart and various types of skeletal muscle from strcptozotocin-diabetic rats. Bio• chem Biophys Acta 1994: 1225:275-282. 25. Khamaisi M. Potashnik R Tirosh A Demshchak E Rudich A Tritschler H Wessel K Bashan N. Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats. Metabolism 1997 46:763- 768. 26. Bray GA. The Zucker-fatty rat: a review. Federation Proc 1977 36: 148-153. 27. Becker-Zimmerman K Berger M. Berchtold P Gries FA. Herberg L Schwenen M. Treadmill training improves intravenous glucose tolerance and insulin sensi• tivity in fatty Zucker rats. Diabetologia 1982: 22:468-474. 28. Ionescu E Sauter JF Jeanrenaud B. Abnormal oral glucose tolerance in geneti• cally obese fa/fa rats. Am J Physiol 1985 248:E500-E506. 29. Turner NC. Gudgeon C. Toseland N. Effects of genetic hyperinsulinemia on vascular reactivity blood pressure. and renal structure in the Zucker rat. J Cardio• vase Pharmacol 1995: 26:714-720. 30. Mathe D. Dyslipidemia and diabetes: animal models. Diab Metabol 1995: 21: 106-111. 31. King PA Horton ED Hirshman MF Horton ES. Insulin resistance in obese Zucker rat fa/fa is associated with a failure of glucose transporter translocation. J Clin Invest 1993 90: 1568-1575. 32. Crettaz M Prentki M Zaninetti D Jeanrenaud B. Insulin resistance in soleus muscle from obese Zucker rats: involvement of several defective sites. Biochem J 1980: 186:525-534. 33. Henriksen EJ Jacob S. Effects of captopril on glucose transport activity in skele• tal muscle of obese Zucker rats. Metabolism 1995 44:267-272. 34. Anai M Funaki M Ogihara T Terasaki J Inukai K Katagiri H Fukushima Y. Yazaki Y Kikuchi M Oka Y Asano T. Altered expression levels and impaired steps in pathways to phosphotidylinositol-3-kinascactivation via insulin receptor substrates I and 2 in Zucker fatty rats. Diabetes 1998 47: 13-23. 35. Pritchard KA Jr. Patel ST. Karpen CW. Newman HA Panganamala RV. Triglyc• eride-lowering effect of dietary vitamin E in stroptozotocin-induceddiabetic rats: increased lipoprotein lipase activity in livers of diabetic rats fed high dietary vitamin E. Diabetes 1986 35:278-281.

slide 374:

Oxidative Stress and Antioxidant Treatment 317 36. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritschler H Low PA. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Dia• betes Care 1995 18:160-1167. 37. Cortez MY Torgan CE Brozinick JT Ivy JL. Insulin resistance of obese Zucker rats exercise trained at two different intensities. Am J Physiol 1991 261 :E6 I 3- E6 I 9. 38. Henriksen EJ Jacob S Fogt DL Youngblood EB Godicke J. Antihypertensive agent moxonidine enhances muscle glucose transport in insulin-resistant rats. Hypertension 1997 30:1560-1565. 39. Nourooz-Zadeh J. Antioxidant and prooxidant profile in diabetes mellitus and its relevance to the onset of the syndrome. Proceedings of the 1998 Oxygen Club of California World Congress Santa Barbara California February 6-8 1998. 40. Paolisso G DAmore A Volpe C Balbi V Saccomanno F Galzerano D Giugli• ano D Varricchio M DOnofrio F. Evidence for a relationship between oxidative stress and insulin action in NIDDM patients. Metabolism 1994 43: 1426-1429. 41. Yijayalingam S Parthiban A Shanmugasundararn KR Mohan Y. Abnormal an• tioxidant status in impaired glucose tolerance and non-insulin-dependent diabetes mellitus. Diabet Med 1996 13:715-719. 42. Salonen JT Nyyssonen K Tuornainen TP Maenpaa PH Korpela H Kaplan G Lynch J Heimrich SP Salonen R. Increased risk of non-insulin-dependent diabe• tes mellitus at low plasma vitamin E concentrations: a four-year follow-up study in men. Br Med J 1995 31 l:l l24-l 127. 43. Nourooz-Zadeh J Rahimi A Tajaddini-Sarmadi J Tritschler H Rosen P Halli• well B Betteridge DJ. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997 40:647-653. 44. Jacob S Streeper RS Fogt DL Hokama JY Tritschler HJ Dietze GJ Henriksen EJ. The antioxidant alpha-lipoic acid enhances insulin-stimulated glucose metab• olism in insulin-resistant skeletal muscle. Diabetes 1996 45:1024-1029. 45. Streeper RS Henriksen EJ Jacob S Hokama JY Fogt DL Tritschler HJ Dietze GJ. Differential effects of stereoisomers of alpha-lipoic acid on glucose metabo• lism in insulin-resistant rat skeletal muscle. Am J Physiol 1997 273:El 85-E 191. 46. Tsakiridis T Estrada DE Tritschler H. Klip A. Thioctic lipoic acid induces protein tyrosine phosphorylation and phosphotidylinositol 3-kinase activation in muscle cells abstr. Diabetologia 1995 38:Al32. 47. Henriksen EJ Jacob S Streeper RS Fogt DL Hokama JY Tritschler HJ. Stimu• lation by alpha-lipoic of glucose transport activity in skeletal muscle of lean and obese Zucker rats. Life Sci 1997 61 :805-812.

slide 375:

This Page Intentionally Left Blank

slide 376:

20 Oxidative Stress and Insulin Action: A Role for Antioxidants Click Here For Best Diabetes Treatment Stephan Jacob Rainer Lehmann Kristian Rett and Hans-Ulrich Haring University of Tiibingen Tiibingen Germany There is increasing evidence that alterations in the capacity to reduce oxidants like superoxide anion radical 02 hydrogen peroxide H202 hydroxyl radi• cal OH- nitric oxide NO and alkyl or peroxyl radicals could play an important role in the pathogenesis of various diseases. The imbalance between oxidants and antioxidants in favor of the oxidants so-called oxidative stress results in a nonenzymatic free radical-mediated oxidation of biological mole• cules membranes and tissues associated with a variety of pathological events. Although it is generally acknowledged that oxidative stress plays a role in the development of angiopathy in diabetes mellitus and its vascular compli• cations l-4 there is little information available about the impact of radical oxygen species in the pathogenesis of insulin resistance and the development of diabetes mellitus 5. Several groups have shown that the levels of free radicals are increased when metabolic control is poor. This is found in both type I 6 and type 2 diabetes 78. Indices of this augmented oxidative stress are reduced or even reversed to normal when glycemia is well controlled and this can be shown even after a very short period of time of improved glycemic control 9. Better metabolic control was also clearly shown to be associated with a drastic reduction of diabetic complications 10-12. These observations could suggest that oxida• tive stress does not play a role in the nonhyperglycemic/euglycemic state.

slide 377:

319

slide 378:

320 Jacob et al. However oxidative stress also seems to be present in uncomplicated type 2 813 or type 1 6 diabetes. Furthermore few reports describe increased radical formation in sev• eral conditions without clinical diabetes mellitus such as dyslipidemia im• paired glucose tolerance hypertension coronary artery disease aging and smoking 1214-20. These conditions have also been found to be asso• ciated with a decrease in insulin sensitivity 21-24 but contrary to the dia• betic state effects mediated by hyperglycemia can be excluded. Therefore it seems that the origin of oxidative stress cannot be solely explained by hyper• glycemia. I. OXIDATIVE STRESS AND INSULIN SENSITIVITY• CLINICAL OBSERVATIONS Paolisso et al. 25 demonstrated close correlations between the presence of 02 and insulin sensitivity in an elderly nondiabetic population. Epidemiologi• cal studies found a close correlation between low levels of antioxidants such as vitamin E or vitamin C and a high risk of developing frank type 2 diabetes 2627. Several groups report a higher prevalence of radical oxygen species in prediabetic individuals who had an impaired oral glucose tolerance test 17-19. An increase in plasma thiobarbituric acid reactive substance TBARS was found in healthy subjects when free fatty acids FFA were experimentally kept elevated by an infusion of intralipid and heparin 28 under these experi• mental conditions insulin sensitivity was markedly reduced 28. It could thus be speculated that the elevation of FFA seen in patients with type 2 diabetes or with insulin resistance 29 could be a source for such an augmented oxida• tive stress. 11. ROLE OF IMPAIRED INSULIN ACTION IN THE PATHOGENESIS OF TYPE 2 DIABETES MELLITUS In type 2 diabetes mellitus plasma glucose levels are elevated as a result of an impairment of several metabolic pathways 29-31 Table 1 . Skeletal mus• cle is the principal organ for postprandial glucose uptake 2930.In the patho• genesis of diabetes mellitus type 2 reduced insulin-stimulated glucose dis• posal insulin resistance plays a key role 2129. When clearance of plasma glucose is impaired blood glucose after a meal will remain slightly elevated

slide 379:

Oxidative Stress and Insulin Action 321 Table 1 Metabolic Alterations in Type 2 Diabetes Diminished insulin-mediated peripheral glucose disposal and metabolism insulin resistance Impaired insulin secretion reduced first-phase response prolonged second phase Decreased insulin-mediated inhibition of lipolysis Increased gluconeogenesis and will thus induce hyperinsulinemia to overcome resistance Fig. l . Hyper• insulinemia however will evoke an alteration of the insulin-signaling cas• cade which will further augment insulin resistance thus leading to a vicious cycle 2930. There is an ongoing scientific discussion as to whether insulin resistance of skeletal muscle or an impairment of insulin secretion is the first and princi• pal disorder. However epidemiological data indicate that reduced insulin sen• sitivity can already be demonstrated when insulin secretion is still adequate Genes Physical inactivity Obesity Saturated rmokl_n_g_/ i_S_N_S _. lnsulin eitance Glucose t Genes B-oel7oo Negative modulation of the insulin signaling chain •----------- Hyperinsulinemia Figure 1 Vicious cycle of insulin resistance and hyperinsulinemia. Due to the re• duced insulin sensitivity peripheral glucose uptake is diminished. Therefore plasma glucose clearance is reduced and postprandial blood glucose will remain slightly higher. This will consequently induce hyperinsulinemia to overcome the insulin resis• tance. Hyperinsulinemia however will evoke a negative modulation of the insulin signaling chain which will further exacerbate the insulin resistance hence leading to a vicious cycle. SNS sympathetic nervous system.

slide 380:

I 322 Jacob et al. nspecies .j. EDNO- formation .Jvasodilation .J.peripheralblood flow r contribution of "I /- alter:t:O:don:bollc radicalox 1 \. insulinresistance ""-----"c lnsuli resistance .J. Glucose-uptake Glucose t B-cell secretion Negative modulation of the insulin signalingchain Hyperinsul Figure 2 Model of the potential pathomechanism induced by oxidative stress. Inter• play between hemodynamic and metabolic alterations. EDNO endothelium-derived nitric oxide. 29. Thus it is currently believed that as long as hyperinsulinemia can com• pensate for insulin resistance glucose tolerance will be norrnal with progres• sive impairment of P-cell function impaired glucose tolerance iGT or frank type 2 diabetes will be the consequence 29 Fig. 2. Ill. NITRIC OXIDE AND INSULIN SENSITIVITY A. Nitric Oxide and Endothelial Dysfunction Several groups have shown that insulin-resistant subjects have a reduced insulin-stimulated increase in leg blood flow the changes in peripheral blood flow were closely associated with the degree of insulin resistance 2232-35. This endothelialdysfunctionis demonstratednot only in patients with type 2 dia• betes but also in nondiabetic subjects such as obese adolescents or first-degree relatives of patients with type 2 diabetes 333536. It seems that peripheral blood flow is augmented via an insulin-mediated NO-dependent process Fig. 3 and inhibition of NO formation prevents the

slide 381:

aired glucose tolerance euglycemia Oxidative Stress and Insulin Action 323 progressive Impairment of B-cell function -------""imp INSULIN RESISTANCE Figure 3 Progress from insulin resistance to type 2 diabetes mellitus. insulin-induced increase in blood flow 37. Thus NO could indirectly affect insulin sensitivity as it increases peripheral blood flow and subsequent sub• strate delivery to the skeletal muscle. However there is still a debate about the clinical relevance of these findings. B. IntracellularNO-A Direct Modulator of Glucose Uptake Very recently it was shown that NO synthase is also present within the skeletal muscle 3839 and NO was found to increase glucose uptake by an insulin• independent mechanism 40-43. Furthermore Roberts et al. 40 reported that exercise-stimulated glucose transport in the skeletal muscle of rats is NO dependent this group provides evidence that NO is markedly involved in the regulation of exercise-induced glucose uptake. Exercise-induced skeletal mus• cle glucose uptake is normal even in insulin-resistant animals 44. Thus the exercised-induced elevation of NO availability 4042 seems to provide an important alternative pathway. Animal studies indicate an improvement of endothelial function after exercise training 45. C. NO-A Radical Scavenger NO is also known to act as a radical scavenger itself 4647. An increased availability of NO was associated with a decrease in the levels of superoxide

slide 382:

324 Jacob et al. 47. Furthermore exercise training was not only found to improve metabo• lism and endothelial function 404245 but also antioxidant defense 48 therefore one of the beneficial effects of exercise could be mediated by the increase of antioxidant defense mechanism. Indirect support for the associa• tion between NO availability and insulin sensitivity emerges from a clinical study in which L-arginine the substrate required for NO synthesis was in• fused and insulin sensitivity and endothelial function was improved 49 the increase of insulin sensitivity however remained significant after adjustment for changes in blood flow 49. It is thus conceivable that an increase in radical oxygen species may reduce the availability of NO and this consequently would contribute to the endothelial dysfunction and possibly to the development of insulin resistance Fig. 3. D. Homocysteine NO and Insulin Sensitivity Homocysteine levels were found to be significantly higher in patients with coronary artery disease and in those with diabetes mellitus 50-54. Elevated homocysteine levels induce oxidative stress and reduce NO availability 55 this can contribute to endothelial dysfunction. It seems possible that hyperho• mocysteinemia could also alter insulin sensitivity by this mechanism. There• fore it remains to be clarified whether there are any interactions between ele• vated homocysteine levels and the development of insulin resistance in nondiabetic subjects. The interesting observation of an augmented oxidative stress insulin resistance and elevated homocysteine levels in smokers 14232450 sug• gests some interactions. However this still remains to be evaluated. IV. INTERVENTIONS KNOWN TO ALTER RADICAL OXYGEN SPECIES A. Lifestyle A diet high in saturated fat and low in fiber and a low level of physical activity are associated with an increased risk of developing type 2 diabetes mellitus 2627. Modifications of these factors are known to improve metabolic control and insulin sensitivity 56 but should be also expected to reduce oxidative stress. Experimental data suggest that exercise training can improve insulin resistance in parallel with a better antioxidant defense 45. One explanation

slide 383:

Oxidative Stress and Insulin Action 325 for this could be the improvement of NO availability in the skeletal muscle see above. Epidemiological data support the protective role of regular exercise: Greater physical activity 57-60 reduces the risk of developing type 2 diabe• tes mellitus even in those with a family history of type 2 diabetes. Because smoking is known to be associated with an increased oxidative stress 14 and insulin resistance 2324 this association could be one mechanism by which smoking cessation improves insulin sensitivity 61 . B. Pharmacological Intervention Several compounds with an antioxidant potential were found to modify insulin sensitivi ty. 1. Troglitazone Troglitazone a thiazolidinedione which is the first compound of the new class of insulin sensitizers 6263 improves glycemia and dyslipidemia by reducing insulin resistance and hyperinsulinemia in type 2 diabetes mellitus 64 and also in normoglycemic subjects with insulin resistance 6566. Experimental data indicate that troglitazone improves insulin action by various mechanisms 67-69 Table 2. Troglitazone has a similar structure to vitamin E and is also known to be a potent radical scavenger 70- 72. At present it is not known whether the radical scavenging ability of troglitazone is relevant for its beneficial effect on insulin resistance. Pioglitazone another thiazolidinedione is also a potent insulin sensitizer but an experimental study recently reported that it had no radical scavenging property 72. 2. Glutathione Administration of glutathione was found to be advantageous in type 2 diabetics and those with impaired glucose tolerance Table 2. Glutathione improved insulin secretion in patients with iGT 73. Because insulin secretion is im• paired in type 2 diabetes 29 and because recent data suggest that lipotoxicity might play a role in decreasing -cell function 7475 glutathione could im• prove -cell function by protecting the -cell. However this hypothesis still needs to be tested. Glutathione also improves insulin sensitivity in patients with type 2 dia• betes after acute 76 and chronic administration 49. In type 2 diabetes a

slide 384:

326 Jacob et al. Table 2 Synopsis of the Effects of Interventions Improving Antioxidant Capacity and Insulin Sensitivity Intervention Direct Indirect Other effects Ref. Diet Exercise yes yes endothelial function t NO-availability I 2-9 Smoking endothelial function 1011 cessation Troglitazone yes endothelial function PPARy Glutathione yes endothelial function J glucose toxicity i LDL-oxidation i insulin secretion in 12-19 20-24 iGT i microviscosity Vitamin E endothelial function J "Iipotoxicity" i vasodilation 25-32 no effect on insulin secretion Vitamin C endothelial function t insulin-stimulated 3133-35 glucose uptake t vasodilation no effect on insulin secretion Thioctic acid yes t insulin-independent t insulin-stimulated 36-45 glucose uptake glucose uptake endothelial function i vasodilation J of adhesion mole- i microcirculation cules NF-K Bl Others ACE inhibi- yes endothelial function insulin-stimulated 46-48 tors glucose uptake is kinin- mediated Vasodilating endothelial function 4950 beta- blockers

slide 385:

Oxidative Stress and Insulin Action 327 reduced plasma GSH/GSSG ratio was found this was negatively associated with the levels of fasting FFA r -0.53 p 0.05 49. In an experimental study with healthy volunteers "metabolic oxidative stress" was induced by infusion of intralipid and heparin resulting in a marked rise of FFA levels. This was associated with an increase of indicators of oxidative stress as re• flected by increased TBARS and a reduced GSH/GSSG ratio 28. In contrast the infusion of glutathione diminished the negative effect of the sustained elevation of FFA although FFA were elevated the alteration of both oxidative stress and insulin-stimulated glucose uptake were markedly attenuated when glutathione was coinfused with intralipid. In addition glutathione even im• proved insulin sensitivity and oxidative stress in the control experiment in which no intralipid was given 28. Ammon et al. 77 showed that the administration of acetyl-cysteine a compound that could increase endogenous formation of glutathione improved glucose disposal in healthy volunteers this was associated with an improved GSH/GSSG ratio. Furthermore experimental studies indicate a protective role of glutathione on endothelial function 78. 3. Vitamin E A large epidemiological study indicates that a low level of vitamin E confers a marked risk for the development of a type 2 diabetes mellitus 26. Low levels of vitamin E are also documented in patients with coronary artery dis• ease 20. A regular intake of higher doses of vitamin E was associated with a marked decline in vascular events in coronary artery disease patients 79. These observations suggest a role for vitamin E and/or oxidative stress in these chronic diseases. In experimental studies it was shown that vitamin E has beneficial ef• fects on insulin sensitivity. Fructose feeding induces insulin resistance and hypertension in rats this is also associated with an increase in radical oxygen species formation 80. Vitamin E administration prevented not only the diet• induced alterations in insulin sensitivity but also reduced oxidative stress. Fi• nally several clinical studies by Paolissos group suggest that vitamin E intake improves insulin sensitivity as measured by the glucose clamp technique in healthy and diabetic subjects 81-84. Vitamin E could also modulate insulin sensitivity by indirect effects because it can improve endothelial function. It was shown that in diabetes mellitus endothelial dysfunction is present 14 and it is suggested that oxida• tive stress reduces vasodilatation 38586. Vitamin E administration restores this defect 38586.

slide 386:

ll 328 Jacob et al. If endothelial dysfunction is involved in modulating insulins action a restoration of endothelial function should also augment insulin sensitivity see Ill.A and Table 2 Fig. 4. Furthermore vitamin E improves insulin secretion in experimentally induced type 1 diabetes mellitus 87. At present however there are no data concerning the effect of vitamin E on -cell function in type 2 diabetes mellitus. 4. Vitamin C There are a few studies indicating a role for vitamin C in modulating insulin sensitivity. Epidemiological data identify low serum levels of the vitamin as a risk factor for the development of type 2 diabetes mellitus 27. Experimental data describe a protective role of vitamin C on the age-associated deterioration of insulin sensitivity 88. Clinical studies describe an enhanced insulin-stimu• lated glucose uptake in a glucose clamp study after acute vitamin C treatment in healthy subjects and in those with type 2 diabetes 8990 the beneficial effects on insulin sensitivity were closely associated with the increases in the plasma levels of the vitamin C 90. Experimental studies indicate also a bene• ficial effect on endothelial function in experimental polyneuropathy 3. Clini• cal studies also describe an improvement of endothelial dysfunction 91. - Oxidative stress -T lation / rlpheral b tc•• •• -r oo---- •J.Glucoaeupta ke ----•""- / I Negative modation of the Insulin sign alling chain Hyperlnsullnomla Figure 4 Decrease of insulin resistance by antioxidants hypothetical mechanisms.

slide 387:

Oxidative Stress and Insulin Action 329 5. a-Lipoic Acid Thioctic acid also known as a-lipoic acid was found to improve insulin action in various experimental models 92-95. Its action seems to involve interac• tion with the insulin receptor signaling cascade and potentially also insulin• independent steps see Chaps. 18 and 19. In vivo experimental studies found an improvement of insulin sensitivity and glucose tolerance after the adminis• tration of the racemic mixture 9596. Clinical pilot trials suggest that this compound might also have benefi• cial effects in humans because insulin resistance was improved after acute or chronic intravenous administration of u-lipoic acid 97-99. Recently a small placebo-controlled pilot trial found an improvement of insulin sensitivity in patients with type 2 diabetes mellitus after oral administration 100. The changes in insulin sensitivity seen after the active treatment was significantly different from that seen in placebo: whereas insulin sensitivity decreased in the control group it improved after o-Iipoic acid. Furthermore thioctic acid improves endothelial function 101 and microcirculation by reducing adhe• sion molecules 102 and preserves endothelial structure 103. 6. Other Compounds There are several other compounds with antioxidant activity also shown to have a beneficial effect on insulin sensitivity. Two groups of antihypertensive agents are known to improve insulin sensitivity and oxidative stress. Angiotensin-converting enzyme inhibitors increase the availability of kinins by inhibition ofkininase II 104 and consequently NO 47. They were shown to augment insulin-stimulated glucose uptake in clinical and experi• mental studies 104105. Recent data suggest that treatment with Ramipril has pronounced effects on NO synthase expression and NO formation and a concomitant decrease in superoxide accumulation which was associated with an extended lifespan in the angiotensin-converting enzyme treated rats 47. The vasodilating beta-blockers carvedilol and celiprolol have a marked antioxidant capacity 46 l 06 l 07 and were found to improve endothelial func• tion 108. In clinical studies they increased insulin sensitivity as documented by the glucose clamp I 09-111 . V. CONCLUSION AND OUTLOOK At present experimental and clinical data suggest but do not prove an associa• tion between insulin sensitivity and oxidative stress. Furthermore experimen-

slide 388:

330 Jacob et al. ta and some clinical studies suggest a beneficial effect on insulin secretion or insulin action after treatment with certain antioxidants. Currently it is still unknown whether the effects on insulin sensitivity are modulated by direct mechanisms for instance on the insulin receptor-signal transduction cascade or whether metabolism improves indirectly such by an improvement of endo• thelial function. It is necessary and seems to be promising to analyze further the associa• tion between oxidative stress and insulin action 112 this involves both the quantitative assessment of insulin sensitivity and the radical oxygen species respective of the oxidant defense system. If an antioxidant is supposed to improve insulin sensitivity by decreasing oxidative stress this should be associated with a decrease in radical oxygen species. Furthermore it would be important to show a dose-response relation• ship that is the more the oxidative stress is reduced the better the insulin resistance is improved. To date only small clinical trials with a short duration of treatment have been conducted Table 2. It is absolutely necessary in the near future to con• duct larger trials involving intensive assessment of the oxidative stress and antioxidant defense and the exact analysis of insulin sensitivity and metabolic control. Therefore there is a need for a "radical initiative" 113. REFERENCES I. Giugliano D Ceriello A Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996 19:257-267. 2. Giugliano D Ceriello A Paolisso G. Diabetes mellitus hypertension and car• diovascular disease: which role for oxidative stress Metabolism 199544:363- 368. 3. Cotter MA Love A Watt MJ Cameron NE Dines KC. Effects of natural free radical scavengers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia 1995 38: 1285-1294. 4. Brownlee M Cerami A Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988 318:1315-1321. 5. Paolisso G Giugliano D. Oxidative stress and insulin action: is there a relation• ship Diabetologia 1996 39:357-363. 6. Santini SA Marra G Giardina B Cotroneo P Mordente A. Martorana GE et al. Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM. Diabetes 1997 46: 1853-1858. 7. Nourooz Zadeh J Tajaddini Sarmadi J McCarthy S Betteridge DJ Wolff SP.

slide 389:

Oxidative Stress and Insulin Action 331 Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995 44: 1054-1058. 8. Ceriello A Bortolotti N Falleti E Taboga C Tonutti L Crescentini A et al. Total radical-trapping antioxidant parameter in NIDDM patients. Diabetes Care 1997 20:194-197. 9. Peuchant E Delmas Beauvieux MC Couchouron A Dubourg L Thomas MJ Perrornat A et al. Short-term insulin therapy and normoglycemia. Effects on erythrocyte lipid peroxidation in NIDDM patients. Diabetes Care 1997 20: 202-207. I 0. Lasker RD. The diabetes control and complications trial. Implications for policy and practice. N Engl J Med 1993 329: 1035-1036. 11. The effect of intensive treatment of diabetes on the development and progres• sion of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993 329:977-986. 12. Implications of the Diabetes Control and Complications Trial. American Diabe• tes Association. Diabetes 1993 42: 1555-1558. 13. Freitas JP Filipe PM Rodrigo FG. Lipid peroxidation in type 2 normolipidemic diabetic patients. Diabetes Res Clin Pract 1997 36:71-75. 14. Marangon K Herbeth B Lecomte E PaulDauphin A Grolier P Chancerelle Y et al. Diet antioxidant status and smoking habits in French men. Am J Clin Nutr 1998 67:231-239. 15. Paolisso G Di Maro G Pizza G D Amore A Sgambato S Tesauro P et al. Plasma GSH/GSSG affects glucose homeostasis in healthy subjects and non• insulin-dependent diabetics. Am J Physiol 1992 263:E435-E440. 16. Paolisso G DAmore A Volpe C Balbi V Saccomanno F Galzerano D et al. Evidence for a relationship between oxidative stress and insulin action in non-insulin-dependent type II diabetic patients. Metabolism 1994 43: 1426- 1429. 17. Vijayalingam S Parthiban A Shanmugasundaram KR Mohan V. Abnormal antioxidant status in impaired glucose tolerance and non-insulin-dependent dia• betes mellitus. Diabet Med 1996 13:715-719. 18. Sadasivudu B Sasikala M Sailaja V Reddy SS. Serum malondialdehyde insu• lin glucose and lipid profile in hypertension. Med Sci Res 1997 25:631- 633. 19. Niskanen LK Salonen JT Nyyssonen K Uusitupa Ml. Plasma lipid peroxida• tion and hyperglycaemia: a connection through hyperinsulinaemia Diabet Med I 995 12:802-808. 20. Regnstrom J Nilsson J Moldeus P Strom K Bavenholm P Tomvall P et al. Inverse relation between the concentration of low-density-lipoprotein vitamin E and severity of coronary artery disease. Am J Clin Nutr 1996 63:377-385. 21. DeFronzo RA Ferrannini E. Insulin resistance. A multifaceted syndrome re• sponsible for NIDDM obesity hypertension dyslipidemia and atherosclerotic cardiovascular disease. Diabetes Care 1991 14: 173-194.

slide 390:

332 Jacob et al. 22. Lind L. Lithell H. Decreased peripheral blood flow in the pathogenesis of the metabolic syndrome comprising hypertension hyperlipidemia and hyperinsuli• nemia. Am Heart J 1993 125:1494-1497. 23. Eliasson B Attvall S Taskinen MR Smith U. The insulin resistance syndrome in smokers is related to smoking habits. Arterioscler Thromb 1994 14: I 946- 1950. 24. Eliasson B Mero N Taskinen MR. Smith U. The insulin resistance syndrome and postprandial lipid intolerance in smokers. Atherosclerosis 1997 129:79- 88. 25. Paolisso G D Amore A Di Maro G Galzerano D Tesauro P Varricchio M ct al. Evidence for a relationship between free radicals and insulin action in the elderly. Metabolism 1993 42:659-663. 26. Salonen JT Nyyssonen K Tuomainen TP Maenpaa PH Korpela H Kaplan GA. et al. Increased risk of non-insulin dependent diabetes mellitus at low plasma vitamin E concentrations: a four year follow up study in men. BMJ 1995 311: 1124-1127. 27. Feskens EJ. Virtanen SM Rasanen L. Tuomilehto J Stengard J Pekkanen J et al. Dietary factors determining diabetes and impaired glucose tolerance. A 20-year follow-up of the Finnish and Dutch cohorts of the Seven Countries Study. Diabetes Care 1995 18:1104-1112. 28. Paolisso G Gambardella A Tagliamonte MR Saccomanno F. Salvatore T Gu• aldiero P et al. Does free fatty acid infusion impair insulin action also through an increase in oxidative stress J Clin Endocrinol Metab 1996 81 :4244- 4248. 29. DeFronzo RA Bonadonna RC Ferrannini E. Pathogenesis of NIDDM. A bal• anced overview. Diabetes Care 1992 15:318-368. 30. Hating H-U. Mehnert H. Pathogenesis of type 2 non-insulin-dependent diabe• tes mellitus: candidates for a signal transmitter defect causing insulin resistance of the skeletal muscle. Diabetologia 1993 36: 176-182. 31. Kellerer M Haring HU. Pathogenesis of insulin resistance: modulation of the insulin signal at receptor level. Diabetes Res Clin Pract 1995 28suppl:S 173- S I 77. 32. Baron AD. Hemodynamic actions of insulin. Am J Physiol 1994 267:El87- E202. 33. Baron AD Brechtel Hook G Johnson A Hardin D. Skeletal muscle blood flow. A possible link between insulin resistance and blood pressure. Hypertension 1993 21:129-135. 34. Petrie JR Ueda S Webb DJ Elliott HL Connell JM. Endothelial nitric oxide production and insulin sensitivity. A physiological link with implications for pathogenesis of cardiovascular disease. Circulation 1996 93: 1331-1333. 35. Rocchini AP Moorehead C Katch V Key J Finta KM. Forearm resistance vessel abnormalities and insulin resistance in obese adolescents. Hypertension 1992 19:615-620. 36. Rittig K Balletshofer B Enderle M Volk A Maerker E Pfohl M et al. Endo-

slide 391:

Oxidative Stress and Insulin Action 333 thelial dysfunction and intima-media thickness in healthy but insulin-resistant first degree realtives of type 2 diabetics abstr. Diabetes 1998 47:AI20. 37. Steinberg HO Brechtel G Johnson A Fineberg N Baron AD. Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J Clin Invest 1994 94: I 172-1179. 38. Kapur S Bedard S Marcotte B Cote CH Marette A. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. Diabetes 1997 46:1691-1700. 39. Frandsen U Lopez Figueroa M Hellsten Y. Localization of nitric oxide syn• thase in human skeletal muscle. Biochem Biophys Res Commun 1996 227: 88-93. 40. Roberts CK Barnard RJ Scheck SH Balon TW. Exercise-stimulated glucose transport in skeletal muscle is nitric oxide dependent. Am J Physiol 1997 273: E220-E225. 41. Young ME Leighton B. Evidence for altered sensitivity of the nitric oxide/ cGMP signalling cascade in insulin-resistant skeletal muscle. Biochem J 1998 329:73-79. 42. Balon TW Nadler JL. Evidence that nitric oxide increases glucose transport in skeletal muscle. J Appl Physiol 1997 82:359-363. 43. Etgen GJ Jr. Fryburg DA Gibbs EM. Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction- and phosphatidylinositol-3- kinase-independent pathway. Diabetes 1997 46:1915-1919. 44. Kusunoki M Storlien LH MacDessi J Oakes ND Kennedy C Chisholm DJ et al. Muscle glucose uptake during and after exercise is normal in insulin• resistant rats. Am J Physiol 1993 264:El67-El72. 45. Sakamoto S Minami K Niwa Y Ohnaka M Nakaya Y Mizuno A et al. Effect of exercise training and food restriction on endothelium-dependent relaxation in the Otsuka Long-Evans Tokushima fatty rat a model of spontaneous NIDDM. Diabetes 1998 47:82-86. 46. Mehta JL Lopez LM Chen L Cox OE. Alterations in nitric oxide synthase activity superoxide anion generation and platelet aggregation in systemic hy• pertension and effects of celiprolol. Am J Cardiol 1994 74:901-905. 47. Wiemer G Linz W Hatrik S Scholkens BA Malinski T. Angiotensin• converting enzyme inhibition alters nitric oxide and superoxide release in nor• motensive and hypertensive rats. Hypertension 1997 30:1183-1190. 48. De Angelis KL Oliveira AR Werner A Bock P Bello Klein A Fernandes TG et al. Exercise training in aging: hemodynamic metabolic and oxidative stress evaluations. Hypertension 1997 30:767- 771. 49. Paolisso G Tagliamonte MR Marfella R Verrazzo G DOnofrio F Giugliano D. t.-Arginine but not n-arginine stimulates insulin-mediated glucose uptake. Metabolism 1997 46: 1068-1073. 50. Welch GN Upchurch GR Jr. Loscalzo J. Homocysteine oxidative stress and vascular disease. Hosp Pract Off Ed 1997 32:81-28588-92. 51. Hoogeveen EK Kostense PJ Beks PJ Mackaay AJ Jakobs C Bouter LM et al.

slide 392:

334 Jacob et al. Hyperhomocysteinemiais associated with an increased risk of cardiovascular disease especially in non-insulin-dependent diabetes mellitus: a population• based study. Arterioscler Thromb Vase Biol 1998 18:133-138. 52. Hofmann MA Kohl B Zumbach MS Borcea V Bierhaus A Henkels M et al. Hyperhomocysteinemia and endothelial dysfunction in IDDM. Diabetes Care 1997 20: 1880-1886. 53. Araki A Sako Y Ito H. Plasma homocysteine concentrations in Japanese pa• tients with non-insulin-dependent diabetes mellitus: effect of parenteral methyl• cobalamin treatment. Atherosclerosis 1993 103: 149-157. 54. Stampfer MJ Malinow MR Willett WC Newcomer LM Upson B Ullmann D et al. A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA 1992 268:877-881. 55. Upchurch GR Jr. Welch GN Fabian AJ Freedman JE Johnson JL Keaney JF Jr. et al. Homocysteine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 1997 272:17012-17017. 56. ODea K. Marked improvement in carbohydrate and lipid metabolism in dia• betic Australian aborigines after temporary reversion to traditional lifestyle. Diabetes 1984 33:596-603. 57. Manson JE Nathan DM Krolewski AS Stampfer MJ Willett WC. Hennekens CH. A prospective study of exercise and incidence of diabetes among US male physicians. JAMA 1992 268:63-67. 58. Manson JE Rimm EB Stampfer MJ. Colditz GA Willett WC Krolewski AS et al. Physical activity and incidence of non-insulin-dependent diabetes mellitus in women. Lancet 1991 338:774-778. 59. Lynch J Heimrich SP Lakka TA Kaplan GA Cohen RD Salonen R et al. Moderately intense physical activities and high levels of cardiorespiratory fit• ness reduce the risk of non-insulin-dependent diabetes mellitus in middle-aged men. Arch Intern Med 1996 156:1307-1314. 60. Heimrich SP Ragland DR Leung RW Paffenbarger RS Jr. Physical activity and reduced occurrence of non-insulin-dependentdiabetes mellitus. N Engl J Med 1991 325:147-152. 61. Eliasson B Attvall S Taskinen MR Smith U. Smoking cessation improves insulin sensitivity in healthy middle-aged men. Eur J Clin Invest 1997 27:450- 456. 62. Bethge H Haring HU. The thiazolidinediones-a novel approach for the treat• ment of type-2 diabetes. Arzneim Forsch Drug Res 1998 48:97-119. 63. Hofmann CA. Colca JR. New oral thiazolidinedioneantidiabetic agents act as insulin sensitizers. Diabetes Care 1992 15: 1075-1078. 64. Inzucchi SE Maggs DG Spollett GR Page SL Rife FS Walton V et al. Effi• cacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 1998 338:867-872. 65. Nolan JJ Ludvik B Beerdsen P Joyce M Olefsky J. Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med 1994 331:1188-1193.

slide 393:

Oxidative Stress and Insulin Action 335 66. Cavaghan MK Ehrmann DA Byrne MM Polonsky KS. Treatment with the oral antidiabetic agent troglitazone improves beta cell responses to glucose in subjects with impaired glucose tolerance. 1 Clin Invest 1997 100:530-537. 67. Kellerer M Kroder G Tippmer S Berti L Kiehn R Mosthaf L et al. Troglita• zone prevents glucose-induced insulin resistance of insulin receptor in rat- I fibroblasts. Diabetes 1994 43:447-453. 68. Peraldi P Xu M Spiegelman BM. Thiazolidinediones block tumor necrosis factor-alpha-induced inhibition of insulin signaling. 1 Clin Invest 1997 100: 1863-1869. 69. Shimabukuro M Zhou YT Lee Y Unger RH. Troglitazone lowers islet fat and restores beta cell function of Zucker diabetic fatty rats. 1 Biol Chem 1998 273: 3547-3550. 70. Cominacini L Garbin U Pasini AF Campagnola M Davoli A Foot E. et al. Troglitazone reduces LDL oxidation and lowers plasma E-selectin concentra• tion in NIDDM patients. Diabetes 1998 47: 130-133. 71. Cominacini L Garbin U Pastorino AM Campagnola M Fratta Pasini A Da• voli A et al. Effects of troglitazone on in vitro oxidation of LDL and HDL induced by copper ions and endothelial cells. Diabetologia 1997 40: 165-172. 72. Inoue I Katayama S Takahashi K Negishi K Miyazaki T Sonoda M et al. Troglitazone has a scavenging effect on reactive oxygen species. Biochem Bio• phys Res Commun 1997 235:113-116. 73. Paolisso G Giugliano D Pizza G Gambardella A Tesauro P Varricchio M et al. Glutathione infusion potentiates glucose-induced insulin secretion in aged patients with impaired glucose tolerance. Diabetes Care 1992 15:1-7. 74. Unger RH. Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Ge• netic and clinical implications. Diabetes 1995 44:863-870. 75. Lee Y Hirose H Ohneda M Johnson JH McGarry JD. Unger RH. Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships. Proc Natl Acad Sci USA 1994 91: I 0878-10882. 76. Laurenti 0 Bravi MC Cassone Faldetta M Ferri C Bianco G Armiento A et al. Glutathione effects on insulin resistance in non-insulin-dependent diabetes mellitus abstr. Diabetologia 1997 40:A305. 77. Ammon HP Muller PH Eggstein M Wintermantel C Aigner B Safayhi H et al. Increase in glucose consumption by acetylcysteine during hyperglycemic clamp. A study with healthy volunteers. Arzneim Forsch Drug Res 1992 42: 642-645. 78. Brigelius Flohe R Friedrichs B Maurer S Schultz M Streicher R. lnterleukin- 1-induced nuclear factor kappa B activation is inhibited by overexpression of phospholipid hydroperoxide glutathione peroxidase in a human endothelial cell line. Biochem 1 1997 328: 199-203. 79. Stephens NG Parsons A Schofield PM Kelly F Cheeseman K Mitchinson MJ. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study CHAOS. Lancet 1996 347:781- 786.

slide 394:

336 Jacob et al. 80. Faure P Rossini E Lafond JL Richard MJ Favier A Halirni S. Vitamin E improves the free radical defense system potential and insulin sensitivity of rats fed high fructose diets. J Nutr 1997 127:103-107. 81. Paolisso G D Amore A Giugliano D Ceriello A Varricchio M DOnofrio F. Phnrmacologic doses of vitamin E improve insulin action in healthy subjects and non-insulin-dependent diabetic patients. Am J Clin Nutr 1993 57:650- 656. 82. Paolisso G DAmore A Galzerano D Balbi V Giugliano D. Varricchio M et al. Daily vitamin E supplements improve metabolic control but not insulin secretion in elderly type 11 diabetic patients. Diabetes Care 1993 16:1433- 1437. 83. Paolisso G Di Maro G Galzerano D Cacciapuoti F Varricchio G Varricchio M et al. Pharmacological doses of vitamin E and insulin action in elderly sub• jects. Am J Clin Nutr 1994 59: 1291-1296. 84. Paolisso G Gambardella A Giugliano D Galzerano D Amato L Volpe C et al. Chronic intake of pharmacological doses of vitamin E might be useful in the therapy of elderly patients with coronary heart disease. Am J Clin Nutr 1995 61:848-852. 85. Rosen P Ballhausen T Bloch W Addicks K. Endothelial relaxation is dis• turbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995 38:1157-1168. 86. Keegan A Walbank H Cotter MA Cameron NE. Chronic vitamin E treatment prevents defective endothelium-dependent relaxation in diabetic rat aorta. Dia• betologia 1995 38:1475-1478. 87. Beales PE Williams AJ Albertini MC Pozzilli P. Vitamin E delays diabetes onset in the non-obese diabetic mouse. Honn Metab Res 1994 26:450-452. 88. Moustafa SA Webster JE Mattar FE. Effects of aging and antioxidants on glucose transport in rat adipocytes. Gerontology 1995 41:301-307. 89. Paolisso G Balbi V Volpe C Varricchio G Gambardella A Saccomanno F et al. Metabolic benefits deriving from chronic vitamin C supplementation in aged non-insulin dependent diabetics. J Am Coll Nutr 1995 14:387-392. 90. Paolisso G D Amore A Balbi V Volpe C Galzerano D Giugliano D et al. Plasma vitamin C affects glucose homeostasis in healthy subjects and in non• insulin-dependent diabetics. Am J Physiol 1994 266:E26 I -E268. 91. Timimi FK Ting HH. Haley EA Roddy MA Ganz P Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with insulin-depen• dent diabetes mellitus. J Am Coll Cardiol 1998 31 :552-557. 92. Streeper RS Henriksen EJ Jacob S Hokama JY Fogt DL Tritschler HJ. Dif• ferential effects of lipoic acid stereoisomers on glucose metabolism in insulin• resistant skeletal muscle. Am J Physiol 1997 273:E 185-E 19 l. 93. Estrada DE Ewart HS Tsakiridis T Volchuk A. Ramlal T Tritschler H et al. Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioc• tic acid: participation of elements of the insulin signalling pathway. Diabetes 1996 45: 1798-1804.

slide 395:

Oxidative Stress and Insulin Action 337 94. Strodter D Lehmann E Lehmann U Tritschler HJ Bretzel RG Federlin K. The influence of thioctic acid on metabolism and function of the diabetic heart. Diabetes Res Clin Pract 1995 29: 19-26. 95. Khamisi M Potashnik R Tirosh A. Demshchak E Rudich A Tritschler H et al. Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats. Metabolism 1997 46:763- 768. 96. Jacob S Streeper RS Fogt DL Hokama JY Tritschler HJ Dietze GJ et al. The antioxidant alpha-lipoic acid enhance insulin-stimulated glucose metabolism in insulin-resistant rat skeletal muscle. Diabetes 1996 45: 1024-1029. 97. Jacob S Henriksen EJ Tritschler HJ Augustin JH Dietze GJ. Improvement of insulin-stimulated glucose-disposal in type 2 diabetes after repeated parenteral administration of thioctic acid. Exp Clin Endocrinol Diabetes 1996 104:284- 288. 98. Jacob S Henriksen EJ Schiemann AL Simon I Clancy DE Tritschler HJ et al. Enhancement of glucose disposal in patients with type 2 diabetes by alpha• lipoic acid. Arzneimittelforschung 1995 45:872-874. 99. Rell K Wicklmayr M Maerker E Ruus P Nehrdich D Herrmann R et al. Effect of acute infusion of thioctic acid on oxidative and non-oxidative metabo• lism in obese subjects with NIDDM abstr. Diabetologia 1995 38:A41. 100. Jacob S Ruus P Rett K et al. Oral lipoic acid improves insulin sensitivity• results of a placebo controled trial in patients with type 2 diabetes. Free Radie Biol Med 1999 273-4:309-314. 10 l. Nagamatsu M Nickander KK Schmelzer JD Raya A Wittrock DA Tritsch I er H et al. Lipoic acid improves nerve blood flow reduces oxidative stress and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care 1995 18:1160-1167. 02. Kusterer K Haak E Ulrich H Haak T. Lipoate prevention of diabetic microan• giopathy. In: Fuchs J Packer L Zimmer G eds. Lipoic Acid in Health and Disease. New York: Marcel Dekker 1997:429-434. 103. Hofmann MA Tritschler HJ Bierhaus A Ziegler R Wahl P Nawroth PP. Lipoate effects on atherogenesis. In: Fuchs J Packer L Zimmer G eds. Lipoic Acid in Health and Disease. New York: Marcel Dekker 1997:321-335. 104. Henriksen EJ Jacob S Augustin HJ Dietze GJ. Glucose transport activity in insulin-resistant rat muscle. Effects of angiotensin-converting enzyme inhibi• tors and bradykinin antagonism. Diabetes 1996 45suppl I :S 125-S 128. 105. Lithell HO. Effect of antihypertensive drugs on insulin glucose and lipid me• tabolism. Diabetes Care 1991 14:203-209. I 06. Yue TL Lysko PG Barone FC Gu JL Ruffolo RR Jr Feuerstein GZ. Carvedi• lol a new antihypertensive drug with unique antioxidant activity: potential role in cerebroprotection. Ann NY Acad Sci 1994 738:230-242. 107. Yue TL Mckenna PJ Gu JL Cheng HY Ruffolo RR Jr Feuerstein GZ. Carvedilol a new antihypertensive agent prevents lipid peroxidation and oxida• tive injury to endothelial cells. Hypertension 1993 22:922-928. 108. Giugliano D Marfella R Acampora R Giunta R Coppola L DOnofrio F.

slide 396:

338 Jacob et al. Effects of perindopril and carvedilol on endothelium-dependent vascular func• tions in patients with diabetes and hypertension. Diabetes Care 1998 21 :631- 636. 109. Jacob S Rett K Wicklmayr M Agrawal B Augustin HJ Dietze GJ. Differen• tial effect of chronic treatment with two beta-blocking agents on insulin sensi• tivity: the carvedilol-metoprolol study. J Hypertens 1996 14:489-494. 110. Malminiemi K Lahtela JT Huupponen R. Effects of celiprolol on insulin sensi• tivity and glucose tolerance in dyslipidemic hypertension. Int J Clin Phannacol Ther 1995 33:156-163. 111. Jacob S Rell K Henriksen EJ. Anti-hypertensive therapy and insulin sensitiv• ity-do we have to redefine the role of beta-blocking agents Am J Hypertension 1998 11:1258-1265. l 12. Rudich A Kozlovsky N Potashnik R Bashan N. Oxidant stress reduces insulin responsiveness in 3T3-LI adipocytes. Am J Physiol 1997 35:E935-E940. 113. Cominacini L Garbin U Lo Cascio V. The need for a "free radical initiative." Diabetologia 1996 39:364-366.

slide 397:

Index Acetylcystein GSH/GSSG 327 Adaptation defined 37 Advanced glycation end products AGEs 80 187-190 diabetes. 188. l 89t formation 22-23 138 glycation 285 production 206f VCAM-1 212 Advanced glycation end products AGEs binding proteins localization l 89t AGE-RAGE interactions 188- 190 Akt u-lipcic acid 294 ALADIN study 176-178 ALADIN 3 study 180 Aldose reductase 138 Angiotensin-converting enzyme ACE inhibitors 135 insulin sensitivity 326t 329 Antioxidant enzymes experimental diabetic neuropathy 122 Antioxidant network 3-6 defined 2 diabetes 6-11 Antioxidants atherogenesis 86 Antioxidants autoimmune disease 86 defined 203 essential fatty acid interactions 140-146 insulin resistance 310-313 plasma ROOH 59-61 Antioxidant therapy diabetic complications 191-195 diabetic polyneuropathy 129-146 dose considerations 135 neurovascular function 132-140 rationale 86 Apoptosis 40 oxidative stress 267 AR inhibitor ARI 95 138 NCV 142 AR pathway activation 113-114 mitochondrial dysfunction 114 Ascorbic acid AA 70- 71 284 NCV 135 Ascorbyl-o-Gl.A NCV 142 Atherogenesis antioxidants 86 Atherosclerosis 219 Autoimmune diabetes thioredoxin 269-270 Autoimmune disease antioxidants 86 Autooxidative glycosylation 113 339

slide 398:

340 BAPTA 21 Basic fibroblast growth factor EON 156 BIM 20 Biomarkers see Oxidative stress markers 13-blockers insulin sensitivity 326t Blood flow DAG-PKC activation 245- 247 neuropathy 129-130 Blood glucose peripheral nerve 99 BM 150639 NCV 142 Body weights peripheral nerve 99 Buthionine sulfoximine-induced cata• lase u-lipoic acid 105 Calcium hyperglycemia-mediated 23- 24 Carbonyl assay proteins 45 Carbonyl compounds detoxification 84-85 Carbonyl stress 84-85 Carboxyethyllsysine CEL 81 Carboxymethyllysine CML 81 Cardillo insulin sensitivity 329 Cardiovascular autonomic neuropathy CAN 174 a- lipoic acid 173-182 Caspases 40 Catalase peripheral nerve 98 I 02- 103 CEL 84 Celiprolol insulin sensitivity 329 Index Cell death 39-40 Cell signaling pathways insulin-dependent 11 lipoate 11 Ciliary neurotrophic factor EON 156 Collagen 248 Cuprozinc superoxide dismutase 122 Cyclooxygenases 21 DAG-PKC activation vascular cell alterations 245-249 Deferoxamine EON 135-136 DE KAN study 178-180 Diabetes oxidative stress 6-11 24-26 77- 87 reactive oxygen species ROS 130-132 Diabetes-induced neurovascular dys• function 93-94 Diabetes mellirus AGE 188 I 89t autoimmune 269-270 defined 303 free radicals 205-208 insulin- dependent 265-270 ischemia- reperfusion 209-211 oxidative stress 186-187 thrombomodulin I 95f type I 305 type 2 see type 2 diabetes Diabetic complications antioxidant therapy 191-195 hyperglycemia 186 a-lipoic acid 192-195 NF-KB activation 186 vitamin C 192 vitamin E 191-192 Diabetic distal symmetric sensorimotor polyneuropathy DPN 115 Diabetic microangiopathy 205-213

slide 399:

Index Diabetic neuropathy 173 experimental 121-127 antioxidant enzymes 122 glucose-mediated oxidative stress 111-115 oxidative stress role 174-175 Diabetic polyneuropathy antioxidant therapy 129-146 diabetes I 1-13 lipoic acid 11-13 Diene conjugation 42 Diet insulin sensitivity 326t ROS 324-325 Diurnal variation plasma ROOHs 54-55 DNA oxidative stress markers 41-42 DPN pathogenesis I 12-113 ROS 114 DRG neuropathology 125-126 vascular perfusion 125-126 ELAM-I 213 Electromobility shift assay EMSA 24-25 Endoneurial ischernia/hypoxia oxidative stress 121 Endothelial dysfunction glycoxidation 45 nitric oxide 322-323 synergistic therapy 141- 142 Endothelial function vitamin E 327 Endothelin-1 193 245 Endothelium EDN 132-135 superoxide anions 20-24 vascular experimental diabetes J 32-135 Endothelium-dependent vasodilation 208-209 341 Endothelium-derived hyperpolarizing factor EDHF 141 Energy metabolism EON 127 8-epiPGF2a 42 ESR signal 4 Essential fatty acids antioxidant interactions 140-146 n-6 142-146 neurovascular dysfunction 141 Ethane breath excretion 44 Evening primrose oil NCY 142 Exercise insulin sensitivity 326t ROS 325 Experimental diabetic neuropathy EDN 121-127 antioxidant dose 135 antioxidant enzymes 122 basic fibroblast growth factor 156 energy metabolism 127 essential fatty acids 141 etiology 155-156 fatty acid/antioxidant interaction 140-146 glucose uptake 127 o-tipoic acid 123-124 nerve growth 136-138 neuropathology 125-127 neurotrophic factors 156-157 neurotrophins 157-158 neurovascular function 132-140 NF-KB 139-140 NGF 156 PKC 139-140 polyol pathway 138-139 sciatic nerve 175 thioctic acid-y-linolenic acid 155- 165 transition metal chelators 135-136 vascular endothelium 132-135

slide 400:

342 Extracellular matrix components 248- 249 Fatty acid/antioxidant interaction EDN 140-146 Fatty acids essential 141-146 free 320 n-6 essential 142-146 Ferrous oxidation FOX assay 54 Fibronectin 248 Free fatty acids FFA insulin resistance 281-282 TBARS 320 Free radicals diabetes mellitus 205-208 generation 1 Fructose peripheral nerve I 00-101 sorbitol 285-287 GLA NCV 142 GLA-a.-lipoic acid NCV 142-146 Glomerular filtration nitric oxide 246 Glomerular filtration rate GFR 246 Glornerular hyperfiltration 246 Glucosamine insulin resistance 282-283 291 Glucose peripheral nerve IOO- IOI Glucose enolization 285 Glucose levels type 2 diabetes mellitus 320-321 Glucose-mediated oxidative stress diabetic neuropathy 111-115 Glucose uptake EDN 127 a.-lipoic acid 292-294 nitric oxide 323 Index Glucotoxicity 284-287 metabolic initiators 111- I 12 sorbitol pathway 206 GLUT 242 translocation n-Iipoic acid 293 GLUT4 isoform 276 translocation 276-277 a.-lipoic acid 293 skeletal muscle glucose transport 304-305 type 2 diabetes 279-280 Glutathione GSH 3 78 86 288 insulin sensitivity 325-326 326t lipid pcroxidation I06 peripheral nerve 97 102 streptozotocin-induced diabetic rats 306 type 2 diabetes mellitus 325-327 xenobiotics 36 Glutathione GSH-containing en• zymes 122 Glutathione GSH/GSSG acetylcystein 327 obese Zucker rat 307-308 streptozotocin-induced diabetic rats 306 Glutathione peroxidase GSH-Px I 22 rx-lipoic acid I05 peripheral nerve l 02-103 Glycoxidation vascular endothelial dysfunction 45 GSSG 86 95 GSSGR peripheral nerve 102-103 Homocysteine insulin sensitivity 324 Human umbilical vein endothelial cells HUVECs ROis 20 Hydroperoxide ROOH antioxidant treatment 59-61

slide 401:

Index Hydroperoxide ROOH diurnal variation 54-55 glycemic control 59 insulin therapy 59 NIDDM 58 plasma levels 55-58 4-hydroxyalkenal peripheral nerve 97-98 JOI 8-hydroxydeoxyguanosine 80HdGO 41 Hyperglycemia 284-287 diabetic complications 186 insulin resistance 291 microvasculature 209 NF-KB 24-26 196f oxidative stress 6- 7 ROS 17-27 sorbitol pathway 207f Hyperglycemia-induced protein kinase C PKC activation 243-245 Hyperglycemia-mediated intracellular calcium 23-24 Hyperglycemic autooxidation oxidative stress 122 Hyperglycemic glycation oxidative stress 122 Hyperglycemic pseudohypoxia sorbitol oxidation 207f Hyperinsulinemia 287 insulin resistance 321r Hypoxia endoneurial oxidative stress 121 ICAM-1 211-213 212 213 lmidazolones 80 Insulin 3T3-Ll adipocytes 290 Insulin-dependent cell signaling path• ways 11 Insulin-dependent diabetes mellitus IDDM oxidative stress 266-267 343 Insulin-dependent diabetes mellitus IDDM pancreatic -cell destruction 265-270 pathogenesis 266 Insulin-mediated glucose transport obese Zucker rat 3 JO Insulin receptor substrate- I IRS-I 304 Insulin resistance antioxidants 310-313 defined 305 glucosamine 291 hyperglycemia 291 hyperinsulinemia 32 l f a-lipoic acid 292-293 molecular basis 276-280 oxidative stress 275-294 288-292 ROS 319-320 3T3-Ll adipocytes. 291-292 triggers 280-283 type 2 diabetes 275-283 in vitro 290-292 in vivo 288-290 Insulin sensitivity ACE inhibitors 329 carvedilol 329 celiprolol 329 clinical observations 320 glutathione 325-326 homocysteine 324 interventions 326t e-lipoic acid 328-329 nitric oxide 322-324 324 pharmacological intervention 325- 329 vitamin C 328 vitamin E 327-328 Insulin signaling type 2 diabetes 279-280 Insulin signaling cascade n-lipoic acid 313 Insulin signaling pathway u-lipoic acid 292-293

slide 402:

344 Insulin therapy plasma ROOH 59 Intracellular calcium hyperglycemia-mediated 23-24 Intracellular signaling thioredoxin 269 IRS proteins 11 Ischemia endoneurial oxidative stress 121 Ischemia-reperfusion diabetes mellitus 209-211 a-lipoic acid 124-125 Isoprostanes measurement 44 JNK 163 Laminin 248 Lifestyle ROS 324-325 Lipid hydroperoxide vitamin E 53-61 Lipid peroxidation diabetes relevance 43 GSH 106 induction 208 measurement 43-44 oxidative stress markers 42- 44 TA 159 Lipoate cell signaling pathways 11 Lipoic acid see also a-Lipoic acid diabetes 11-13 diabetic polyneuropathy 11-13 hypoglycemic effects 12 ischemia-reperfusion 211 NADH/NAD+ 7 redox potential 4 therapeutic aspects 86 therapeutic effects 7-9 a-Lipoic acid 159 160 288 Akt 294 Index a-Lipoic acid buthionine sulfoximine-induced cata- lase 105 CAN 178-180 clinical trials 173-182 diabetic complications 192-195 diabetic neuropathy 176-178 EON 123-124 efficacy 177 glucose uptake 292-294 GLUT translocation 293 GLUT4 translocation 293 GSH-Px 105 insulin resistance 292-293 insulin sensitivity 326t 328-329 insulin signaling cascade 313 insulin signaling pathway 292- 293 ischemia reperfusion injury 124- 125 lipid peroxidation 159 neurovascular function 132-133 NF-KB DNA binding 193-194 obese Zucker rat 308-310 polyneuropathy 180 safety 178 streptozotocin-induced diabetic rats 293 306-307 therapeutic aspects 292 Lipoproteins low-density oxidation 26 Lipoxygenases 21 Low-density lipoprotein LDL 53 oxidation 26 vitamin E 66-68 Maillard reaction 187 MDA peripheral nerve 97-98 101 MDA-Lys 81 Metabolic initiators glucose toxicity 111-112 Metal chelators 135-136

slide 403:

Index Metatyrosines 45 Methionine sulfoxide 80 Methionine sulfoxide reductase 2 Microangiopathy 2 IO 21 Of Microvascular dysfunction streptozotocin diabetic rats 209 Mitochondrial dysfunction AR pathway 114 Mitogen-activated protein MAP ki- nases 163 25 mM ryanodine I 0-1 I MOLD 84 Muscle glucose transport regulation 304-305 N-acetylcysteine therapeutic aspects 86 137 NADH/NAD+ lipoic acid 7 Na+-K+ ATPase 248 Natural oxidants 287-288 Necrosis 40 Neovascularization PKC 247 Nerve blood flow neuropathy 129-130 Nerve function 132- 135 Nerve growth 136-138 Nerve regeneration antioxidant therapy 136-138 N-6 essential fatty acids 142-146 Neurological Assessment of Thioctic Acid in Diabetic Neuropathy 181 Neuropathy nerve blood flow 129-130 Neuropeptide Y NPY 160-161 Neurotrophic factors EDN 156-157 Neurotrophic growth factor NGF EDN. 156 oxidative stress 158 sciatic nerve 157-158 345 Neurotrophins EDN 157-158 oxidative stress 158 Neurovascular dysfunction 138-139 141 diabetes-induced 93-94 ROS-induced 94-95 Neurovascular function oxidative stress and antioxidant ther• apy 132-140 NF-KB EDN 139-140 hyperglycemia 24-25 NF-KB activation 190-191 diabetic complications 186 hyperglycemia I 96f NF-KB DNA binding n-lipoic acid 193-194 Nitric oxide NO 130-132 calcium 208-209 endothelial dysfunction 322-323 glomerular filtration 246 glucose uptake 323 insulin sensitivity 322-324 324 radical scavenger 323-324 reactive oxygen species ROS 130-132 Noninsulin-dependent diabetes rnelli• tus NIDDM complications 53 ROOH 58 Nonobese diabetic NOD mice pancreatic -cell destruction 265-270 pathogenesis 266 Normoglycemia 173 NT-3 mRNA leg muscle 157-158 Obese Zucker rat characteristics of. 306 GSH/GSSG 307-308 insulin-mediated glucose transport 310 u-lipoic acid. 308-310

slide 404:

346 Orthotyrosines 45 Overadaptation 39 Oxidants natural 287-288 Oxidative damage 79-81 secondary 80-81 Oxidative stress apoptosis 267 defined 1-2 34-36 77 detection 283-284 diabetes 6-1 I 77-87 diabetes consequences 24-26 disease 34-40 endoneurial ischemia/hypoxia 121 factors affecting 78- 79 glucose-mediated diabetic neuropathy I I 1-1 15 hyperglycemia 6- 7 hyperglycemic glycation and autoox• idation 122 increased 82 insulin-dependent diabetes mellitus 266-267 insulin resistance 275-294. 288- 292 measurement 78- 79 mechanisms 36-37 121- 122 neurotrophins 158 neurovascular function 132-140 NGF 158 parameters 39t peripheral nerve 158-159 polyol pathway 138-139 tissue injury 33-34 Oxidative stress markers 33-46 38f 80t 82-83 DNA 41-42 lipid peroxidation 42-44 need for 40 protein damage 44-45 ROS/RNS/RCS 40 Oxygen ROS/RNS/RCS 36 Index P38 163 Pancreatic -cells endogenous thioredoxin 269 insulin-dependent diabetes mellitus 265-270 PDTC. 10 Pentane breath excretion 44 Pentosidine 81 Peripheral nerve MDA 97-98 IOI oxidative stress 158-159 Peripheral nerve studies 93-107 animals 95-96 autooxidative defense enzyme mea- surement 98-99 biochemical measurements 96-99 experimental procedure 96 GSH measurement 97 4-hydroxyalkenal measurement 97- 98 IOI materials and methods 95-99 reagents 96 results 99-104 sorbitol pathway intermediates mea• surement 96-97 statistical analysis 99 total MDA measurement 97-98 Peroxynitrite 22 diabetic vascular complications 26 Physical activity ROS 325 Pioglitazone 325 PKC isoforms in A7r5 smc o-tocopherol. 226-227 Polyol pathway EDN 138-139 oxidative stress 138-139 PP2A. a.-tocopherol 236 -tocopherol 236 Primaquine 133 Probucol 133

slide 405:

Index Protein kinase C PKC EON 139-140 inhibitors 20 n-tocopherol 220-221 vitamin E 249-251 Protein kinase C PKC activation hyperglycemia-induced 243-245 Protein kinase C PKC a inhibition u-tocopherol 227-228 232-233 Protein kinase C PKC P-isofonn inhi• bition 249 Protein kinase C PKC isoforms. 242 cc-tocopherol. 229-231 Protein kinases stress-activated thioctyl-y-linolenic acid 163-164 Protein phosphatase PP2A. u-tocopherol 233-235 Proteins carbonyl assay 45 diabetes relevance. 45-46 oxidative stress markers 44-45 ROS/RNS/RCS 44-45 Proteoglycans 248 P-selectin 212 Pseudohypoxia 107 Pyrraline 80 Reactive oxygen intermediates ROI generation 22-23 HUVEC 20 hyperglycemia 20- 24 streptozotocin diabetic rats 19-20 vasculature source 18-20 vitamin E 18-19 Reactive oxygen species ROS. 37t alterations 324-329 diabetes NO and vasorelaxation 130-132 diet 324-325 DPN 114 exercise 325 hyperglycemia 17-27 347 Reactive oxygen species ROS insulin resistance 319-320 lifestyle 324-325 sources 130-132 Reactive oxygen species ROS• induced neurovascular dys• function 94-95 Receptor for AGE RAGE 188-189 206 R-lipoic acid 7 11-12 Rochester Diabetic Neuropathy Study 180-181 ROS/RNS/RCS oxidative stress markers 40 oxygen 36 proteins 44-45 RRR-a-tocopherol see Vitamin E Sciatic nerve EON 125 nerve conduction studies 126 Skeletal muscle glucose transport GLUT4 translocation 304-305 regulation 304-305 Smoking cessation insulin sensitivity 326t Smooth muscle cell proliferation o-tocopherol 220 Smooth muscle cells smc 219 SNAP23. 277 Sorbitol fructose levels 285-287 peripheral nerve 100- IO I Sorbitol oxidation hyperglycemic pseudohypoxia 207f Sorbitol pathway glucotoxicity 206 hyperglycemia 207f Sorbitol pathway intermediates peripheral nerve 96-97 Stauroporine 20 Streptozotocin diabetic rats ROls 19-20

slide 406:

348 Streptozotocin-induced diabetic rats characteristics of 305 glutathione 306 GSSG/GSH 306 u-lipoic acid 293 306-307 microvascular dysfunction 209 sciatic nerve blood flow 129-130 Stress-activated protein kinases SAPKs thioctyl-y-linolenic acid 163-164 Suicide mechanism 40 Suicide response 40 Superoxide anions endothelial formation 20-24 Superoxide dismutase SOD 2 peripheral nerve 98 I 02-103 Syntaxin 4. 277 TBA-reactive substances TBARS 43 TBA test 42 Thiobarbituric acid reactive substances TBARS 53-54 free fatty acids. 320 Thioctic acid-v-linolenic acid EON. 155-165 Thioctyl-y-linolenic acid stress-activated protein kinases 163-164 in vivo studies 160-163 Thiol antioxidants IO Thioredoxin TRX antioxidative effects 267-269 autoimmune diabetes 269-270 intracellular signaling. 269 pancreatic P-cells 269 Thiotic acid see a-Lipoic acid Thrombomodulin diabetes rnellitus I 95f Tissue injury 39 oxidative stress 33-34 3T3-Ll adipocytes insulin. 290 insulin resistance 291-292 Index Tocopherol see u-Tocopherol: P• Tocopherol n-tocopherol cell proliferation inhibition 226 molecular basis 219-237 PKC inhibition 226-235 PKC a inhibition 227-228 232- 233 PKC isoforms autophosphorylation 229-231 PKC isoforms in A7r5 smc 226- 227 plasma levels 36 58-59 PP2A 236 protein phosphatase PP2A 233- 235 smooth muscle cell proliferation 220 P-Tocopherol PP2A 236 o-Tocopherol studies 219-237 animals 219 cell cultures 219 cell proliferation 219 immunoprecipitated PKCa. 224 methods 219-225 PKC activity 219-220 PKC isoform autophosphorylation 223-224 PKC isoform immunoprecipitation 223 PKCa/protein phosphatase assay 225 protein phosphatase activity assay 225 in vivo cell labeling 224 Western blot analyses. 223 Torglitazone insulin sensitivity 325 Transition metal chclators 135-136 Transretinol 69- 70 Trien tine EON. 135-136 Triphenylphosphine TPP 54

slide 407:

Index Troglitazone insulin sensitivity 326t Trytophan hydroxylation 45 Tumor necrosis factor-ex insulin resistance 281 Type I diabetes animal models 305 Type 2 diabetes animal models 3-6 305 glutathione 325-327 insulin resistance 275-283 metabolic alterations 321 t oxidative stress origin 284-288 plasma glucose levels 320-32 I vitamin E 289-290 327 Tyrosine 80 Van der W aerden rest 99 Vascular cell adhesion molecule VCAM-1 212 213 Vascular endothelial growth factor VEGF 246 formation 23-24 Vascular endothelium 132-135 Vascular permeability PKC 247 Vasorelaxation reactive oxygen species ROS. 130-132 349 Vesperlysine 81 Vitamin A 69- 70 plasma 69- 70 Vitamin B diabetic neuropathy 176-178 Vitamin C 3 70- 71 288 diabetic complications 192 insulin sensitivity 326t 328 NCV 135 plasma 70- 71 Vitamin E 3 66-68. 288 diabetic complications 191-192 endothelial function. 327 insulin sensitivity 326t 327-328 LDL 66-68 PKC inhibition 249-251 plasma 36 58-59 66-68 plasma lipid hydroperoxide 53-61 ROI 18-19 standardization 68-69 type 2 diabetes 289-290 type 2 diabetes mellitus 327 Vitamin E deficiency premature cell aging 220 Vitamin E radical 5 Wistar rats 95-96 Xenobiotics GSH 36

authorStream Live Help