Diabetes Ebook: A new approach in type 2 diabetes mellitus treatment

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A new approach in type 2 diabetes mellitus treatment is an informative ebook that helps who are in type 2 diabetes.

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Want to Cure Diabetes Click Here Ali Mennatallah Ahmed Ismail: A new approach in Type 2 diabetes mellitus treatment: Evaluation of the beneficial effect of L-cysteine in the treatment of type 2 diabetes mellitus. Hamburg Anchor Academic Publishing 2015 Buch-ISBN: 978-3-95489-353-9 PDF-eBook-ISBN: 978-3-95489-853-4 Druck/Herstellung: Anchor Academic Publishing Hamburg 2015 Bibliografische Information der Deutschen Nationalbibliothek: Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie detaillierte bibliografische Daten sind im Internet über http://dnb.d-nb.de abrufbar. Bibliographical Information of the German National Library: The German National Library lists this publication in the German National Bibliography. Detailed bibliographic data can be found at: http://dnb.d-nb.de

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All rights reserved. This publication may not be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior permission of the publishers. Das Werk einschließlich aller seiner Teile ist urheberrechtlich geschützt. Jede Verwertung außerhalb der Grenzen des Urheberrechtsgesetzes ist ohne Zustimmung des Verlages unzulässig und strafbar. Dies gilt insbesondere für Vervielfältigungen Übersetzungen Mikroverfilmungen und die Einspeicherung und Bearbeitung in elektronischen Systemen. Die Wiedergabe von Gebrauchsnamen Handelsnamen Warenbezeichnungen usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürften. Die Informationen in diesem Werk wurden mit Sorgfalt erarbeitet. Dennoch können

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Fehler nicht vollständig ausgeschlossen werden und die Diplomica Verlag GmbH die Autoren oder Übersetzer übernehmen keine juristische Verantwortung oder irgendeine Haftung für evtl. verbliebene fehlerhafte Angaben und deren Folgen. Alle Rechte vorbehalten © Anchor Academic Publishing Imprint der Diplomica Verlag GmbH Hermannstal 119k 22119 Hamburg http://www.diplomica-verlag.de Hamburg 2015 Printed in Germany Want to Cure Diabetes Click Here LIST OF CONTENTS Chapter Page LIST OF TABLES ................................................................................... ii LIST OF FIGURES ................................................................................. iv LIST OF ABBREVIATIONS ................................................................. vii

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I. INTRODUCTION ........................................................................ 1 II. AIM OF THE WORK ................................................................ 67 III. MATERIALS AND METHODS ............................................... 68 IV. RESULTS .................................................................................... 97 V. DISCUSSION ............................................................................. 161 VI. CONCLUSIONS AND RECOMMENDATIONS .................. 187 VII. SUMMARY ................................................................................ 190 VIII. REFERENCES ........................................................................... 195

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ii LIST OF TABLES Want to Cure Diabetes Click Here Table Page 1 Effect of STZ-induced type 2 diabetes on fasting serum glucose fasting serum insulin and HOMA-IR in male albino rats 103 2 Effect of STZ-induced type 2 diabetes on lipid profile in male albino rats 105 3 Effect of STZ-induced type 2 diabetes on oxidative stress parameters in male albino rats 108 4 Effect of STZ-induced type 2 diabetes on inflammatory parameters in male albino rats 110 5 Effect of treatment with the studied drugs for 2 weeks on fasting serum glucose in male albino rats mg/dl 113 6 Effect of treatment with the studied drugs for 2 weeks on fasting serum insulin in male albino rats ng/ml 116 7 Effect of treatment with the studied drugs for 2 weeks on HOMA-IR in male albino rats 119 8 Effect of treatment with the studied drugs for 2 weeks on serum triglycerides in male albino rats mg/dl 122 9 Effect of treatment with the studied drugs for 2 weeks on serum total cholesterol in male albino rats mg/dl 125 10 Effect of treatment with the studied drugs for 2 weeks on serum HDL-C in male albino rats mg/dl 128 11 Effect of treatment with the studied drugs for 2 weeks on serum LDL-C in male albino rats mg/dl 131

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iii 12 Effect of treatment with the studied drugs for 2 weeks on serum free fatty acids in male albino rats mmol/L 134 13 Effect of treatment with the studied drugs for 2 weeks on non- HDL-cholesterol in male albino rats mg/dl 137 14 Effect of treatment with the studied drugs for 2 weeks on triglycerides to HDL-cholesterol ratio in male albino rats 140 15 Effect of treatment with the studied drugs for 2 weeks on hepatic malondialdehyde in male albino rats nmol/gm wet tissue 143 16 Effect of treatment with the studied drugs for 2 weeks on hepatic reduced glutathione in male albino rats μg/mg protein 146 Table Page 17 Effect of treatment with the studied drugs for 2 weeks on serum monocyte chemoattractant protein-1 in male albino rats pg/ml 149 18 Effect of treatment with the studied drugs for 2 weeks on serum C- reactive protein in male albino rats mg/L 152 19 Effect of treatment with the studied drugs for 2 weeks on serum nitric oxide in male albino rats nmol/ml 155

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iv LIST OF FIGURES Want to Cure Diabetes Click Here Figure Page 1 Model for the effects of adipocytes on pancreatic -cell function/mass and insulin sensitivity in the pathogenesis of type 2 diabetes 10 2 Mitochondrial overproduction of superoxide activates the major pathways of hyperglycemic damage by inhibiting glyceraldehyde-3- phosphate dehydrogenase GAPDH 16 3 Development of type 2 diabetes 19 4 The cellular origins of reactive oxygen species their targets and antioxidant systems. 23 5 Schematic of the effects of chronic oxidative stress on the insulin signaling pathway 25 6 Structure of GSH -glutamylcysteinyl glycine where the N- terminal glutamate and cysteine are linked by the -carboxyl group of glutamate 29 7 Chemical structure of L-cysteine 32 8 The transsulfuration pathway in animals. 33 9 Sources and actions of cysteine and glutathione GSH 36 10 The role of serine kinase activation in oxidative stressinduced insulin resistance and the protective effect of some antioxidants by preserving the intracellular redox balance 37 11 Chemical structure of biguanides 41 12 Structure of human proinsulin and some commercially available insulin analogs. 57 13 Model of control of insulin release from the pancreatic -cell

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v by glucose and by sulfonylurea drugs 59 14 Schematic diagram of the insulin receptor heterodimer in the activated state. 60 15 Standard curve of insulin 73 16 Standard curve of Monocyte chemoattractant protein-1 82 MCP- 1 17 Standard curve of nitric oxide 85 18 Standard curve of MDA 88 19 Standard curve of reduced glutathione 91 20 Standard curve of Protein 93 21 Effect of STZ-induced type 2 diabetes on fasting serum glucose in male albino rats 104 Figure Page 22 Effect of STZ-induced type 2 diabetes on fasting serum insulin in male albino rats 104 23 Effect of STZ-induced type 2 diabetes on HOMA-IR in male albino rats 104 24-a Effect of STZ-induced type 2 diabetes on serum triglycerides in male albino rats 106 24-b Effect of STZ-induced type 2 diabetes on serum total cholesterol in male albino rats 106 24-c Effect of STZ-induced type 2 diabetes on serum HDL-C in male albino rats 106 24-d Effect of STZ-induced type 2 diabetes on serum LDL-C in male albino rats 106 24-e Effect of STZ-induced type 2 diabetes on serum free fatty acids in male albino rats 107

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vi 24-f Effect of STZ-induced type 2 diabetes on non- HDL-C in male albino rats 107 24-g Effect of STZ-induced type 2 diabetes on TGs/HDL ratio in male albino rats 107 25 Effect of STZ-induced type 2 diabetes on hepatic malondialdehyde in male albino rats 109 26 Effect of STZ-induced type 2 diabetes on hepatic reduced glutathione in male albino rats 109 27 Effect of STZ-induced type 2 diabetes on serum monocyte chemoattractant protein-1 in male albino rats 111 28 Effect of STZ-induced type 2 diabetes on serum C-reactive protein in male albino rats 111 29 Effect of STZ-induced type 2 diabetes on serum nitric oxide in male albino rats 111 30 Effect of treatment with the studied drugs for 2 weeks on fasting serum glucose in male albino rats 114 31 Effect of treatment with the studied drugs for 2 weeks on fasting serum insulin in male albino rats 117 32 Effect of treatment with the studied drugs for 2 weeks on HOMA-IR in male albino rats 120 33-a Effect of treatment with the studied drugs for 2 weeks on serum triglycerides in male albino rats 123 33-b Effect of treatment with the studied drugs for 2 weeks on serum total cholesterol in male albino rats 126 Figure Page 33-c Effect of treatment with the studied drugs for 2 weeks on serum high density lipoprotein cholesterol in male albino rats 129

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vii 33-d Effect of treatment with the studied drugs for 2 weeks on serum low density lipoprotein cholesterol in male albino rats 132 33-e Effect of treatment with the studied drugs for 2 weeks on serum free fatty acids in male albino rats 135 33-f Effect of treatment with the studied drugs for 2 weeks on non- HDL-cholesterol in male albino rats 138 33-g Effect of treatment with the studied drugs for 2 weeks on triglycerides to HDL-cholesterol ratio in male albino rats 141 34 Effect of treatment with the studied drugs for 2 weeks on hepatic malondialdehyde in male albino rats 144 35 Effect of treatment with the studied drugs for 2 weeks on hepatic reduced glutathione in male albino rats 147 36 Effect of treatment with the studied drugs for 2 weeks on serum monocyte chemoattractant protein-1 in male albino rats 150 37 Effect of treatment with the studied drugs for 2 weeks on serum C- reactive protein in male albino rats 153 38 Effect of treatment with the studied drugs for 2 weeks on serum nitric oxide in male albino rats 156 39 Histopathological evaluation of pancreatic sections stained with hematoxylin and eosin HE stain X 10. 158 40 Comparison of mean percentage change in biochemical metabolic oxidative stress and inflammatory parameters between untreated and treated metformin L-cysteine and their combination experimentally induced type 2 diabetic adult male rats 159 41 Comparison of mean percentage change in lipid profile between untreated and treated metformin L-cysteine and their combination experimentally induced type 2 diabetic adult male rats 160

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viii LIST OF ABBREVIATIONS 8-OH-Guanine : 8-hydroxy Guanine ACC : Acetyl-CoA carboxylase ACEI : Angiotensin converting enzyme inhibitors ACOD : Acyl-CoA oxidase Acyl CS : Acyl CoA synthetase ADP : Adenosine diphosphate AGEs : Advanced glycation endproducts AIDS : Acquired immunodeficiency syndrome Akt : Apoptosis serine/therionine kinase AMP : Adenosine monophosphate AMPK : Adenosine monophosphate-activated kinase protein ARB : Angiotensin receptor blocker ATP : Adenosine triphosphate ATPase : Adenosinine triphosphatase enzyme BH4 : Tetrahydrobiopterin cofactor Ca2+ : Calcium ion CAT : Catalase Ccl2 : Chemokine ligand 2 CCl4 : Carbon tetrachloride Ccr2 : Cognate receptor chemokine receptor 2 CD4 : Cluster of differentiation 4 cNOS : Constitutive nitric oxide synthase CoA : Coenzyme A CoQ10 : Coenzyme Q 10 CRP : C-reactive protein Cu : Cupper Cu/Zn SOD : Cupper zinc superoxide dismutase DAG : Diacylglycerol DHAP : dihydroxyacetone phosphate DM : Diabetes mellitus

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ix DNA : Deoxyribonucleic acid DPP-4 : Dipeptidyl peptidase-4 DTNB : 55’-Dithiobis-2-nitrobenzoic acid ECS : Endocannabinoid system EDTA : Disodium salt of ethylene diamine tetraacetic acid ELISA : Enzyme linked immunosorbent assay eNOS : Endothelial nitric oxide synthase ESRF : End stage renal failure ETC : Electron transport chain FADH2 : Reduced flavin-adenine dinucleotide FAS : Fatty acid synthase FBPase : Fructose 16-bisphosphatase FDA : Food and drug administration Fe : Iron FFAs : Free fatty acids FSG : Fasting serum glucose FSI : Fasting serum insulin GADA : Glutamic acid decarboxylase autoantibodies GAPDH : Glyceraldehyde-3-phosphate dehydrogenase GDM : Gestational diabetes mellitus GFAT : Glutamine:fructose-6-phosphate amidotransferase GIP : Gastric inhibitory polypeptide Gln : Glutamine GLP-1 : Glucagon-like peptide-1 Glu : Glutamate GLUT1 : Glucose transporter-1 GLUT2 : Glucose transporter-2 GLUT3 : Glucose transporter-3 GLUT4 : Glucose transporter-4 GPx : Glutathione peroxidase GSH : Reduced glutathione GSSG : Oxidized glutathione

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x GST : Glutathione transferase H2O : Water molecule H2O2 : Hydrogen peroxide HBA1C : Glycated Hemoglobin A 1C HCl : Hydrochloric acid HDL-C : High density lipoprotein cholesterol HFD : High fat diet HIV-1 : Human immunodeficiency virus-1 HLA : Human leukocyte antigen HMG-CoA : 3-hydroxy-3-methyl-glutaryl-Coenzyme A HNF-1 : Hepatic nuclear factor-1 HNF-4 : Hepatic nuclear factor-4 HNO2 : Nitrous oxide HOCL : Hypochlorous acid HOMA-IR : Homeostasis model assessment resistance of insulin • HRO2 : Hydroperoxyl HRP : Horseradish peroxidase enzyme IAAs : Insulin autoantibodies ICAM-1 : Intercellular adhesion molecule-1 ICAs : Islet-cell autoantibodies IDL : Intermediate density lipoprotein IGT : Impaired glucose tolerance IKK : Inhibitor of nuclear factor-B kinase beta IL-1 : Interleukin-1 IL-10 : Interleukin-10 IL-2 : Interleukin-2 IL-6 : Interleukin-6 IL-8 : Interleukin-8 iNOS : Inducible nitric oxide synthase IPF-1 : Insulin promoter factor-1 IR : Insulin resistance

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xi IRS-1 : Insulin receptor substrate-1 K + /Na + tartarate : Potassium sodium tartarate K + ATP channels : Potassium channels adenosine triphosphate KCl : Potassium chloride LADA : Latent autoimmune diabetes in adults LDL-C : Low density lipoprotein cholesterol LPL : Lipoprotein lipase LSD : Least significant difference MAOIs : Monoamine oxidase inhibitors MCP-1 : Monocyte chemoattractant protein-1 MDA : Malondialdehyde microRNA : micro-ribonucleic acid Mn-SOD : Manganese superoxide dismutase MODY : Maturity onset diabetes of the young mRNA : Messenger ribonucleic acid NAC : N-acetyl cysteine NAD+ : Oxidized nicotinamide-adenine dinucleotide NADH : Reduced nicotinanide adenine dinucleotide NADP : Oxidized Nicotinamide adenine dinucleotide phosphate NADPH : Reduced Nicotinamide adenine dinucleotide phosphate NaOH : Sodium hydroxide NED : N-1-naphthyl ethylenediamine NEFA : Non-esterified fatty acids NF- B : Nuclear factor kappa B nNOS : Neural nitric oxide synthase NO : Nitric oxide NO2- : Nitrite • NO2 - : Nitrogen dioxide NO3- : Nitrate Non-HDL-C : Non-high density lipoprotein cholesterol

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xii NPH : Neutral protamine NSAIDs : Non steroidal anti-inflammatory drugs O2 : Oxygen molecule - O2 : Superoxide radical OCT1 : Organic cation transporter-1 OH : Hydroxyl radical ONOO - : Peroxynitre P : Phosphate PAI-1 : Plasminogen-activator inhibitor -1 PCOS : Polycystic ovarian syndrome PDX-1 : Pancreas duodenum homeobox-1 PEPCK : Phosphenolpyruvate carboxykinase PI3K : Phosphatidylinositol 3-kinase PKC : Protein kinase C PPAR- : Peroxisome proliferator-activated receptor alpha PPAR- : Peroxisome proliferator-activated receptor gamma Prx : Peroxiredoxin PUFAs : Polyunsaturated fatty acids RAGE : Receptor for advanced glycation endproducts rDNA : Recombinant deoxyribonucleic acid RNS : Reactive nitrogen species RO2 : Proxyl radical RONOO : Alkyl peroxynitrates ROS : Reactive oxygen species rpm : Rotation per minute SDS : Sodecyl sulphate SGLT2 : Sodium-glucose cotransporter-2 SH : Thiol or sulfhydryl group SOD : Superoxide dismutase SREBP : Sterol regulatory element-binding protein STZ : Streptozotocin

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xiii SUR1 : Sulfonylurea receptor 1 SUs : Sulfonylureas T1DM : Type 1 diabetes mellitus T2DM : Type 2 diabetes mellitus TBA : Thiobarbituric acid TBARS : Thiobarbituric acid reactive substances TBHB : 244-tribromo-3-hydroxy-benzoic acid TC : Total cholesterol TCA : Trichloroacetic acid TGs : Triglycerides TMB : 33’55’ tetramethylbenzidine TMP : 11 33-tetramethoxypropane TNB : 5-thionitrobenzoic acid TNF- : Tumor necrosis factor- Trx : Thioredoxin TxA2 : Thromboxane A2 Tyr : Tyrosine TZDs : Thiazolidinediones UDP-GlcNac : Uridine diphospho-N-acetylglucosamine VCAM-1 : Vascular cell adhesion molecule-1 VEGF : Vascular endothelial growth factor VLDL : Very low density lipoproteins WHO : World Health Organization ZDF : Zucker diabetic fatty Zn : Zinc

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BACKGROUND OF THE STUDY Want to Cure Diabetes Click Here Diabetes mellitus DM is a chronic multisystem disorder with biochemical consequences and serious complications that affect many organs. There are complex interactions between genetic epigenetic environmental and behavioural factors that contribute to the development of diabetes. Non-pharmacological and pharmacological interventions have been used for diabetic management. Over the past few years research has started to focus on the use of novel adjuvant drugs as antioxidants and antiinflammatory drugs for better management as it was revealed that both oxidative stress and inflammation play a critical role in the disease pathogenesis. Metformin is a widely used oral antidiabetic agent for the management of type 2 diabetes. Its primary mode of action appears to be through improvement of insulin sensitivity and suppression of hepatic gluconeogenesis and glycogenolysis. Moreover it affects glucose transport system increases glucose utilization and delays its absorption from the

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intestine. It also shows beneficial effects on diabetes as weight reduction and improvements in lipid profile inflammation and endothelial function. L-cysteine is a semi-essential sulfur containing amino acid. One important function of L-cysteine is that it is a precursor of glutathione which is pivotal for the detoxification of cellular oxidative stress. Dietary intake of cysteine-rich proteins lowers the oxidative stress and insulin resistance. It improves glycemic control shows an anti-inflammatory effect and implies a protective effect on pancreatic -cells. Taking the above mentioned data in consideration it seems that combined therapy of metformin and an antioxidant like L-cysteine may be of value in treatment of the diabetic state and amelioration of the oxidative stress and inflammation associated with diabetes mellitus.

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1 INTRODUCTION Diabetes mellitus is the most common endocrine metabolic disorder affecting about 170 million people worldwide 1 . It represents a group of diseases with complex heterogeneous etiology characterized by chronic hyperglycemia with carbohydrate fat and protein metabolic abnormalities 2 which are due to insulin deficiency and/or insulin resistance 3 . These abnormalities result in the impairment of uptake and storage of glucose and reduced glucose utilization for energy purposes. Defects in glucose metabolizing machinery and consistent efforts of the physiological system to correct the imbalance in glucose metabolism place an over-exertion on the endocrine system. Continuing deterioration of endocrine control exacerbates the metabolic disturbances and leads primarily to hyperglycemia 4 then proceeds to the development of longterm complications such as microangiopathy nephropathy neuropathy and retinopathy. The basis of these complications is a subject of great debate and research. Hyperglycemia and metabolic derangement are accused as the main causes of these long- standing changes in various organs. Hyperglycemia may also lead to increased generation of free radicals and reduced antioxidant defense system 3 . Epidemiology of diabetes mellitus Want to Cure Diabetes Click Here Diabetes mellitus is a common growing disease which is considered epidemic by WHO. Its incidence in adults and adolescents have been alarmingly rising in developed countries with estimate for an increase of 60 in the adult population above 30 years of age in 2025 with a higher prevalence in the 45 to 64 years-old adults 5 . These increases are expected because of population ageing and urbanization. According to the WHO undiagnosed diabetes in Egypt will be about 8.8 million by the year 2025 6 . Diabetes mellitus classification The current classification includes four main categories 7 :

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2 I- Type 1 diabetes either type 1A immune-mediated e.g. latent autoimmune diabetes in adults LADA or type 1 B idiopathic II- Type 2 diabetes III- Other specific types 1. Genetic defects of -cell function maturity onset diabetes of the young MODY. These defects may be in genes of hepatic nuclear factor HNF-1 or HNF-4 or insulin promoter factor-1 IPF-1. 2. Genetic defects in insulin action Type A insulin resistance lipoatrophic diabetes. 3. Diseases of the exocrine pancreas pancreatitis neoplasia cystic fibrosis hemochromatosis. 4. Endocrinopathies acromegaly Cushings syndrome glucagonoma pheochromocytoma hyperthyroidism. 5. Drug or chemical induced vacor streptozotocin alloxan glucocorticoids thyroid hormone diazoxide thiazide diuretics minoxidil oral contraceptives L-dopa. 6. Infections congenital rubella cytomegalovirus. 7. Uncommon forms of immune-mediated diabetes “Stiff-man” syndrome anti- insulin receptor antibodies. 8. Other genetic syndromes sometimes associated with diabetes Down syndrome Klinefelter syndrome Turner syndrome. IV-Gestational diabetes mellitus Gestational diabetes mellitus GDM is defined as any abnormal carbohydrate intolerance that begins or is first recognized during pregnancy 8 . It is associated with an increased risk of perinatal mortality and congenital abnormalities which is further increased by impaired glycemic control 9 . It occurs in approximately 7 of all pregnancies and if occurred once it is likely to occur in subsequent pregnancies. Up to 70 of women with GDM have a potential risk of developing type 2 diabetes mellitus. The risk factors for developing gestational diabetes are similar to those for type 2

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3 diabetes including family history age obesity and ethnicity 10 . It is known that pregnancy is a diabetogenic state characterized by impaired insulin sensitivity particularly in the second and third trimester. This is due to changes in some hormones such as human placental lactogen progesterone prolactin and cortisol that antagonize the effects of insulin and decrease phosphorylation of insulin receptor substrate-1 IRS-1 triggering a state of insulin resistance. Logically the pancreas should compensate for this demand by increasing insulin secretion. However in GDM there is deterioration of beta cell function particularly the first phase insulin secretion 8 . An intermediate group of individuals with impaired fasting glucose and/or impaired glucose tolerance was classified as “pre-diabetics”. Their progression to diabetes is common particularly when non-pharmacological interventions such as lifestyle changes are not provided 7 . I-Type 1 diabetes mellitus Type 1 diabetes mellitus T1DM is an organ-specific progressive cellular-mediated autoimmune disease characterized by a defect in insulin production as a result of selective and massive destruction of islet -cells 80– 90. It accounts for only about 5–10 of all cases of diabetes however its incidence continues to increase worldwide. The progression of the autoimmune process is generally slow and may take several years before the onset of the clinical diabetes 11 . Markers of the immune -cell destruction including circulating insulin autoantibodies IAAs islet-cell autoantibodies ICAs and glutamic acid decarboxylase autoantibodies GADA are present in 90 of patients at the time of diagnosis 7 . Two forms are identified: • Type 1A DM which results from a cell-mediated autoimmune attack on -cells and has a strong genetic component inherited through the human leukocyte antigen HLA complex mainly HLA-DR3 and HLA-DR4 12 but the factors that trigger onset of clinical disease remain largely unknown.

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4 • Type 1B DM idiopathic is a far less frequent form has no known cause and occurs mostly in individuals of Asian or African descent 7 . This form of diabetes lacks evidence for -cell autoimmunity and is not HLA associated. While the disease is often manifested by severe insulinopenia and/or ketoacidosis -cell function often recovers rendering almost normal glucose levels 13 . Starting generally at a young age T1DM is also referred to as ‘juvenile diabetes’. Indeed it affects children and young adults in particular and generally occurs before the age of 40 with incidence peaks at 2 4-6 and 10- 14 years 11 . However it can also occur at any age even as late as in the eighth and ninth decades of life. The slow rate of -cell destruction in adults may mask the presentation making it difficult to distinguish from type 2 diabetes. This type of diabetes is known as “Latent Autoimmune Diabetes in Adults” 13 . Patients with type 1 diabetes are severely insulin deficient and are dependent on insulin replacement therapy for their survival 13 . II-Type 2 diabetes mellitus Type 2 diabetes mellitus T2DM is a complex metabolic disorder of polygenic nature that is characterized by defects in both insulin action and insulin secretion 14 . T2DM affects nowadays more than 150 million people worldwide and is projected to increase to 439 million worldwide in 2030 15 . By the end of the 20 th century its incidence has increased dramatically in children and adolescents as a result of the rise in childhood obesity and is now continuing to rise changing the demographics of the disease in this group 16 . Genetic epigenetic and environmental factors have been implicated in type 2 diabetes mellitus pathogenesis with increasing evidences that epigenetic factors play a key role in the complex interplay between them 17 . Epigenetic mechanisms are commonly associated to gene silencing and transcriptional regulation of genes 18 . The epigenetic control of gene expression is based on modulation of chromatin structure and accessibility to transcription factors which is achieved by multiple mechanisms. These mechanisms involve methylation–demethylation of cytidine–guanosine

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5 sequences in the promoter regions acetylation– deacetylation of lysine residues of core histones in the nucleosome and presence of microRNA molecules which bind to their complementary sequences in the 3 end of mRNA and reduce the rate of protein synthesis 19 . Actions of major pathological mediators of diabetes and its complications such as hyperglycemia oxidative stress and inflammation can lead to the dysregulation of these epigenetic mechanisms 17 . Insulin resistance in peripheral tissues such as muscle and fat is often the earliest recognizable feature of T2DM results in a compensatory hyperinsulinemia that promotes further weight gain. This occurs until the cells can no longer compensate for the increased insulin resistance then cell failure and hyperglycemia ensue 20 . It is also associated with comorbidities such as hypertension hyperlipidemia and cardiovascular diseases which taken together comprise the ‘Metabolic Syndrome’ 20 . Similar to adults obesity in children appears to be a major risk factor for type 2 diabetes 21 . Many studies show a strong family history among affected youth with 45-80 having at least one parent with diabetes and 74- 100 having a first- or second-degree relative with type 2 diabetes 22 . Until now type 2 diabetes was typically regarded as a disease of the middle-aged and elderly. While it is still true that this age group maintains a higher risk than the younger adults do evidence is accumulating that onset in children and adolescents is increasingly common. Onset of diabetes in childhood or adolescence around the time of puberty heralds many years of disease and increases the risk of occurrence of the full range of both micro- and macrovascular complications 23 . Although a mother could transmit genetic susceptibility to her offspring it is more likely that maternal diabetes increases the risk of diabetes in children by altering the intrauterine environment which can impair the normal -cell development and function as well as the insulin sensitivity of skeletal muscle 24 . Moreover malnutrition during fetal or early life and low birth weight appear to be associated with an increased risk of adulthood insulin resistance glucose intolerance T2DM dyslipidemia and hypertension 25 .

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6 Normal glucose homeostasis Maintenance of serum glucose concentrations within a normal physiological range fasting blood glucose level 70-110 mg/dl is primarily accomplished by two pancreatic hormones insulin secreted by the -cells and glucagon secreted by -cells 26 . Derangements of glucagon or insulin regulation can result in hyperglycemia or hypoglycemia respectively. In the postabsorptive state the majority of total body glucose disposal takes place in insulin-independent tissues 27 . Approximately 50 of all glucose utilization occurs in the brain and 25 of glucose uptake occurs in the splanchnic area liver and the gastrointestinal tissue. The remaining 25 of glucose metabolism takes place in insulin-dependent tissues primarily muscle with only a small amount being metabolized by adipocytes 28 . Approximately 85 of endogenous glucose production is derived from the liver and the remaining amount is produced by the kidney. Glycogenolysis and gluconeogenesis contribute equally to the basal rate of hepatic glucose production 27 . In the postprandial state the maintenance of whole-body glucose homeostasis is dependent upon a normal insulin secretory response and normal tissue sensitivity to the effects of hyperinsulinemia and hyperglycemia to augment glucose disposal. This occurs by three tightly coupled mechanisms: i suppression of endogenous glucose production and increase glycogen synthesis ii stimulation of glucose uptake by the splanchnic tissues and iii stimulation of glucose uptake by peripheral tissues primarily muscle 27 . The route of glucose administration also plays an important role in the overall glucose homeostasis. Oral glucose ingestion has a potentiating effect on insulin secretion that the insulin concentrations in the circulation increase very rapidly by at least two to threefold after oral glucose when compared to a similar intravenous bolus of glucose. This potentiating effect of oral glucose administration is known as “the incretin effect” and is related to the release of glucagon-like peptide-1 GLP-1 and glucosedependent insulinotropic polypeptide also called gastric inhibitory polypeptide GIP from the gastrointestinal tissues 29 . Glucose homeostasis in type 2 diabetes mellitus

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7 Type 2 diabetic individuals are characterized by defects in insulin secretion insulin resistance involving muscle liver and the adipocytes and abnormalities in splanchnic glucose uptake 28 . Insulin secretion in insulin resistant non-diabetic individuals is increased in proportion to the severity of the insulin resistance and glucose tolerance remains normal. Thus their pancreas is able to "read" the severity of insulin resistance and adjust its secretion of insulin. However the progression to type 2 diabetes with mild fasting hyperglycemia 120-140 mg/dl is heralded by an inability of the -cell to maintain its previously high rate of insulin secretion in response to a glucose challenge without any further or only minimal deterioration in tissue sensitivity to insulin 27 . The relationship between the fasting plasma glucose concentration and the fasting plasma insulin concentration resembles an inverted U or horseshoe. As the fasting plasma glucose concentration rises from 80 to 140 mg/dl the fasting plasma insulin concentration increases progressively peaking at a value that is 2-2.5 folds greater than in normal weight nondiabetic age-matched controls. The progressive rise in fasting plasma insulin level can be viewed as an adaptive response of the pancreas to offset the progressive deterioration in glucose homeostasis. However when the fasting plasma glucose concentration exceeds 140 mg/dl the beta cell is unable to maintain its elevated rate of insulin secretion and the fasting insulin concentration declines precipitously. This decrease in fasting insulin level has important physiologic implications since at this point hepatic glucose production begins to rise which is correlated with the severity of fasting and postprandial hyperglycemia 27 . Moreover the largest part of the impairment in insulin-mediated glucose uptake is accounted for a defect in muscle glucose disposal. Thus in the basal state the liver represents a major site of insulin resistance 30 however in the postprandial state both decreased muscle glucose uptake and impaired suppression of hepatic glucose production contribute to the insulin resistance together with defect in insulin secretion are the causes of postprandial hyperglycemia. It should be noted that brain glucose uptake occurs at the same rate during absorptive and postabsorptive periods and is not altered in type 2 diabetes 28 .

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8 Pathophysiology of type 2 diabetes mellitus \ To Cure Diabetes Click Here Insulin resistance: Insulin resistance IR is the impaired sensitivity and attenuated response to insulin in its main target organs adipose tissue liver and muscle leading to compensatory hyperinsulinemia 31 . In adipose tissue insulin decreases lipolysis thereby reducing FFAs efflux from the adipocytes. However intra-abdominal fat is metabolically distinct from subcutaneous fat as it is more lipolytically active and less sensitive to the antilipolytic effects of insulin. In liver insulin inhibits gluconeogenesis by reducing key enzyme activities. While in skeletal muscle insulin predominantly induces glucose uptake by stimulating the translocation of the GLUT4 glucose transporter to the plasma membrane and promotes glycogen synthesis 32 . Insulin resistance leads to increased lipolysis of the stored triacylglycerol molecules with subsequent increase circulating FFAs concentrations and ectopic fat accumulation. This results in increases in the flux of FFAs from fat to liver and periphery. Excess delivery of FFAs stimulates liver glucose and triglycerides production 33 impedes insulin mediated glucose uptake and decreases glycogen synthesis in skeletal muscle 34 as well as impairs vascular reactivity and induces inflammation 35 . This is shown in Figure 1. At the molecular level impaired insulin signaling results from reduced receptor expression or mutations or post-translational modifications of the insulin receptor itself or any of its downstream effector molecules. These reduce tyrosine-specific protein kinase activity or its ability to phosphorylate substrate proteins IRS 36 resulting in phosphatidylinositol3-kinase PI3K/Akt pathway impairment 37 . To date several methods for evaluating insulin resistance in humans have been reported such as fasting serum insulin levels homeostasis model assessment of insulin resistance HOMA-IR and insulin tolerance test 38 . Because abnormalities in insulin action are poorly detected by a single

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9 determination of either glucose or insulin levels the insulin resistance is commonly evaluated by HOMA-IR which is likely to be the most simple and repeatable index 38 39 . Glucotoxicity: Insulin resistance results in chronic fasting and postprandial hyperglycemia. Chronic hyperglycemia may deplete insulin secretory granules from -cells leaving less insulin ready for release in response to a new glucose stimulus 40 . This has led to the concept of glucose toxicity which implies the development of irreversible damage to cellular components of insulin production over time. Hyperglycemia stimulates the production of large amounts of reactive oxygen species ROS in -cells. Due to ROS interference loss of pancreas duodenum homeobox-1 PDX-1 has been proposed as an important mechanism leading to -cell dysfunction. PDX- 1 is a necessary transcription factor for insulin gene expression and glucose- induced insulin secretion besides being a critical regulator of -cell survival. Additionally ROS are known to enhance NFB activity which potentially induces -cell apoptosis 41 . Lipotoxicity: Chronically elevated free fatty acids FFAs level results from the resistance to the antilipolytic effect of insulin. It is known that FFAs acutely stimulate insulin secretion but chronically impair insulin secretion induce further hepatic and muscle insulin resistance stimulate gluconeogenesis and cause a decrease -cell function and mass an effect referred to as -cell lipotoxicity 42 . In the presence of glucose fatty acid oxidation in -cells is inhibited and accumulation of long-chain acyl coenzyme A occurs. This mechanism has been proposed to be an integral part of the normal insulin secretory process. However its excessive accumulation can diminish the insulin secretory process by opening -cell potassium channels. Another mechanism might involve apoptosis of -cells possibly via generation of nitric oxide through inducible nitric oxide synthase iNOS activation which results in great production of toxic peroxynitrite ONOO - 43 .

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10 Thus glucolipotoxicity may play an important role in the pathogenesis of hyperglycemia and dyslipidemia associated with type 2 diabetes 44 . Dyslipidemia: Diabetic dyslipidemia is typically defined by its characteristic lipid ‘triad’ profile known as atherogenic dyslipidemia which is usually an increase in plasma triglycerides a decrease in high-density lipoprotein cholesterol and a concomitant increase in small dense oxidized low-density lipoproteins 45 46 . These lipid abnormalities may be a more important risk factor for atherosclerosis and cardiovascular diseases than hyperglycemia 46 .

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11 Figure 1: Model for the effects of adipocytes on pancreatic -cell function/mass and insulin sensitivity in the pathogenesis of type 2 diabetes 47 . In diabetic patients there is typically a preponderance of smaller denser oxidized LDL particles which may increase atherogenicity and cardiovascular risk even if the absolute concentration of LDL cholesterol is not elevated 45 . Non-HDL cholesterol non-HDL-C is a new measure that reflects the combined lipid profile change. It encompasses all cholesterol present in the potentially atherogenic lipoprotein particles VLDL remnants IDL and LDL. Non-HDL-C has been shown to correlate with coronary artery disease severity and progression as well as predicts cardiovascular morbidity and mortality in patients with diabetes 48 . Another simple tool Triglycerides to HDL-cholesterol ratio TGs: HDL-C has been proposed as an atherogenic index that has proven to be a highly significant predictor of myocardial infarction even stronger than total cholesterol to HDL-C ratio and LDL-C to HDL-C ratio 49 . Moreover a significant negative relationship between TGs: HDL-C ratio and insulin sensitivity was observed. Thus a TGs: HDL-C ratio 3.5 provides a simple mean of identifying insulin resistant dyslipidemic patients who are at increased risk of cardiovascular diseases 50 . The precise pathogenesis of diabetic dyslipidemia is not fully known nevertheless a large body of evidence suggests that insulin resistance has a central role in the development of this condition as a result of the increased influx of free fatty acids from insulin-resistant fat cells into the liver in the presence of adequate glycogen stores 51 . Diabetic complications and their pathogenesis Hyperosmolar hyperglycemic non-ketotic state Want to Cure Diabetes Click Here

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12 It is one of the major acute complications which is a life-threatening condition commonly occurs in elderly patients with type 2 diabetes. There is almost always a precipitating factor which include the use of some drugs acute situations and chronic diseases. Abnormal thirst sensation and limited access to water also facilitate development of this syndrome. It is associated with four major clinical features which are severe hyperglycemia blood glucose more than 600 mg/dl absent or slight ketosis plasma hyperosmolarity and profound dehydration. Treatment of this state should be started immediately with the determination and correction of the precipitating event and lifesaving measures while the other clinical manifestations should be corrected with the use of appropriate fluids and insulin 52 . However chronic complications can be divided into microvascular affecting eyes kidneys and nerves and macrovascular affecting the coronary cerebral and peripheral vascular systems 53 . Microvascular complications In fact microvascular complications can begin in developing at least 7 years before the clinical diagnosis of type 2 diabetes. Conversely type 1 patients may not develop signs of microvascular complications until 10 years after diagnosis of diabetes 54 . Nephropathy: Diabetic nephropathy is a frequent complication of type 1 and type 2 diabetes mellitus characterized by excessive urinary albumin excretion hypertension and progressive renal insufficiency. The natural history of diabetic nephropathy has 5 stages which include hyperfiltration with normal renal function histological changes without clinically evident disease incipient diabetic nephropathy or microalbuminuria overt diabetic nephropathy macroalbuminuria and reduced renal function and renal failure requiring dialysis end stage renal disease 55 . Neuropathy: Diabetic peripheral neuropathy is one of the most prevalent and complicated conditions to manage among diabetic patients. Diabetes is the major contributing reason for non-traumatic lower extremity amputations more than 60 of cases. Ischemia occurs because of compromised vasculature that fails to deliver oxygen and nutrients to nerve

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13 fibers. This results in damage to myelin sheath covering and insulating nerve. The most common form involves the somatic nervous system however the autonomic nervous system may be affected in some patients. Sensorimotor neuropathy is characterized by symptoms such as burning tingling sensations and allodynia. Autonomic neuropathy can cause gastroparesis sexual dysfunction and bladder incontinence 54 . Retinopathy: Diabetic retinopathy is the most frequent cause of new cases of blindness among adults aged 20-74 years 56 . Non-proliferative retinopathy produces blood vessel changes within the retina which include weakened blood vessel walls leakage of fluids and loss of circulation. It generally does not interfere with vision 54 . However if left untreated it can progress to proliferative retinopathy that is very serious and severe. It occurs when new blood vessels branch out or proliferate in and around the retina 56 . It can cause bleeding into the fluid-filled center of the eye or swelling of the retina leading to blindness. The duration of diabetes and the degree of hyperglycemia are probably the strongest predictors for development and progression of retinopathy 54 . Macrovascular complications The hallmark of diabetic macrovascular disease is the accelerated atherosclerosis involving the aorta and the large and medium-sized arteries which is a leading cause of morbidity and mortality in diabetes 54 . Accelerated atherosclerosis caused by accumulation of lipoproteins within the vessel wall resulting in the increased formation of fibrous plaques 53 . Hyperglycemia also affects endothelial function resulting in increased permeability altered release of vasoactive substances increased production of procoagulation proteins and decreased production of fibrinolytic factors 53 . All these changes result in atherosclerotic heart disease myocardial infarction and sudden death peripheral vascular disease and cerebrovascular disease including cerebral hemorrhage infarction and stroke. Hyperglycemia causes tissue damage through four major mechanisms. Several evidences indicate that all these mechanisms are activated by a single upstream event which is the mitochondrial overproduction of reactive oxygen species 57 Figure 2.

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14 Increased polyol pathway flux The polyol pathway is based on a family of aldo-keto reductase enzymes which can use as substrates a wide variety of carbonyl compounds and reduce them by NADPH to their respective sugar alcohols polyols. Glucose is converted to sorbitol by the enzyme aldose reductase which is then oxidized to fructose by the enzyme sorbitol dehydrogenase using NAD + as a cofactor. Aldose reductase is found in tissues such as nerve retina lens glomerulus and vascular cells. In many of these tissues glucose uptake is mediated by insulin-independent GLUTs intracellular glucose concentrations therefore rise in parallel with hyperglycemia 57 . Several mechanisms include sorbitol-induced osmotic stress increased cytosolic NADH/NAD + and decreased cytosolic NADPH have been proposed to explain tissue damage resulted from this pathway 58 . The most cited is an increase in redox stress caused by the consumption of NADPH a cofactor required to regenerate reduced glutathione GSH which is an important scavenger of ROS. This could induce or exacerbate intracellular oxidative stress 57 . Increased intracellular advanced glycation endproducts AGEs formation and increased expression of the receptor for AGEs RAGE AGEs are formed by the non-enzymatic reaction of glucose and other glycating compounds derived both from glucose and fatty acids with proteins 59 . Intracellular production of AGE precursors can damage cells by altering protein functions and binding of plasma proteins modified by AGE precursors to RAGE on cells such as macrophages and vascular endothelial cells. RAGE binding induces the production of ROS which in turn activates the pleiotropic transcription factor nuclear factor NF-B causing multiple pathological changes in gene expression 60 . These effects induce procoagulatory changes and increase the adhesion of inflammatory cells to the endothelium. In addition this binding appears to mediate in part the increased vascular permeability induced by diabetes probably through the induction of VEGF 57 .

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15 Increased protein kinase C PKC activation PKC activation results primarily from enhanced de-novo synthesis of diacylglycerol DAG from glucose via triose phosphate. Evidence suggests that the enhanced activity of PKC isoforms could also result from the interaction between AGEs and their cell-surface receptors 57 . PKC activation implicated in many processes such as increased vascular permeability angiogenesis blood flow abnormalities capillary and vascular occlusion which are involved in the pathology of diabetic complications 58 . Increased hexosamine pathway flux Hyperglycemia and elevated free fatty acids also appear to contribute to the pathogenesis of diabetic complications by increasing the flux of glucose and fructose-6-phophate into the hexosamine pathway leading to increases in the transcription of some key genes and alteration in protein functions such as eNOS inhibition 57 . Mitochondrial superoxide overproduction It has now been established that all of the different pathogenic mechanisms described above stem from a single hyperglycemia-induced process namely overproduction of superoxide by the mitochondrial electron- transport chain that can damage cells in numerous ways. It is hypothesized that excess ROS inhibits GAPDH glyceraldehyde-3phosphate dehydrogenase a glycolytic key enzyme promoting shunting of upstream glucose metabolites into the aforementioned pathways 57 . This overproduction of ROS can be prevented by manganese superoxide dismutase 61 .

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16 Figure 2: Mitochondrial overproduction of superoxide activates the major pathways of hyperglycemic damage by inhibiting glyceraldehyde-3-phosphate dehydrogenase GAPDH 62 .

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17 Obesity inflammation and insulin resistance To Cure Diabetes Click Here Although genetic predisposition to insulin resistance exists it is widely accepted that the increasingly sedentary lifestyle such as consumption of a high caloric diet and lack of exercise have increased the global prevalence of not only insulin resistance and diabetes but also of obesity 63 . Between 60 and 90 of cases of type 2 diabetes now appear to be related to obesity 64 . The close association of these two common metabolic disorders has been referred to as “diabesity” 65 . Adipocytes are not merely a site for storage of energy in the form of triglycerides but also a source of many adipokines 63 that have effects on many peripheral tissues including skeletal muscles and liver. As body weight increases there is expansion of the adipose tissue mass particularly visceral intraabdominal adipose tissue resulting in not only excessive free fatty acids release but also altered release of these adipokines 66 . Increased release of various inflammatory cytokines such as tumor necrosis factor- TNF- IL-6 MCP-1 and resistin mainly from visceral fat and leptin mainly from subcutaneous fat together with decreased release of adiponectin contribute to the whole body insulin resistance 67 . Figure 3 shows how inflammation contributes to develop insulin resistance and type 2 diabetes mellitus. The inflammation is triggered in the adipose tissue by macrophages which form ring-like structures surrounding dead adipocytes. As adipose tissue expands during the development of obesity certain regions become hypoperfused leading to adipocyte microhypoxia and cell death. Adipocyte hypoxia and death trigger a series of proinflammatory program which in turn recruit new macrophages. Another proposed mechanism is the activation of inflammatory pathway by oxidative stress. Hyperglycemia and high fat diet have been shown to increase ROS production via multiple pathways such as NADPH oxidase activation which in turn activates nuclear factor-B triggering inflammatory response in adipose tissue 68 . TNF- resistin IL-6 and other cytokines appear to participate in the induction and maintenance of the chronic low-grade inflammatory state which is one of the hallmarks of obesity and type 2 diabetes 69 . IL-6 may

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18 interfere with insulin signaling inhibit lipoprotein lipase activity and increase concentrations of non-esterified fatty acids NEFA contributing to dyslipidemia and insulin resistance 70 . In addition IL-6 stimulates the secretion of further proinflammatory cytokines such as IL-1 and increases the hepatic production of CRP thus explaining its increase in the metabolic syndrome and diabetes 71 . C-reactive protein CRP monocyte chemoattractant protein-1 MCP1 and other chemokines have essential roles in the recruitment and activation of macrophages in the adipose tissue and in the initiation of inflammation 69 72 . CRP activation of monocytes increases the expression of Ccr2 the receptor for MCP-1 72 . Overexpression of MCP-1 causes inhibition of Akt and tyrosine phosphorylation in liver and skeletal muscle which contributes to insulin resistance 73 74 . This demonstrates a clear association between increased levels of MCP-1 and CRP with the decreased insulin sensitivity and increased vascular inflammation 75 explaining the increased risk of atherosclerosis cardiovascular disease and stroke in diabetic patients 76 77 .

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19 Figure 3: Development of type 2 diabetes insulin resistance that precedes the development of hyperglycemia is associated with obesity and is induced by adipokines FFAs and chronic inflammation in adipose tissue. Pancreatic -cells compensate for insulin resistance by hypersecretion of insulin. However at some point -cell compensation is followed by -cell failure and diabetes ensues 63 . This state of proatherogenesis and low-grade inflammation is known to cause induction of inducible nitric oxide synthase iNOS increasing nitric oxide production 78 . Nitric oxide NO is a free radical known to act as a biological messenger in mammals. It has a dual role as a mediator of physiological and pathophysiological processes.

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20 In pancreatic islets excess NO is produced on exposure to cytokines which mediates -cell injury and leads to diabetes mellitus. Nitric oxide can also combine with oxygen to produce potent cellular killers such as the highly toxic hydroxyl radical OH and peroxynitrite ONOO - . In diabetes mellitus there is increased breakdown of NO by superoxide resulting in the excessive formation of peroxynitrite a potent oxidant that can attack many types of biological molecules. High levels of peroxynitrite cause initation of lipid peroxidation sulfhydryl oxidation nitration of some amino acids direct DNA damage and oxidation of antioxidants 3 . Oxidative stress in diabetes mellitus Oxidative stress refers to a situation of a serious imbalance between free radical-generating and radical-scavenging systems i.e. increased free radical production or reduced activity of antioxidant defenses or both leading to potential tissue damage 79 . There is currently great interest in the potential contribution of reactive oxygen species ROS in pathogenesis of diabetes and more importantly in the development of secondary complications of diabetes 80 . Free radical species include a variety of highly reactive molecules such as ROS and reactive nitrogen species RNS. ROS include free radicals such as superoxide O 2 •- hydroxyl OH • peroxyl RO 2 • hydroperoxyl HRO 2 •- as well as non-radical species such as hydrogen peroxide H 2 O 2 and hypochlorous acid HOCl. RNS include free radicals like nitric oxide NO • and nitrogen dioxide NO 2 •- as well as non-radicals such as peroxynitrite ONOO - nitrous oxide HNO 2 and alkyl peroxynitrates RONOO 81 . Production of one ROS or RNS may lead to the production of others through radical chain reactions 82 . Of these reactive molecules O 2 •- NO • and ONOO - are the most widely studied species as they play important roles in diabetic complications 83 . To avoid free radical overproduction antioxidants are synthesized to neutralize free radicals. Antioxidants include a manifold of enzymes such as superoxide dismutase SOD catalase glutathione peroxidase and glutathione reductase as well as many non-enzymatic antioxidants as vitamin A C and E 84 . This is shown in Figure 4.

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21 Free radicals at physiological levels play a key role in defense mechanisms as seen in phagocytosis and neutrophil function. They are also involved in gene transcription and to some extent acts as signaling molecules. However excess generation of free radicals in oxidative stress has pathological consequences including damage to nucleic acid proteins and lipids causing tissue injury and cell death 83 . Oxidative damage to DNA lipids and proteins To Cure Diabetes Click Here 1- Nucleic acid: The hydroxyl radical is known to react with all components of the DNA molecule damaging both the purine and pyrimidine bases and the deoxyribose backbone causing base degeneration single strand breakage and cross-linking to proteins. The most extensively studied DNA lesion is the formation of 8-OH-Guanine. Permanent modification of genetic material resulting from these oxidative damage incidents represents the first step involved in mutagenesis carcinogenesis and ageing 85 86 . 2- Proteins: Collectively ROS can lead to oxidation of the side chain of amino acids residues of proteins particularly methionine and cysteine residues forming protein-protein cross-linkages and oxidation of the protein backbone 87 resulting in protein fragmentation denaturation inactivation altered electrical charge and increased susceptibility to proteolysis 88 . The concentration of carbonyl groups is a good measure of ROS-mediated protein oxidation 89 . 3- Membrane lipids: ROS attack polyunsaturated fatty acids PUFAs of phospholipids in the cell membranes which are extremely sensitive to oxidation because of double and single bonds arrangement 90 . The removal of a hydrogen atom

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22 leaves behind an unpaired electron on the carbon atom to which it was originally attached. The resulting carbon-centered lipid radical can have several fates but the most likely one is to undergo molecular rearrangement followed by reaction with O 2 to give a peroxyl radical which are capable of abstracting hydrogen from adjacent fatty acid side chains and so propagating the chain reaction of lipid peroxidation. Hence a single initiation event can result in conversion of hundreds of fatty acid side chains into lipid hydroperoxides 91 . Further decomposition of these lipid hydroperoxides produces toxic aldehydes in particular 4hydroxynonenal and malondialdehyde 92 . The occurrence of lipid peroxidation in biological membranes causes impairment of membrane functioning changes in fluidity inactivation of membrane-bound receptors and enzymes and increased non-specific permeability to ions 93 . Thus lipid peroxidation in-vivo has been implicated as the underlying mechanisms in numerous disorders and diseases such as cardiovascular diseases atherosclerosis liver cirrhosis cancer neurological disorders diabetes mellitus rheumatoid arthritis and aging 89 . Malondialdehyde MDA is a major highly toxic by-product formed by PUFAs peroxidation. MDA can react both irreversibly and reversibly with proteins DNA and phospholipids resulting in profound mutagenic and carcinogenic effects 92 94 . The determination of plasma urine or other tissue MDA concentrations using thiobarbituric acid TBA reaction continues to be widely used as a marker of oxidative stress as its level correlates with the extent of lipid peroxidation 95 .

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23 Figure 4: The cellular origins of reactive oxygen species their targets and antioxidant systems 68 83 .

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24 Sources of oxidative stress in diabetes Multiple sources of oxidative stress in diabetes including enzymatic non- enzymatic and mitochondrial pathways have been reported 81 . Enzymatic sources of augmented generation of reactive species in diabetes include NOS NADPH oxidase and xanthine oxidase . If NOS lacks its substrate L-arginine or one of its cofactors NOS may produce O 2 •- instead of NO • and this is referred to as the uncoupled state of NOS. NADPH oxidase is a membrane associated enzyme that consists of five subunits and is a major source of O 2 •- production 81 . There is plausible evidence that protein kinase C PKC which is stimulated in diabetes via multiple mechanisms activates NADPH oxidase 82 . Non-enzymatic sources of oxidative stress originate from hyperglycemia which can directly increase ROS generation. Glucose can undergo auto-oxidation and generate OH • radicals. In addition glucose reacts with proteins in a non-enzymatic manner leading to the development of Amadori products followed by formation of advanced glycation endproducts AGEs. ROS is generated at multiple steps during this process 96 . Once AGEs are formed they bind to various receptors termed RAGE and this step is also generating ROS 97 . Moreover cellular hyperglycemia in diabetes leads to the depletion of NADPH through the polyol pathway resulting in enhanced production of O 2 • 98 . The mitochondrial respiratory chain is another source of nonenzymatic generation of reactive species. During the oxidative phosphorylation process electrons are transferred from electron carriers NADH and FADH 2 through four complexes in the inner mitochondrial membrane to oxygen generating ATP and O 2 • which is immediately eliminated by natural defense 83 . However in the diabetic cells more glucose is oxidized by Krebs cycle which pushes more NADH and FADH 2 into the electron transport chain ETC thereby overwhelming complex III of ETC where the transfer of electrons is blocked. Thus the generated electrons are directly donated to molecular oxygen one at a time generating excessive superoxide 57 . Therefore in diabetes electron transfer and oxidative phosphorylation are uncoupled resulting in excessive O 2 • formation and inefficient ATP synthesis 99 .

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25 Antioxidants Reactive oxygen species can be eliminated by a number of antioxidant defense mechanisms which involve both enzymatic and non-enzymatic strategies. They work in synergy with each other and against different types of free radicals 100 . Hyperglycemia not only engenders free radicals but also impairs the endogenous antioxidant defense system and causes inflammation in many ways in diabetes mellitus 101 Figure 5. Decreases in the activities of SOD catalase and glutathione peroxidase decreased levels of glutathione and elevated concentrations of thiobarbituric acid reactants are consistently observed in diabetic patients and in experimentally-induced diabetes 100 . Figure 5: Schematic of the effects of chronic oxidative stress on the insulin signaling pathway 102 I- Enzymatic antioxidants

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26 • Superoxide dismutase SOD Isoforms of SOD are variously located within the cell. Cu/Zn-SOD is found in both the cytoplasm and the nucleus. Mn-SOD is confined to the mitochondria but can be released into the extracellular space 100 . SOD converts superoxide anion radicals produced in the body to hydrogen peroxide which is then detoxified to water either by catalase or by glutathione peroxidase in the lysosomes and mitochondria respectively 96 thereby reducing the likelihood of superoxide anion interacting with nitric oxide to form reactive peroxynitrite 100 . However H 2 O 2 can also be converted to the highly reactive OH • radical in the presence of transition elements like iron and copper 103 . II- Non-enzymatic antioxidants 1- Vitamins Vitamins A C and E are diet-derived and detoxify free radicals directly. They also interact in recycling processes to generate their reduced forms. - tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical generating dihydroascorbic acid which is recycled by glutathione 100 . Vitamin E a fat soluble vitamin reacts directly with peroxyl and superoxide radicals to protect membranes from lipid peroxidation 100 . It exists in eight different forms of which -tocopherol is the most active form in humans. Hydroxyl radical reacts with tocopherol forming a stabilized phenolic radical which is reduced back to the phenol by ascorbate and NADPH dependent reductase enzymes 96 . The deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. However there have been conflicting reports about vitamin E levels in diabetic animals and human subjects that its plasma and/or tissue levels are reported to be unaltered increased or decreased in diabetes 100 . Vitamin C ascorbic acid is an important potent water soluble antioxidant vitamin in human plasma acting as an electron donor it is capable of scavenging oxygen-derived free radicals and sparing other endogenous antioxidants from consumption 104 . It can increase NO production in endothelial cells by stabilizing NOS cofactor

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27 tetrahydrobiopterin BH4 96 . Vitamin C itself is oxidized to dehydroascorbate which is considered as a marker of oxidative stress as in smoking and diabetes mellitus 105 . Plasma and tissue levels of vitamin C are 40–50 lower in diabetic compared with non-diabetic subjects 106 . 2- Coenzyme Q10 CoQ10 It is an endogenously synthesized lipid soluble antioxidant that acts as an electron carrier in the complex II of the mitochondrial electron transport chain and in higher concentrations it scavenges O 2 •- and improves endothelial dysfunction in diabetes 83 96 . 3- -Lipoic acid It is an antioxidant which can exert beneficial effects in both aqueous and lipid environments. -lipoic acid is reduced to another active compound dihydrolipoate which is able to regenerate other antioxidants such as vitamin C vitamin E and reduced glutathione through redox cycling 83 96. 4- Trace elements Selenium an essential trace element is involved in the complex defense system against oxidative stress through selenium-dependent glutathione peroxidases and other selenoproteins 107 . It has insulinmimetic properties on glucose metabolism both in-vitro and in-vivo by stimulating the tyrosine kinases involved in the insulin signaling cascade 108 . Within the context of diabetes mellitus controversially data on selenium levels in biological fluids can be found. Lower similar and even higher selenium levels were reported in diabetic patients with respect to healthy subjects 109 . Zinc magnesium and chromium are of special interest. Severe Zn deficiency is not frequent but concerns have been raised about Zn levels in diabetic patients. Some studies have reported Zn deficiency in type 2 diabetes others failed to find significant differences with healthy subjects 110 . Low magnesium levels have been associated with increased severity of type 2 diabetes whereas controversy exists about the importance of hypomagnesaemia in pre-diabetic states 110 . Previous studies also reported

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28 that diabetic patients have a significantly lower plasma chromium levels with higher urinary levels than in healthy subjects. This combination of abnormalities suggests a chronic renal loss of chromium 111 . Vanadium compounds are one of the most studied substances for the long-term treatment of diabetes. Vanadium exhibits insulin-mimetic effects in-vitro and in the streptozotocin diabetic rat with some insulin-enhancing effects 112 . 5- Glutathione Glutathione -glutamyl-L-cysteinylglycine GSH Figure 6 is a small intracellular ubiquitous tripeptide which is a sulfhydryl SH antioxidant antitoxin and enzyme cofactor 113 present in both prokaryotes and eukaryotes 114 . Being water soluble it is found mainly in the cell cytosol and other aqueous phases of the living system 113 . Glutathione antioxidant system predominates among other antioxidants systems due to its very high reduction potential and high intracellular concentrations compared to other antioxidants in tissues. Glutathione is found almost exclusively in its thiol-reduced active form GSH comprises 90 of the total low molecular weight thiol in the body 115 . GSH often attains millimolar levels inside cells especially highly concentrated in the liver and in lens spleen kidney erythrocytes and leukocytes however its plasma concentration is in micromolar range 116 . Glutathione is an essential cofactor for antioxidant enzymes namely the GSH peroxidases which serve to detoxify hydrogen peroxide and other peroxides generated in water phase as well as the cell membranes and other lipophilic cell phases by reacting them with GSH which then becomes in the oxidized form GSSG. The recycling of GSSG to GSH is accomplished mainly by the enzyme glutathione reductase using the coenzyme NADPH as its source of electrons. Therefore NADPH coming mainly from the pentose phosphate shunt is the predominant source of GSH reducing power 117 . Moreover GSH is an essential component of the glyoxalase enzyme system which is responsible for catabolism of the highly reactive aldehydes methylglyoxal and glyoxal. It can also bind to these aldehydes causing them

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29 to be excreted in bile and urine 115 . These effects have particular implications for preventive health as lipid peroxidation has been found to contribute to the development of many chronic diseases in humans. The ratio of reduced to oxidized glutathione GSH/GSSG within cells is often used as a measure of cellular toxicity or vice versa as a predictor of the antioxidative capacity and redox state of the cells 114 . GSH in the body is synthesized mostly de-novo with cysteine being the limiting amino acid so increasing cysteine supply is necessary to raise GSH synthesis and concentration. GSH may be a good reservoir for cysteine as its concentration in tissues is 5-7 times higher than free cysteine 118 . Figure 6: Structure of GSH -glutamylcysteinyl glycine where the Nterminal glutamate and cysteine are linked by the -carboxyl group of glutamate 119 . GSH makes major contributions to the recycling of other antioxidants that have become oxidized such as -tocopherol vitamin C and perhaps also the carotenoids 117 . Moreover GSH is important in the synthesis and repair of DNA as it is required in the conversion of ribonucleotides to deoxyribonucleotides 120 . A major function of GSH is the detoxification of xenobiotics and/or their metabolites. It is also involved in maintaining the essential thiol status of many important enzymes and proteins 117 . It participates in some cellular functions as amino acid translocation across the cell membrane 121 and folding of newly synthesized proteins 122 . In addition GSH is essential for the proliferation growth differentiation and activation of immune cells 117 and is implicated in the modulation of cell death cellular apoptosis and necrosis 123 .

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30 Some oxidative stressors are known for their ability to deplete GSH. These include smoking alcohol intake some over the counter drugs as acetaminophen household chemicals strenuous aerobic exercise dietary deficiency of methionine an essential amino acid and GSH precursor ionizing radiation tissue injury surgery trauma bacterial or viral infections as HIV-1 and environmental toxins 117 . GSH reduction has been associated with the pathogenesis of a variety of diseases therefore systemic GSH status could serve as an index of general health. Glutathione in liver diseases GSH depletion is involved in liver injury and enhanced morbidity related to liver hypofunction. Studies had been demonstrated a decrease in plasma and liver GSH increase in GSSG and a significant decrease in cysteine present in cirrhotic patients chronic alcoholic and non-alcoholic liver disease fatty liver acute and chronic hepatitis as compared with the healthy subjects 117 . Glutathione in immunity and HIV disease Adequate GSH is essential for mounting successful immune responses when the host is immunologically challenged. Healthy humans with relatively low lymphocyte GSH were found to have significantly lower CD4 counts 117 . It was postulated that GSH deficiency could lead to the progression of immune dysfunction weight loss cachexia and wasting syndrome which are known AIDS stigmas. GSH depletion is also seen in many autoimmune diseases as Crohns disease an inflammatory immunomediated disorder in which low GSH elevated GSSG levels and altered GSH enzymes were found in the affected ileal zones 114 . Glutathione in diabetes mellitus Low blood thiol status and reduced systemic GSH content were reported in diabetic and glucose intolerant patients as a result of insulin deficiency. It was reported that chronic hyperglycemia resulted in enhanced apoptosis in human endothelial cells which was attenuated by insulin due to its ability to

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31 induce glutamate cysteine ligase expression 119 . Platelets from diabetics have lower GSH levels and make excess thromboxane TxA2 thus having a lowered threshold for aggregation. This may contribute to the increased atherosclerosis seen in the diabetic population 117 . Furthermore glutathione deficiency is associated with aging and many other diseases as neurodegenerative diseases including Parkinsons disease schizophrenia and Alzheimer’s disease atherosclerosis and cardiovascular diseases human pancreatic inflammatory diseases and metal storage diseases as Wilson’s disease 117 124 . Strategies for repleting cellular glutathione In light of the copious evidence supporting the importance of GSH for homeostasis and for resistance to toxic attack as well as the contribution of its deficiency in many diseases a number of researchers had been stimulated to find new potential approaches and methods for maintaining or restoring GSH levels 125 . Optimizing GSH would likely augment antioxidant defenses and stabilize or raise the cell’s threshold for susceptibility to toxic attack 117 . • Oral glutathione Oral GSH was reported to replete GSH in subjects with depleted GSH but not healthy ones. Intact GSH can be absorbed slowly by intestinal lumen enterocytes and epithelial cells such as lung alveolar cells thus intact GSH can be also delivered directly into the lungs as an aerosol 117 . Circulating GSH is safe and soluble in plasma. It reacts only slowly with oxygen and is less susceptible to auto-oxidative degradation. However currently the use of GSH as a therapeutic agent is limited by its unfavorable pharmacokinetic properties. GSH has a short half-life in human plasma and difficulty in crossing cell membranes so administration of high doses is necessary to reach a therapeutic value 125 which will not be a particularly cost-effective way to accomplish GSH repletion. • L-cysteine It is a sulfur containing semi-essential amino acid as humans can synthesize it from the essential amino acid methionine only to a limited and

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32 generally not sufficient extent 126 . Its chemical structure is illustrated in Figure 7. One important function of L-cysteine is being a precursor that limits the synthesis of glutathione. It also serves as a very important precursor for synthesis of proteins coenzyme A and inorganic sulphate 127 . Cysteine is catabolized in the gastrointestinal tract and plasma 128 so it is relatively unstable in the blood. When substituted into the diet in place of the total protein allowance it can replete GSH 117 . Figure 7: Chemical structure of L-cysteine 115 Biosynthesis In animals L-cysteine is synthesized from L-methionine and L-serine via trans- sulfurtion reaction 127 . The sulfur is derived from methionine which is converted to homocysteine through the intermediate S- adenosylmethionine. Cystathionine -synthase then combines homocysteine and serine to form the asymmetrical thioether cystathionine. The enzyme cystathionine -lyase converts the cystathionine into cysteine and ketobutyrate 129 . This is shown in Figure 8.

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33 Figure 8: The transsulfuration pathway in animals. The first three reactions involve methyl group transfer via Sadenosylmethionine 129 . Biological functions of L-cysteine The chemical structure of cysteine contains a free sulfhydryl group which is the reactive entity that contributes to many of cysteine’s biological activity serving as a nucleophile with susceptibility to be oxidized to the disulfide derivative cystine 130 . As a moderately powerful redox pair cysteine and its disulfide partner cystine have an important physiological function as antioxidants. Cysteines antioxidant properties are typically expressed in the glutathione where the free SH group of cysteine within glutathione confers its functional properties. Cysteine and glutathione form a major part of the endogenous thiol pool that reacts with the vasoregulatory molecule NO to form nitrosothiol which acts as NO-carrier molecules stabilizing this normally volatile molecule. S- nitrosothiols have potent relaxant activity antiaggregatory and anti- inflammatory functions. They have greater halflives than free NO and are more resistant than free NO to degradation by superoxide. In this way nitrosothiol increases the bioavailability of NO and potentiates its effects 115 . All actions of cysteine and GSH are shown in Figure 9. Cysteine is a component of many structural and functional proteins. It is able to stabilize protein structures by forming disulfide covalent crosslinks which add stability to the three-dimensional structures of protein increase the rigidity of proteins affect their susceptibility to denaturation and provide proteolytic resistance 115 131 . The precise location of cysteine within a protein also plays a direct role in the protein’s function. For example cysteine is found at the active site of several enzymes including eNOS regulating its catalytic activity 115 . Proteins containing cysteine such as metallothionein can bind to heavy metals tightly because of the high affinity of thiol group to these metals thus cobalt cupper inorganic arsenic and selenium toxicities can be ameliorated

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34 by oral cysteine ingestion 132 . L-cysteine has been proposed as a preventative or antidote for some of the negative effects of alcohol including liver damage and hangover. It counteracts the poisonous effects of acetaldehyde the major by-product of alcohol metabolism by supporting its conversion into the relatively harmless acetic acid 133 . Aside from its oxidation to cystine cysteine participates in numerous posttranslational modifications 134 . Cysteine and insulin resistance An early study demonstrated that cysteine has an insulin-like action promoting the entry of glucose into adipose cells mediated by its free SH group. Cysteine has been subsequently shown to increase the levels of GLUT3 and GLUT4 with a marked enhancement of glucose uptake in mouse muscle and human neuroblastoma cells 135 . Moreover Cysteine may improve glucose metabolism by preventing oxidative or nitrosative inhibition of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6- phosphate dehydrogenase 115 . In cultured adipocytes it was demonstrated that cysteine supplementation reverses the increased intracellular oxidative stress after AGE-RAGE interaction which causes a decrease in glucose uptake 136 and also prevents the methylglyoxal induced decrease in IRS-1 tyrosine phosphorylation and PI3K activity that impair insulin signaling 137 . This is illustrated in Figure 10. It was also reported that cysteine analogues potentiate the glucoseinduced insulin release in pancreatic islets isolated from female Wistar rats 138 . Dietary intake of whey protein and -lactoalbumin cysteine- rich proteins lowers the oxidative stress and insulin resistance induced by sucrose in rats 139 . Other studies have reported that N-acetylcysteine supplementation reduces fructose-induced insulin resistance in rats 140 and also improves insulin sensitivity in women with polycystic ovaries 141 . Other effects of L-cysteine 1- L-cysteine administration prevents liver fibrosis by direct inhibition of activated hepatic stellate cells proliferation and transformation 142

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35 . It also shows a cytoprotective effect against carbon tetrachloride CCl 4 -induced hepatotoxicity by reversal of CCl 4 induced lactate dehydrogenase release and decreased cellular thiols mainly glutathione 143 . 2- Previous clinical studies suggest that the acquired immunodeficiency syndrome AIDS may be the consequence of a virus-induced cysteine deficiency. HIV-infected persons were found to have abnormally high TNF- and IL-2 receptor alpha-chain. All the corresponding genes are associated with NF-B whose transcription is negatively regulated by cysteine or cysteine derivatives thus they may be considered as adjuvant therapy for the treatment of patients with HIV-1 infection 144 145 . Side effects of L-cysteine Gastrointestinal problems as indigestion flatulence diarrhea nausea and vomiting are the main side effects of L-cysteine. Allergic reactions include itching and facial swelling are another possible side effects. Copious amount of water should be taken with cysteine to prevent cystine renal stones formation. It was showed by in-vitro studies that cysteine mimics many of the chemical properties of homocysteine which is known to increase the risk of the cardiovascular diseases 146 . N-acetylcysteine NAC N-acetyl-L-cysteine is a cysteine precursor that is rapidly absorbed and converted to circulating cysteine by deacetylation. It is used as an antioxidant and as a mucolytic due to its ability to break disulphide bonds in the mucous. It has liver protecting effects so it is a well-established antidote for acetaminophen overdose 147 . It also has antimutagenic and anticarcinogenic properties. In addition NAC can prevent apoptosis and promote cell survival a concept useful for treating certain degenerative diseases 148 . NAC can scavenge ROS and increase depleted glutathione levels. Activation of redox-sensitive NF-B in response to a variety of signals IL1 TNF- and ROS can be also inhibited by NAC. NAC can interfere with cell adhesion smooth muscle cell proliferation stability of rupture-prone

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36 atherosclerotic plaques in the cardiovascular system reduce lung inflammation and prolong survival of transplants 148 . Figure 9: Sources and actions of cysteine and glutathione GSH 115 .

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37 Figure 10: The role of serine kinase activation in oxidative stressinduced insulin resistance and the protective effect of some antioxidants by preserving the intracellular redox balance 149 .

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38 Management of diabetes mellitus To Cure Diabetes Naturally Click Here I- Non-pharmacological management of diabetes Lifestyle modifications are the cornerstone of management of diabetes mellitus and include the prescription of a healthy diet regular exercise management of stress and avoidance of tobacco 150 . 1-Diet The aims of dietary management are to achieve and maintain ideal body weight euglycemia and desirable lipid profile prevent and postpone complications related to diabetes and provide optimal nutrition during pregnancy lactation growth old age and associated conditions such as hypertension and catabolic illnesses 151 . However there is no single description for diet composition that can achieve these goals in all patients. Thus the dietary recommendations should be individualized according to the person’s ethnicity cultural and family background personal preferences and associated co-morbid conditions 150 . Diet that contains 60 carbohydrates high dietary fiber low to moderate dietary fat and moderate high biological value proteins as well as vitamins and minerals especially chromium vitamin E and C is considered proper for management of diabetic patients 152 . 2- Physical activity Exercise program should be individualized according to patient’s capacity and disabilities. Diabetic patient must wear appropriate footwear. It should also be noted that poorly controlled patients may develop hyperglycemia during exercise whereas patients treated with insulin and insulin secretagogues could develop hypoglycemia 153 . The best form of exercise recommended to diabetic is a stepwise increase of aerobic exercises. There are several benefits from a regular exercise schedule. These include reduction of hypertension and weight increase in bone density improvement in insulin sensitivity cardiovascular

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39 function and lipid profile reduces serum triglycerides and increases HDLC as well as improvement in the sense of physical and mental well-being and the overall quality of life 152 . 3- Stress management Diagnosis of diabetes mellitus is a stressful situation in life of an individual and appropriate management requires an approach that includes behavioural modification to develop positive attitude and healthy life style. A satisfactory treatment plan should include special attention to person with diabetes quality of life coping skills optimal family support and a healthy workplace environment. Appropriate support and counseling is an essential component of the management at the time of diagnosis and throughout life 150 . II- Pharmacological management of type 2 diabetes mellitus A- Antidiabetic agents Even when non-pharmacological measures are successfully implemented the progressive natural history of the disease dictates that the majority of patients will later require pharmacologic therapy and this should be introduced promptly if the glycemic target is not met or not maintained. Preserving -cell function and mass are important considerations in maintaining long-term glycemic control. If -cell function deteriorates beyond the capacity of oral agents to provide adequate glycemic control then the introduction of insulin should not be delayed 154 . Terminology within the field of antidiabetic agents may simplify the usage of the different agents. Hypoglycemic agents have the capacity to lower blood glucose below normal level to the extent of frank hypoglycemia e.g. sulfonylureas. Antihyperglycemic agents euglycemic agents can reduce hyperglycemia but when acting alone they do not have the capability to lower blood glucose below normoglycemia to the extent of frank hypoglycemia e.g. metformin thiazolidinediones gliptins glucosidase inhibitors 154 .

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40 They are classified into: - Oral antidiabetic agents: • Insulin sensitizers: o Biguanides including metformin o Thiazolidinediones or glitazones including rosiglitazone and pioglitazone • Insulin secretagogues: o Sulfonylureas including gliclazide glipizide glimepiride and glibenclamide o Meglitinides non-sulfonylurea secretagogues including nateglinide and repaglinide • Alpha-glucosidase inhibitors including acarbose miglitol and voglibose - Novel treatments: oral and non-insulin parenteral agents o Gliptins including sitagliptin and vildagliptin o Glucagon-like peptide-1 receptor agonist including exenatide and liraglutide o Amylin and amylin analogs including pramlintide o Rimonabant - New experimental agents 1. Insulin sensitizers 1.1. Biguanides The history of biguanides stems from a guanidine-rich herb Galega officinalis goat’s rue or French lilac that was used as a traditional treatment for diabetes in Europe because of its glucose lowering effect 154 155 . Its structural formula is illustrated in Figure 11.

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41 Several guanidine derivatives were adopted for the treatment of diabetes in the 1920s. These agents all disappeared as insulin became available but three biguanides – metformin phenformin and buformin – were introduced in the late 1950s 155 . However phenformin and buformin were withdrawn in many countries in the late 1970s because of a high incidence of lactic acidosis 156 . Metformin remained and was introduced into the USA in 1995 and since then it became most widely prescribed first line antidiabetic agent worldwide 157 . Figure 11: Chemical structure of biguanides 158 . Pharmacological effects of metformin 1- Antihyperglycemic effect Metformin exerts a range of actions that counter insulin resistance and decrease hyperglycemia by reducing fasting and postprandial blood

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42 glucose 159 . The glucose-lowering efficacy of metformin requires a presence of at least some insulin because metformin does not mimic or activate the genomic effects of insulin. The precise mechanisms through which metformin exerts its glucose lowering effects are not entirely understood. However its primary mode of action appears to be increasing hepatic insulin sensitivity resulting in decreased hepatic glucose output through suppression of gluconeogenesis and glycogenolysis. Metformin may also modestly augment glucose uptake in peripheral tissues increase fatty acid oxidation and increase glucose metabolism in the splanchnic bed. Metformin’s molecular effects appear to be at least in part mediated by adenosine monophosphate-activated protein kinase AMPK but it is unclear if this pathway represents the drug’s specific or unique target 160 . AMPK activation determines a wide variety of physiological effects including increased fatty acid oxidation and enhanced glucose uptake by skeletal muscle by increasing translocation of GLUT1 and insulin-sensitive glucose transporters GLUT4 into the cell membrane 161 . Administration of metformin to obese subjects was also found to increase levels of active GLP-1 after a glucose load this phenomenon appears to occur through mechanisms other than DPP-4 inhibition and may instead be due to direct stimulation of GLP-1 secretion or a reduction in DPP-4 secretion 160 . Interestingly these incretin-sensitizing effects of metformin appear to be mediated by PPAR- dependent pathway as opposed to the more commonly described AMPK activation pathway 162 . Importantly the likelihood of hypoglycemia induced by metformin monotherapy is quite low as the drug does not exert its effects through an increase in insulin secretion 160 . A new insight on the mechanism of action of metformin is its ability to decrease plasma glucose through the release of -endorphin from adrenal gland which activates peripheral opoid 1 receptors. -endorphin acts as a positive regulator in glucose utilization and a negative modulator in hepatic gluconeogenesis in the insulin-deficient state. These actions are mediated by the amelioration of GLUT4 gene expression and the attenuation of raised hepatic phosphenolpyruvate carboxykinase PEPCK gene expression a rate-controlling enzyme of gluconeogenesis 163 . Metformin also has cardioprotective benefits and offers some protection against vascular complications independently of its antihyperglycemic

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43 effect 164 . It was reported that metformin is associated with a decrease in myocardial infarction due to its effect on various atherothrombotic risk markers and factors including reduced carotid intima-media thickness increased fibrinolysis and reduced concentrations of the anti-thrombolytic factor plasminogen activator inhibitor-1 PAI-1 154 . 2-Anti-inflammatory effect Metformin also offers some protection against vascular inflammation and complications independently of its antihyperglycemic effect 165 . This may be mediated through the reduction of thrombotic factor and inflammatory markers 166 . 3-Antioxidative effect Metformin may also exert antioxidative effects as it prevents hyperglycemia-induced PKC activation and protects against high glucoseinduced oxidative stress through a mitochondrial permeability transition dependent pathway that is involved in cell death 167 . This may be in relation to metformin’s ability to inhibit non-toxically complex I in the mitochondrial respiratory chain 165 . Metformin is also able to react in-vitro with OH • radical. However it is not a very good scavenger of ROS at molecular level. Thus it seems that metformin exerts its in-vivo antioxidant activity by different pathways other than the simple free radical scavenging action. These pathways include increasing the antioxidant enzyme activities decreasing the markers of lipid peroxidation 168 and inhibiting the formation of advanced glycation end products by its ability to react directly with and neutralize highly reactive - dicarbonyl intermediates involved in AGEs formation such as methylglyoxal 164 . Metformin can also increase the activity of glyoxalase an enzyme which deactivates methylglyoxal to D-lactate 164 . Pharmacokinetics of metformin Metformin is an orally administered medication which is 50–60 bioavailable. Administration with food may decrease its absorption the

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44 clinical significance of which is unknown. The drug is minimally proteinbound and has few known drug interactions other than that known to occur with cimetidine which increases metformin levels in plasma by up to 40. Metformin is not metabolized prior to its complete excretion in the urine via glomerular filtration and tubular secretion. The drug has an elimination halflife of approximately 6 hours. Decreases in renal function will decrease clearance of the medication. It is generally dosed 2–3 times daily but is available in an extended release preparation which may be administered once a day. 85 of the maximal glucose-lowering effect is seen at a daily dose of 500 mg 3 times daily while the most effective glucose lowering occurs with a total daily dose of 2000 mg 160 . Indications Because metformin does not cause weight gain it is often preferred for overweight and obese people with T2DM. It can be introduced in insulin- resistant states before the development of hyperglycemia 163 . Metformin can resume ovulation in women with anovulatory polycystic ovarian syndrome PCOS which is an unlicensed application of the drug in the absence of diabetes 154 . Adverse effects and contraindications The main tolerability issue with metformin is abdominal discomfort and other gastrointestinal adverse effects including diarrhea nausea vomiting flatulence stomach upset and metallic taste in approximately 30 of patients 169 . Anorexia and stomach fullness are likely part of the reason for weight loss noted with metformin. These effects are often transient and can be ameliorated by taking the drug with meals and using a small initial dose which is then gradually titrated slowly until target level of blood glucose control is attained or using extended-release preparations of metformin 170 however around 5 of patients cannot tolerate the drug at any dose 171 . It can reduce gastrointestinal absorption of vitamin B 12 which rarely causes frank anemia 172 . The most serious adverse event associated with metformin is lactic acidosis that is typically characterized by a raised blood lactate concentration decreased arterial pH and/or bicarbonate concentration with

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45 an increased anion gap. It is rare but about half of cases are fatal 154 . The true likelihood of lactic acidosis occurring as a result of metformin accumulation is unclear. However given these concerns the drug is contraindicated in the setting of renal dysfunction or in those at risk for lactic acidosis such as individuals with kidney hypoperfusion due to hypotension or septicemia congestive heart failure chronic cardiopulmonary dysfunction significant hepatic dysfunction or alcohol abuse. Renal function must be assessed prior to and periodically during metformin therapy particularly in the elderly 160 . Presenting symptoms of lactic acidosis are generally non-specific flulike symptoms but often include hyperventilation malaise and abdominal discomfort. Treatment should be commenced promptly bicarbonate remains the usual therapy. Hemodialysis to remove excess metformin can be helpful and may assist restoration of fluid and electrolyte imbalance occurred during treatment with high dose intravenous bicarbonate 154 . Metformin also should be temporarily stopped when using intravenous radiographic contrast media or during surgery with general anaesthesia 154 . 1.2. Thiazolidinediones TZDs TZDs are pharmacological ligands for the nuclear receptor peroxisome proliferator- activated receptor- PPAR- which is highly expressed in adipose tissue and to a lesser extent in muscle pancreatic cells vascular endothelium and macrophages 173 . Therefore thiazolidinediones can affect responsive genes at these locations giving rise to “pleiotropic effects” 174 . Many of these genes participate in lipid and carbohydrate metabolism. Troglitazone was the first thiazolidinedione to enter routine clinical use however it was associated with fatal cases of idiosyncratic hepatotoxicity and was withdrawn in 2000 175 . Two other thiazolidinediones rosiglitazone and pioglitazone were then introduced which did not show hepatotoxicity indicating that troglitazone’s hepatotoxicity has presumably a compound specific phenomenon 176 . However rosiglitazone was withdrawn in 2010 from market as the clinical investigations revealed its implication in cardiovascular side effects 177 .

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46 Mode of action TZDs stimulate PPAR- promoting differentiation of pre-adipocytes into mature adipocytes 178 these new small adipocytes are particularly sensitive to insulin and show increased uptake of fatty acids with increased lipogenesis 179 . This in turn reduces circulating free fatty acids facilitating glucose utilization and restricting fatty acid availability as a source for hepatic gluconeogenesis. By reducing circulating fatty acids ectopic lipid deposition in muscle and liver is reduced which further contributes to improvements of glucose metabolism. TZDs also increase glucose uptake into adipose tissue and skeletal muscle via increased availability of GLUT4 glucose transporters 154 . Pharmacokinetics Absorption of rosiglitazone and pioglitazone is rapid and almost complete with peak concentrations at 1-2 hours but slightly delayed when taken with food. Both drugs are metabolized extensively by the liver and are almost completely bound to plasma proteins but their concentrations are not sufficient to interfere with other protein-bound drugs 154 . Indications TZDs are indicated as monotherapy in T2DM associated with no risk for hypoglycemia development. They are often used to gain additive efficacy in combination with other antidiabetic drugs particularly metformin in overweight patients 180 . Interestingly because of the effects of thiazolidinediones on hepatic fat metabolism these drugs might even be useful for the treatment of non-alcoholic steatohepatitis 181 . Adverse effects and contraindications Fluid retention leading to weight gain anemia and development of heart failure as well as increased incidence of bone fractures are the major adverse effects of TZDs 153 182 . Their use is contraindicated in patients with evidence of heart failure or pre-existing liver disease 183 and they should be used with caution in patients

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47 with osteoporosis and pre-existing macular edema 184 . A debate on the risk of tumor development upon stimulation of PPAR- in colonic cells has been reported thus familial polyposis coli is a contraindication to TZDs on the theoretical grounds 183 . 2. Insulin secretagogues 2.1. Sulfonylureas Since their introduction in the 1950s sulfonylureas SUs have been used extensively as insulin secretagogues for the treatment of T2DM. Sulfonylureas were developed as structural variants of sulfonamides after the latter were reported to cause hypoglycemia. Early sulfonylureas such as carbutamide tolbutamide and chlorpropamide are often referred to as “first generation”. These have been largely superceded by the more potent probably safer “second generation” sulfonylureas notably glibenclamide glyburide gliclazide glipizide and then followed by glimepiride which is considered “third generation” of SUs 183 . Mode of action Sulfonylureas act directly on the -cells of the islets of Langerhans to stimulate insulin secretion. They enter -cell and bind to the cytosolic surface of the sulfonylurea receptor 1 SUR1 which forms part of voltage dependent K + ATP channels leading to its closure and reducing the efflux of potassium enabling membrane depolarization which in turn opens adjacent voltage- dependent L-type calcium channels increasing calcium influx and causing release of insulin 185 . They are ineffective in totally insulin-deficient patients requiring about 30 of normal -cells function for successful therapy 186 . SUs don’t increase insulin formation but stimulate the release of stored insulin in response to glucose concentrations which are below the normal threshold for glucose-stimulated insulin release approximately 5 mmol/L thus they are capable of causing hypoglycemia in normal and diabetic subjects 183 .

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48 Sulfonylureas appear to enhance insulin-stimulated glucose utilization in liver muscle and adipose tissue through increasing insulin receptor number and enhancing the post-receptor complex enzyme reactions mediated by insulin 187 . They are capable of suppressing hepatic glucose production and potentiating adipose tissue glucose transport and lipogenesis as well as skeletal muscle glucose uptake and glycogen synthesis 154 . It has been advocated that sulfonylurea drugs have extrapancreatic effects in addition to their insulin secretory effect on pancreatic -cells as they effectively improve peripheral insulin resistance through activation of peroxisome proliferator- activated receptor- PPAR- like activity 188 . Pharmacokinetics Sulfonylureas vary considerably in their pharmacokinetic properties which in turn affects their clinical suitability for different patients. Longer acting sulfonylureas can be given once daily but carry greater risk of hypoglycemia especially those with active metabolites. Sulfonylureas are highly bound to plasma proteins which can lead to interactions with other protein-bound drugs such as salicylates NSAIDs sulfonamides and warfarin increasing the risk of hypoglycemia 154 . Other drug interactions include: • Interactions that increase glucose lowering effect of SUs as with some antifungals and MAOIs by reducing hepatic metabolism and with probencid by decreasing excretion. • Interactions that decrease glucose lowering effect of SUs as with rifampicin and other microsomal enzyme inducers. Indications Sulfonylureas are widely used as monotherapy and in combination with metformin a thiazolidinedione or an -glucosidase inhibitor 189 . These combinations afford an additive glucose-lowering efficacy at least initially but increase the risk of hypoglycemia. Adverse effects and contraindications

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49 Weight gain reflects the anabolic effects of increased plasma insulin concentrations. Hypoglycemia is the most common and potentially most serious adverse effect of sulfonylurea therapy. Very occasionally sulfonylureas produce sensitivity reactions 183 . 2.2. Meglitinides short-acting prandial insulin releasers Nowadays postprandial hyperglycemia is widely recognized as a central feature of early diabetes and impaired glucose tolerance IGT. It is caused primarily by the impairment of first phase insulin secretion and its correction is important for long-term glycemic control 190 . Meglitinide analogs known as non-sulfonylurea secretagogues were evaluated as potential antidiabetic agents after an observation in the 1980s that meglitinide the non-sulfonylurea moiety of glibenclamide could stimulate insulin secretion similar to sulfonylureas. Repaglinide which was the first approved member of this group and nateglinide were introduced as “prandial insulin releasers” 191 . Mode of action Prandial insulin releasers act similar to SUs. However they activate a different potassium channel in the pancreatic -cell leading to membrane depolarization and insulin release 192 . By generating a prompt increase of insulin to coincide with meal digestion these agents help to restore partially the first phase glucose-induced insulin response that is lost in T2DM. Specifically targeting postprandial hyperglycemia might also address the vascular risk attributed to prandial glucose excursions and reduce the risk of interprandial hypoglycemia as less insulin is secreted several hours after meal 154 . Pharmacokinetics The pharmacokinetic properties of these compounds favored a rapid but short-lived insulin secretory effect that suited administration with meals to promote prandial insulin release. Repaglinide is almost completely and rapidly absorbed with peak plasma concentrations after about 1 hour. It is quickly metabolized in the liver to inactive metabolites and rapidly eliminated in the bile with a terminal elimination half-life of 1 to 1.7 hours 193 . Taken

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50 about 15 minutes before a meal repaglinide produces a prompt insulin response which lasts about 3 hours coinciding with the duration of meal digestion 183 . Repaglinide may be more suitable than nateglinide in patients with moderate renal insufficiency where metformin and some SUs are contraindicated 183 192 . Indications They are theoretically safer in older adults particularly if other agents are contraindicated because of their short half-life and lower risk of hypoglycemia however the need for multiple daily dosages may be a disincentive. They can be used in patients who have an allergy to SUs medication 192 . Prandial insulin releasers can be used as monotherapy in patients inadequately controlled by non-pharmacological measures or as add- ons to metformin or TZDs to produce a synergistic effect 154 . Adverse effects Fewer and less severe hypoglycemic episodes occur with meglitinides than with sulfonylureas. They have a similar risk for weight gain as SUs 192 183 . Sensitivity reactions are uncommon . 3. -Glucosidase inhibitors Acarbose the first -glucosidase inhibitor was introduced in the early 1990s followed by two further agents miglitol and voglibose 194 . Mode of action Their mechanism of action is unique. This is the sole dug class not targeted at a specific pathophysiological defect of type 2 DM. They competitively inhibit the activity of -glucosidase enzymes in the brush border of enterocytes lining the intestinal villi preventing the enzymes from cleaving disaccharides and oligosaccharides into monosaccharides the final steps of carbohydrate digestion delaying glucose absorption. By moving glucose absorption more distally along the intestinal tract glucosidase inhibitors may alter the release of glucose-dependent intestinal hormones GIP and GLP-1 which probably reduce postprandial insulin concentrations concurrently with the attenuated rise in postprandial glucose levels 183 .

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51 Thus -glucosidase inhibitors can effectively reduce postprandial glucose excursions and improves glycemic control 195 . Pharmacokinetics Acarbose is degraded by amylases in the small intestine and by intestinal bacteria less than 2 of the unchanged drug is absorbed along with some of the intestinal degradation products. Absorbed material is mostly eliminated in the urine within 24 hours 183 194 . Miglitol is almost completely absorbed and eliminated unchanged in the urine and faeces 194 . Indications and contraindications -Glucosidase inhibitors can be used rarely as monotherapy due to comparatively mild efficacy 189 for type 2 diabetic patients with postprandial hyperglycemia but only slightly raised fasting glycemia however they are more commonly used as add-on to other therapies again to target postprandial hyperglycemia 196 . They should be taken with meals they are most effective when given with a starchy complex digestible carbohydrate high-fiber diet with restricted amounts of glucose and sucrose 154 . -Glucosidase inhibitors are contraindicated for patients with a history of chronic intestinal disease inflammatory bowel disease predisposition to bowel obstruction and malabsorption syndromes 196 . Moreover high dosages of acarbose can occasionally increase liver enzyme concentrations 183 . Adverse effects -Glucosidase inhibitors have a good safety record but their application has been limited by gastrointestinal side effects which represent the main problem including flatulence abdominal bloating and discomfort and sometimes diarrhea. They do not cause weight gain or frank hypoglycemia 154 . 4. Novel treatment for diabetes mellitus 4.1. Gliptins

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52 Briefly incretin hormones glucose-dependent insulinotropic polypeptide GIP and glucagon-like peptide 1 GLP-1 are secreted from the intestine in response to meal digestion one of their key actions is to increase glucose-induced insulin secretion by the pancreatic islet -cells thereby reducing prandial glucose excursions 154 . Moreover GLP-1 also suppresses glucagon secretion from the islet -cells and in preclinical models proliferation and neogenesis of the -cell and prevention of -cell apoptosis has been observed. GLP-1 also delays gastric emptying and suppresses food intake and appetite 197 . It was noted in the 1980s that the incretin effect is reduced in T2DM. However the peptides cant be administrated straightforward because they are rapidly degraded by the enzyme dipeptidyl peptidase-4 DPP-4. Alternatively gliptins DPP-4 inhibitors such as sitagliptin vildagliptin and more recently saxagliptin were introduced to enhance incretin levels 154 . Mode of action Gliptins inhibit the aminopeptidase activity of DPP-4 an enzyme found free and in epithelial cells in most tissues especially in the intestinal mucosa which cleaves the N-terminal of the incretins GLP-1 and GIP 198 . Raised endogenous incretin concentrations enhance nutrient-induced insulin secretion decreasing postprandial hyperglycemia. Pharmacokinetics Sitagliptin and vildagliptin are each highly bioavailable rapidly absorbed and show relatively low plasma protein binding 199 . Most of their doses are eliminated unchanged in the urine so they are contraindicated in patients with renal impairment or may require dose adjustment 200 . Indications Gliptins are not licensed to be used as monotherapy in T2DM. Currently as newly available agents gliptins tend to be preferred as add-on therapy in patients inadequately controlled by metformin or TZDs. Lack of weight gain makes gliptins suitable for overweight and obese patients 154 . Adverse effects

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53 As there are many natural substrates for DPP-4 including neuropeptide Y bradykinin gastrin releasing polypeptide substance P insulin-like growth factor I and several chemokines such as monocyte chemotactic protein-1 gliptins have the potential to influence the hungersatiety system gastrointestinal motility growth vascular reactivity and immune mechanisms 201 . 4.2. GLP-1 receptor agonists As GLP-1 shows important functions in glycemic control GLP-1 mimetics have been developed. They are designed to be DPP-4 resistant to prolong their plasma half-life. GLP-1 receptor agonists taken subcutaneously improve glucose metabolism mainly by increasing insulin secretion inhibiting glucagon secretion and delaying gastric emptying 197 . This delay in gastric emptying decreases caloric intake promoting weight loss. It is unknown whether GLP-1 receptor agonists can delay the progression of T2DM or not. At present two GLP-1 receptor agonists exenatide and liraglutide are approved for the treatment of diabetes 202 . Exenatide a synthetic exendin-4 has shorter half-life than liraglutide which is a once daily human GLP-1 analog 203 . They both produce a reduction in glucose concentrations predominately affecting postprandial glycemic excursion with only modest effects on fasting blood glucose and low risk of hypoglycemia 204 . Adverse effects The main side effects as nausea and vomiting are dose dependent 205 . About 40-50 of treated subjects develop antibodies to exenatide. Exenatide is not recommended for patients with severe kidney failure because it is predominantly eliminated by glomerular filtration 206 . Future GLP-1 receptor agonists now in phase 3 clinical development Albiglutide is a long-acting GLP-1 receptor agonist developed by genetic fusion of a DPP-4 resistant GLP-1 dimer to recombinant human albumin 207 . Despite the large size of the molecule albiglutide inhibits gastric emptying

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54 and appetite although its anorectic effect may be weaker than that of native GLP-1 because of an impaired blood brain barrier permeability 208 . Taspoglutide is a modified human GLP-1 receptor agonist taken once weekly 209 . Lixisenatide is a novel modified exendin-4 molecule 210 . 4.3. Amylin and amylin analogs Amylin is a 37 amino acid peptide co-secreted with insulin. It delays gastric emptying suppresses postprandial glucagon secretion and increases satiety 211 . Human amylin has an inherent tendency to self-aggregate forming fibrils and to adhere to surfaces. Thus pramlintide has been developed as an amylin analog with some amino acid replacement. Pramlintide was approved by the FDA in 2005 for use subcutaneously in insulin treated subjects with either T1DM or T2DM. Long-term clinical trials have shown quite promising results that the use of pramlintide as an adjunct to insulin minimizes postprandial glucose excursions and reduces both HbA 1C and body weight when compared to placebo 212 . It is primarily eliminated by the kidneys 213 . Side effects The most common side effects are gastrointestinal often nausea and vomiting but these are generally transient. Hypoglycemia can also occur. However when the dose is titrated especially with a reduction in insulin dose both side effects have been reduced significantly 214 . 4.4. Rimonabant Rimonabant is the first agent of the class of drugs that act on the novel endocannabinoid system ECS. The ECS is a novel physiological neuroendocrine system that plays a key role in appetite and metabolism both in brain and adipose tissue. Animal studies have shown that blocking the ECS leads to weight loss and improved insulin sensitivity. Due to this effect agents that block receptors CB1 and CB2 in this system have been developed for the management of human obesity. By blocking CB1 receptors in the brain rimonabant has been shown to reduce weight by suppressing

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55 appetite and by modifying glucose and fat metabolism. In the adipose tissue the drug increases concentrations of adiponectin improving insulin sensitivity 215 . The most common side effects were nausea vomiting diarrhea dizziness anxiety depression and suicidal thoughts 216 217 . These mental disorders result in its withdrawal from the market in 2008 217 . 5. New Experimental Agents Many potential drugs are currently in investigation. They are undergoing phase I/II studies.  PPAR/ ligands muraglitazar and tesaglitazar - development stopped due to adverse risk profile aleglitazar - is now under clinical development 218 .  Sodium-dependent glucose transporter 2 inhibitors SGLT2 inhibitors increase urinary glucose excretion 218 .  Fructose 16-bisphosphatase inhibitors FBPase inhibitors decrease gluconeogenesis in the liver 218 .  Imeglimin is an indirect activator of AMP-kinase acting at the mitochondrial level as an oxidative phosphorylation blocker to inhibit hepatic gluconeogenesis increase muscle glucose uptake and restore normal insulin secretion. It will be the first of a new class of antidiabetic agents Glimins if it is approved. It is in development for use both as monotherapy and in combination with other antidiabetic agents 218 . Treatment strategies for initiation of oral therapy 219 In patients with newly diagnosed type 2 diabetes • Initiate pharmacologic therapy with an oral agent preferably an insulin sensitizer. It is recommended to start with metformin the optimal first line agent especially in obese patients or a thiazolidinedione or a sulfonylurea as monotherapy as long as no contraindication is present. The meglitinides and the -glucosidase inhibitors are less effective and are less commonly

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56 used to initiate therapy. If the blood glucose level is especially high 280- 300 mg/dl and the patient is symptomatic insulin should be considered as first-line therapy. • If monotherapy fails to achieve the desired level of glycemic control a second oral agent should be added. Various combination tablets are available including Metaglip glipizide plus metformin and Glucovance glyburide plus metformin. • In diabetic patients in whom glycemic control is not achieved with two oral agents several options are available. These include 1 addition of a third oral antidiabetic agent 2 addition of bedtime insulin to oral agent therapy if this option is chosen to avoid hypoglycemia it is preferred to stop sulfonylurea and continue the insulin sensitizer 3 switching the patient to a mixed-split insulin regimen with or without an insulin sensitizer. It is important to note that ultimately most patients with type 2 diabetes will require treatment with insulin either alone or in combination with an oral agent. B- Insulin Insulin is a hormone secreted by pancreatic -cells affects a wide range of physiological processes although it is best known for its important regulatory role in glucose homeostasis. Insulin secretion is increased in response to elevated plasma glucose stimulating glucose uptake and glycogen synthesis and inhibiting glycogenolysis and gluconeogenesis thus maintaining normoglycemia. In addition to these well-established short-term actions insulin exerts a number of other important metabolic effects as it regulates the expression of genes involved in amino acid uptake lipid metabolism in muscle and adipose tissue and in cell growth development and survival 220 . With the introduction of several new insulins since 1996 insulin therapy became the treatment of type 1 diabetes and under certain conditions for type 2 diabetic patients. Insulin therapies are now able to more closely mimic physiologic insulin secretion and thus achieve better glycemic control in patients with diabetes 221 .

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57 In 1955 insulin was the first protein to be fully sequenced. The insulin molecule consists of 51 amino acids arranged in two chains an A chain 21 amino acids and B chain 30 amino acids that are linked by two disulfide bonds. Proinsulin is the insulin precursor that is first processed in the golgi apparatus of the -cell and then packaged into granules. Proinsulin a single- chain 86 amino acid peptide shown in Figure 12 is cleaved into insulin and C-peptide a connecting peptide both are secreted in equimolar portions from the -cell upon stimulation from glucose and other insulin secretagogues. Although proinsulin may have some mild hypoglycemic action C-peptide has no known physiologic function 222 . Figure 12: Structure of human proinsulin and some commercially available insulin analogs. Insulin is shown as the shaded peptide chains A and B. differences in the A and B chains and amino acid modifications for insulin aspart lispro and glulisine are noted 222 . In general insulin should be initiated in patients with type 2 diabetes under the following circumstances 223 : • Symptomatic diabetes thirst weight loss visual impairment or severe hyperglycemia i.e. fasting plasma glucose 250 mg/dl • Advanced renal or hepatic disease

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58 • Intolerance or contraindications to oral agents or increased risk of a major side effect such as lactic acidosis with metformin treatment • Intercurrent events that require hospitalization such as myocardial infarction cardiovascular accidents cerebrovascular accidents acute illness or surgery • Hypoperfusion states such as sepsis or hypotension • Triglyceride level higher than 700 mg/dl especially in patients with coronary artery disease as hypertriglyceridemia worsens insulin resistance and cardiovascular diseases • Corticosteroid therapy • Pregnancy • Ketoacidosis or hyperosmolar states • Inability to control blood glucose level or reduce HbA 1C less than 7 after using combination oral hypoglycemic agents for 4 months or longer • Latent autoimmune diabetes in adults LADA misdiagnosed as type 2 diabetes mellitus Insulin secretion Insulin is released from pancreatic -cells at a low basal rate and at a much higher stimulated rate in response to a variety of stimuli especially glucose Figure 13. Other stimulants such as other sugars e.g. mannose certain amino acids e.g. leucine arginine fatty acids amylin and hormones such as glucagon-like polypeptide-1 are recognized 222 . When evoked by glucose insulin secretion is biphasic the first phase reaches a peak after 1 to 2 minutes and is short-lived for 10 minutes which suppresses hepatic glucose production and facilitates the second phase which has a delayed onset but a longer duration lasts two hours and covers mealtime carbohydrates. Between meals a low continuous insulin level called basal insulin serves the ongoing metabolic needs. In type 2 diabetes first phase release is absent and second phase release is delayed and inadequate 224 .

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59 Figure 13: Model of control of insulin release from the pancreatic -cell by glucose and by sulfonylurea drugs 222 . The insulin receptor The full insulin receptor consists of two covalently linked heterodimers each containing an -subunit which is entirely extracellular and constitutes the recognition site and a -subunit that spans the membrane and contains a tyrosine kinase. The binding of an insulin molecule to the -subunits at the outside surface of the cell activates the receptor and through a conformational change brings the catalytic loops of the opposing cytoplasmic -subunits into closer proximity. This facilitates mutual phosphorylation of tyrosine residues on the -subunits and activates tyrosine kinase directed at cytoplasmic proteins 222 . This is illustrated in Figure 14. After tyrosine phosphorylation at several critical sites the IRS molecules bind to and activate other kinases most significantly phosphatidylinositol-3-kinase which produce further phosphorylations to adaptor proteins. This network of phosphorylations within the cell represents insulins second message and results in multiple effects. These include translocation of glucose transporters to the cell membrane with a resultant increase in glucose uptake increased glycogen synthase activity and increased glycogen formation multiple effects on protein synthesis and lipogenesis and activation of transcription factors that enhance DNA synthesis and cell growth and division. Aberrant serine phosphorylation of

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60 the insulin receptor -subunits or IRS molecules may result in insulin resistance and functional receptor down-regulation 222 . Figure 14: Schematic diagram of the insulin receptor heterodimer in the activated state 222 . Like any receptor insulin receptors are subjected to up-regulation and increase in responsiveness to insulin in low insulin concentrations while subjected to down-regulation and decrease in responsiveness to insulin in high insulin concentrations. Insulin actions on carbohydrate fat and protein metabolism Insulin influences glucose metabolism in most tissues especially the liver where it inhibits glycogenolysis and gluconeogenesis decreasing hepatic glucose output and stimulates glycogen synthesis increasing hepatic glycogen stores. In muscle unlike liver uptake of glucose is slow and is the rate-limiting step in carbohydrate metabolism. The main effects of insulin in muscles are to increase facilitated transport of glucose via GLUT4 and to stimulate glycogen synthesis and glycolysis 225 . Insulin increases glucose uptake by GLUT4 in adipose tissue as in muscle enhancing glucose metabolism. Moreover it increases synthesis of fatty acid and triglyceride in adipose tissue and in liver. It inhibits lipolysis partly via dephosphorylation and hence inactivation of lipases 225 .

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61 Insulin stimulates uptake of amino acids into muscle and increases protein synthesis. It also decreases protein catabolism and inhibits oxidation of amino acids in the liver 225 . In addition insulin increases the permeability of many cells to potassium magnesium and phosphate ions. The effect on potassium is clinically important. Insulin activates sodium-potassium ATPases in many cells causing a flux of potassium into cells. Under certain circumstances injection of insulin can kill patients because of its ability to acutely suppress plasma potassium concentrations 225 . Insulin pharmacokinetics Endogenous insulin passes through the portal vein to the liver where extensive metabolism occurs. The rest enters the systemic circulation where its concentration is only about 15 of that entering the liver in fasting state. In contrast when insulin is injected subcutaneously intramuscularly or intravenously it enters the systemic circulation and then distributes to all tissues the liver and other peripheral organs in the same concentration. Therefore during insulin treatment liver is relatively hypoinsulinized and the peripheral tissues are relatively hyperinsulinized. Intravenous administration continuous or pulse is not practical but permits much more physiological insulin profiles. Other routes of insulin delivery have been proposed including the intraperitoneal route which allows insulin to enter at least in part the portal vein similarly to the endogenously secreted insulin transdermal insulin patches yielded disappointing results and rectal suppositories unable to induce a physiological profile of insulinemia 226 . Research into oral administration of insulin has been ongoing for several years. Oral delivery of insulin is restricted mainly due to its susceptibility to denaturation and proteolysis as well as its inability to traverse across biological barriers 227 228 . Various approaches have been adopted to overcome the inherent barriers for oral insulin including chemical modification of insulin and co-administration of adjuvants either in the form of absorption enhancers or protease inhibitors. Among the promising effective approaches towards developing oral insulin delivery systems is the use of polymeric nanoparticles 228 . The insulin loaded nanoparticles coated

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62 with the mucoadhesive chitosan an intestinal permeation enhancer may prolong their residency in the gut protect them from gastric enzymes and enhance permeability by disrupting tight junctions between gut epithelial cells thus allowing the drug to reach its ultimate bloodstream destination 227 228 . Inhaled insulin is a powder form of recombinant DNA rDNA human insulin that is administered through an inhaler device. Insulin is readily absorbed into the bloodstream through alveolar walls but the challenge has been to create particles that are small enough to pass through the bronchial tree without being trapped and still enter the alveoli in sufficient amounts to have a clinical effect 222 . Insulin preparations Effects of insulin therapy and its mechanism of action are the same as normal insulin. Insulin for clinical use was once either porcine or bovine but is now almost entirely human made by recombinant DNA technology. Porcine and bovine insulins differ from human insulin in their amino acid sequence and are liable to elicit an immune response a problem that is avoided by the use of recombinant human insulin. Commercial insulin preparations are highly purified and differ in a number of ways such as differences in the recombinant DNA production techniques amino acid sequence concentration solubility and the time of onset and duration of their biologic action 222 . 1 Ultra-short-acting with very fast onset and short duration taken before meals e.g. insulin lispro insulin aspart and insulin glulisine 229 . 2 Short-acting with rapid onset of action Regular insulin which is a soluble crystalline zinc insulin. Its effect appears within 30 minutes and peaks between 2 and 3 hours after subcutaneous injection and generally lasts 5-8 hours 222 . 3 Intermediate-acting NPH Neutral Protamine Hagedorn or Isophane its absorption and the onset of action are delayed by combining appropriate amounts of insulin and protamine so that neither is present in an

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63 uncomplexed form "isophane" 222 . Another intermediate acting analogue is “Lente insulin” which is a mixture of 3:7 semilente and ultralente. They have an onset of approximately 1-2 hours and duration of 18-24 hours 219 . 4 Long-acting with slow onset of action Ultralente insulin has an onset of action 4-6 hours a peak effect 16-18 hours after injection and a duration of action up to 36 hours 219 . 5 Ultra-long-acting insulin analog Insulin Glargine Lantus® is a soluble "peakless" i.e. having a broad plasma concentration plateau. This product was designed to provide reproducible and convenient insulin replacement 222 through providing a constant basal insulin supply that mimics the physiological insulin secretion 230 . Insulin detemir is a most recently developed long-acting insulin analog designed to prolong its availability by increasing both self-aggregation in subcutaneous tissue and reversible albumin binding 231 . Insulin degludec is a novel insulin analog in clinical development that forms soluble multihexamer assemblies after subcutaneous injection resulting in an ultra-long duration of action 232 . Uses of insulin 225 • For treatment of type 1 and type 2 diabetes mellitus which are the primary indications for insulin therapy • For hyperkalemia as insulin promotes the passage of potassium simultaneously with glucose into cells • In anterior pituitary function test the insulin stress test Adverse effects of insulin 1- Hypoglycemia is the most frequent and feared complication of insulin treatment with potentially serious sequelae 233 . Poor timing of meals exercise and insulin treatment can lead to hypoglycemia which may lead to coma convulsions and even death mainly due to glucose deprivation in brain 234 .

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64 2- Weight gain is also among insulin adverse effects. Improved glycemic control decreases glucosuria thereby decreasing the loss of calories through the urine and the direct lipogenic effects of insulin on adipose tissue both contribute to weight gain 233 . 3- Insulin lipodystrophy lipoatrophy or lipohypertrophy can occur after repeated administration of insulin at the same injection site however these are rare with purified human insulin. Lipoatrophy might be the result of a repeated mechanical trauma. In addition insulin impurities can stimulate immune factors which lead to local release of lipolytic substances 235 . On the other hand lipohypertrophy is due to a possible growth factor effect of insulin on cellular elements of subcutaneous tissue 226 . Both may alter the absorption rate of insulin thus possibly affecting the metabolic control. 4- Local allergy or generalized allergy ranging from a simple urticaria to more severe reactions such as anaphylaxis may occur 226 . Management of diabetic dyslipidemia: 236 To Cure Diabetes Naturally Click Here In type 2 diabetes mellitus an increased prevalence of lipid abnormalities contributes to the accelerated atherosclerosis thus aggressive screening of these abnormalities are essential. A fasting lipid profile is recommended at the initial evaluation and at least annually for adults with T2DM because frequent changes in glycemic control may affect lipoprotein levels. In adults with low risk lipid values repeated assessments can be done every 2 years. The target lipid profile is to reduce LDL to 100 mg/dl or to 70 mg/dl in high risk patients and triglycerides to 150 mg/dl as well as increase HDL to 40 mg/dl for men and to 50 mg/dl for women. NonHDL-C can be targeted in patients with triglycerides 200 mg/dl its goal is 130 mg/dl. • Achieve optimal glycemic control and maximal adherence to therapeutic and lifestyle changes.

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65 • Lowering LDL-C to the target level is the primary goal of therapy. For patients more than 40 years of age statin therapy HMG-CoA reductase inhibitors is recommended to achieve a LDL-C reduction of 30-40 regardless of baseline LDL levels. Statins are the most effective LDL-C lowering medication with an excellent safety profile. They are effective in lowering cardiovascular events independent of baseline LDL pre-existing vascular disease type or duration of diabetes or adequacy of glycemic control. In patients who do not achieve LDL cholesterol targets bile acid sequestrants and cholesterol absorption inhibitors can be used for further reduction of LDL-C levels. • Fibrates particularly fenofibrate and niacin are primarily used to lower triglycerides and raise HDL-C levels to achieve target levels. In addition they can reduce LDL-C levels as well. • Combination therapy using statins and fibrates or niacin may be necessary to achieve lipid targets in some patients. • In the presence of dyslipidemia characterized predominantly by severely elevated triglycerides 500 mg/dl it should be aggressively managed as it is considered a risk factor for pancreatitis 51 . A fibrate is recommended but additional therapy with niacin and omega-3- fatty acid may be required. Management of diabetic complications: 236 Cure Diabetes in 21 Days • Concerning cardiovascular diseases low dose aspirin and an angiotensin converting enzyme inhibitors ACEI or an angiotensin receptor blocker ARB are recommended to diabetic patients with hypertension as they decrease myocardial infarction risk and can confer unique protective effects. • Guidelines advocate for the use of ACEI or ARBs for microalbuminuria even in non-hypertensive patients because of their ability to slow its progression to clinical proteinuria as well as the latter to end stage renal failure ESRF. In further progression in renal

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66 insufficiency the essential approaches are managing the secondary complications as anemia and hyperparathyroidism as well as managing protein intake hyperphosphatemia hyperkalemia and the overall nutrition. However in ESRF dialysis and transplantation are the available managing options. • Panretinal photocoagulation is considered the treatment of choice for patients with proliferative retinopathy which is used to stop neovascularization before recurrent hemorrhages into the vitreous causing irreparable damage. • Infected foot ulcers usually require intravenous antibiotics bed rest with foot elevation and surgical debridement. In addition reducing plantar pressure using specialized footwear accelerates healing. • Intensive control of hyperglycemia and hypertension as well as control the pain if presents represent the primary tools in the prevention and management of neuropathy. Tricyclic antidepressants antiseziure medications with analgesics may be helpful in some patients with painful neuropathy. Physical therapy is often helpful. • Metoclopramide and domperidone are effective in gastroparesis. • Patients with orthostatic hypotension can benefit from nonpharmacological and pharmacological interventions midodrine and caffeine used to treat this condition.

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67 AIM OF THE WORK Cure Diabetes in 21 Days The aim of present study is to evaluate the possible effects of oral treatment with the antidiabetic drug metformin and the antioxidant amino acid L-cysteine when used alone or in combination in streptozotocin experimentally-induced diabetic rats. Moreover it was aimed to shed a light on the glycemic control lipid profile inflammatory markers and hepatic tissue oxidative stress changes occurring in streptozotocin experimentally- induced diabetic rats.

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68 MATERIALS AND METHODS Cure Diabetes in 21 Days 1-Experimental animals In the present study 50 adult male albino Wistar rats 3-4 months old weighing between 170-200 gm were used. The rats were obtained from the animal house of the Medical Research Institute Alexandria University. All rats were kept under observation for at least one week prior to study with free access to food and water for acclimatization. Rats were exposed to alternate cycle of 12 hours light and 12 hours darkness. They were put in pairs in transparent cages under good sanitary conditions and normal humidity. All procedures were performed in accordance with regulations of the National Research Council’s guide for the care and use of laboratory animals. 2-Induction of type 2 diabetes mellitus in rats To develop a rat model of experimentally-induced type 2 diabetes mellitus which resembles that occurring in human population overnight fasting rats were injected intravenously with low dose streptozotocin 15 mg/kg after high fat diet for two months. The solution was freshly prepared and was injected via the caudal vein. Diabetes was confirmed 3 days later by blood glucose level 200-300 mg/dl for 2 consecutive days in fed animals. 3-Drugs and doses 1- Streptozotocin Sigma Aldrich Co-USA dissolved in 0.1M citrate buffer pH 4.5 and injected intravenously through the caudal vein in a dose 15 mg/kg 237 . 2- Metformin HCl Pharco Pharmaceuticals Alexandria Egypt dissolved in distilled water and given by oral gavage to the rats in a dose 300 mg/kg/day 238 .

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69 3- L-cysteine Sigma Co Ltd-England dissolved in distilled water and given by oral gavage to the rats in a dose 300 mg/kg/day 239 . 4- Experimental design In the present study rats were divided into five groups 10 rats each. In all groups except group V rats were kept on a high fat diet 30 fat for 2 months after which they were rendered diabetic by streptozotocin injection intravenously. After induction of diabetes drug treatment was carried out for 2 weeks as follows: Group I: Received distilled water orally and served as untreated diabetic control. Group II: Treated orally with 300mg/kg/day metformin HCl dissolved in distilled water. Group III: Treated orally with 300mg/kg/day L-cysteine dissolved in distilled water. Group IV: Treated orally with both metformin HCl 300mg/kg/day and L-cysteine 300mg/kg/day in distilled water. Group V: Fed conventional rat chow and served as normal non-diabetic control. After 2 weeks of treatment overnight fasting rats were sacrificed by decapitation. Blood was collected and serum was separated for the determination of the following biochemical metabolic parameters: 1- Fasting glucose level 240 . 2- Insulin 241 . 3- Triglycerides 242 . 4- Total cholesterol 243 . 5- HDL 244 . 6- LDL 245 . 7- Free fatty acids 246 . 8- Monocyte chemoattractant protein-1 MCP-1 247 248 . 9- C-reactive protein 249 . 10- Nitric oxide 250 .

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70 Immediately after collection of blood livers were excised washed with ice-cold saline and preserved for the assessment of: 1- Malondialdehyde level 251 . 2- Reduced glutathione 252 . 3- Protein content 253 . I- Biochemical metabolic parameters 1- Determination of fasting serum glucose levels 240 Reaction principle According to Trinder’s method serum glucose was determined after enzymatic oxidation in the presence of glucose oxidase. The formed hydrogen peroxide reacts under catalysis of peroxidase with phenol and 4aminophenazone to a red-violet quinoneimine as an indicator. Glucose + O 2 +H 2 O ⎯ Glucose ⎯⎯⎯ Oxidase ⎯⎯→ Gluconic acid + 4H 2 O 2 2H 2 O 2 + 4-aminophenazone + phenol ⎯ Peroxidase ⎯⎯⎯⎯→ Quinoneimine + 4H 2 O Reagents 1- Enzyme reagent Phosphate buffer pH 7.5 0.1 mmol/L 4-aminophenazone 0.25 mmol/L Phenol 0.75 mmol/L Glucose oxidase 15 KU/L Peroxidase 1.5 KU/L Mutarotase 2.0 KU/L 2- Standard 100 mg/dl 5.55mmol/L Method

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71 In a spectrophotometer cuvette the following reagents were pipetted - 20 μl sample - 2000 μl enzyme reagent The contents were mixed and then incubated for 5 minutes at 37ºC. The absorbance was read at 500 nm within 60 minutes against a blank containing only 2000 μl of the enzyme reagent. The same procedure was repeated using 20 μl of the standard instead of the sample. Calculation Absorbance of sample Concentration of fasting serum glucose mg/dl X 100 Absorbance of standard 2- Determination of fasting serum insulin 241 Serum insulin was determined according to Bank’s method by an invitro enzyme linked immunosorbent assay using ALPCO ® insulin rat ELISA kit. Principle of the assay: This assay is a sandwich type immunoassay based on the immobilization of monoclonal antibodies specific for insulin as the solid phase. Insulin molecules present in the sample become bound to the wells by the immobilized antibody then reacts with a horseradish peroxidase enzyme labeled monoclonal antibody conjugate resulting in insulin molecules being sandwiched between the solid phase and the conjugate. On using 33’55’ tetramethylbenzidine TMB as a substrate a blue color is formed which then changes to yellow color after addition of a stop solution. The intensity of the color generated is directly proportional to the amount of insulin in the sample. Reagents - 96 well insulin microplate coated with mouse monoclonal anti-insulin

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72 - Zero standard 0 ng/ml - Standards A E 0.15 0.4 1 3 5.5 ng/ml - Mammalian insulin high and low controls reconstituted each in 0.6 ml distilled water. - Conjugate stock 11X HRP labeled monoclonal anti-insulin antibody diluted with 10 parts conjugate buffer - Wash buffer concentrate 21X diluted with 20 parts distilled water. - TMB substrate 33’55’ tetramethylbenzidine - Stop solution 0.3 M HCl Method All reagents samples and microplate strips were brought to room temperature. 1. 10 μl of each standard reconstituted control or sample were pipetted into its respective well. 2. 75 μl of working strength conjugate was pipetted into each well. 3. The microplate was incubated for 2 hours at room temperature 1825°C on a horizontal microplate shaker at 700-900 rpm. 4. After incubation the microplate was washed 6 times with working strength wash buffer then 100 μl of TMB substrate was pipetted into each well. 5. The microplate was again incubated with shaking at 700-900 rpm for 15 minutes at room temperature 18-25°C. 6. 100 μl of stop solution was pipetted into each well with gentle shaking to stop the reaction. 7. The absorbance was read at 450 nm within 30 minutes following the addition of stop solution. Calculation

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73 The concentration of serum insulin in the samples was determined in ng/ml from a calibration curve which was constructed from the standards. The zero standard was used as a blank with its average value subtracted from each well. The calibration curve of insulin is illustrated in Figure 15. 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 0 1 2 3 4 5 6

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74 Concentration ng/ ml Figure 15: Standard curve of insulin 3- Determination of serum triglycerides 242 Reaction principle The determination of serum triglycerides was carried out according to Rifai’s method. Triglycerides were determined after enzymatic hydrolysis with lipases by a colorimetric method. The indicator is a quinoneimine derivative formed from hydrogen peroxide 4-aminoantipyrine and pchlorophenol under the catalytic influence of peroxidase. Triglycerides + H 2 O ⎯ Lipases ⎯⎯⎯→ Glycerol + Fatty acids Glycerol + ATP ⎯Glycerol ⎯⎯⎯ Kinase ⎯⎯ → Glycerol-3-phosphate + ADP Glycerol-3-phosphate + O2 ⎯Gycerol ⎯⎯⎯-3⎯P- ⎯ oxidase ⎯ ⎯→ Dihydroacetone phosphate + H 2 O 2 2H 2 O 2 + 4-aminoantipyrine + p-chlorophenol ⎯ Peroxidase ⎯⎯ ⎯ ⎯→ Quinoneimine + 4 H 2 O + HCl Reagents 1- Reagent 1

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75 Pipes buffer 50 mmol/L pH 7.00 p-Chlorophenol 2.7 mmol/L Magnesium ions 14.8 mmol/L ATP 3.15 mmol/L Potassium ferrocyanide 10 μmol/L 4-aminoantipyrine 0.31mmol/L Lipoprotein lipases 2000 U/L Glycerol kinase 500 U/L Glycerol-3-phosphate oxidase 4000 U/L Peroxidase 500 U/L 2- Reagent 2 Standard: Glycerol triglycerides equivalent 200 mg/dl 2.28 mmol/L Method In a spectrophotometer cuvette the following reagents were pipetted - 3 μl sample - 300 μl reagent 1 The contents were mixed then incubated for 10 minutes at 37ºC. The absorbance was read at 500 nm against a blank containing 300 μl of reagent 1 with 3 μl distilled water. The same procedure was repeated using 3 μl of the standard instead of the sample. Calculation Absorbance of sample Concentration of serum triglycerides mg/dl X 200 Absorbance of standard 4-Determination of serum total cholesterol 243 Reaction principle

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76 Serum total cholesterol was determined according to Allian’s method. After enzymatic hydrolysis and oxidation of cholesterol the reaction product hydrogen peroxide forms a red violet indicator quinoneimine with 4-aminoantipyrine and phenol under the catalytic action of peroxidase. Cholesterol ester + H 2O ⎯ Cholestero ⎯⎯⎯ l ⎯ Esterase ⎯⎯→ Cholesterol + fatty acids Cholesterol + O 2 ⎯ Cholestero ⎯⎯⎯ l ⎯ Oxidase ⎯⎯→ 3-Cholesterolone + H 2 O 2 2H 2 O 2 + 4-aminoantipyrine + phenol ⎯Peroxidase ⎯⎯⎯⎯→ Quinoneimine + 4H 2 O Reagents Reagent 1 Color reagent: - Good’s buffer pH 6.7 50 mmol/L - Phenol 5mmol/L - 4-aminoantipyrine 0.3 mmol/L - Cholesterol esterase 200 U/L - Cholesterol oxidase 50 U/L - Peroxidase 3000 U/L Reagent 2 Standard: 200 mg/dl 5.2 mmol/L Method In a spectrophotometer cuvette the following reagents were pipetted - 10 μl sample - 1000 μl color reagent R 1 The contents were mixed then incubated for 10 minutes at 37ºC. The absorbance was read at 500 nm within 60 minutes against a blank containing 1000 μl of color reagent R 1 with 10 μl distilled water. The same procedure was repeated using 10 μl of the standard R 2 instead of the sample. Calculation

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77 Absorbance of sample Concentration of serum total cholesterol mg/dl X 200 Absorbance of standard 5-Determination of serum HDL-cholesterol 244 Reaction principle The determination of serum HDL-cholesterol was carried out according to Grove’s method. Very low-density lipoproteins VLDL and low-density lipoproteins LDL in the sample are precipitated with phosphotungestate and magnesium ions. The supernatant contains highdensity lipoproteins. The HDL-cholesterol is then spectrophotometerically measured by means of the coupled reactions described below: Cholesterol ester + H 2 O ⎯ Cholestero ⎯⎯⎯ l ⎯ Esterase ⎯⎯→ Cholesterol + fatty acids Cholesterol + ½ O 2 + H 2O ⎯ Cholestero ⎯⎯⎯ l ⎯ Oxidase ⎯⎯→ Cholestenone + H 2 O 2 2H 2 O 2 + 4-aminoantipyrine + phenol ⎯Peroxidase ⎯⎯⎯⎯→ Quinoneimine + 4H 2 O Reagents Reagent A Phosphotungestate 0.4 mmol/L Magnesium chloride Reagent B Color reagent 20 mmol/L HDL-cholesterol standard 15 mg/dl Method 1- A mixture of 0.2 ml sample and 0.5 ml reagent A was left to stand for 10 minutes at room temperature and then centrifuged at a minimum of 4000 rpm for 10 minutes. The supernatant was then carefully collected.

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78 2- 0.1 ml of the clear supernatant was pipetted into a spectrophotometer cuvette followed by the addition of 1 ml of color reagent B and incubated for 10 minutes at 37°C. 3- The absorbance was read at 500 nm within 30 minutes against a blank containing 100 μl of distilled water and 1 ml of reagent B. The sample procedure was repeated using 100 μl of the HDL-cholesterol standard instead of the sample. Calculation Absorbance of sample Concentration of serum HDL-cholesterol mg/dl X 52.5 Absorbance of standard 6-Determination of serum LDL-cholesterol 245 Reaction principle Low-density lipoproteins LDL were determined according to Friedewald’s method. LDL in the sample is precipitated with polyvinyl sulphate. Their concentration is calculated from the difference between the serum total cholesterol and the cholesterol in the supernatant after centrifugation. The cholesterol is spectrophotometerically measured by means of the coupled reaction described below: Cholesterol esters + H 2O ⎯ Cholestero ⎯⎯⎯ l ⎯ Esterase ⎯⎯→ Cholesterol + Fatty acids Cholesterol + ½ O 2 + H 2O ⎯ Cholestero ⎯⎯⎯ l ⎯ Oxidase ⎯⎯→ Cholestenone + H 2 O 2 2H 2 O 2 + 4-aminoantipyrine + phenol ⎯ Peroxidase ⎯⎯⎯⎯→ Quinoneimine + 4H 2 O Reagents Reagent A Polyvinyl sulphate 3 gm/L Polyethylene glycol 3 gm/L Reagent B Color reagent

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79 Reagent C Standard: 200 mg/dl 5.2 mmol/L Method 1- 0.4 ml of sample and 0.2 ml of reagent A were mixed thoroughly and was left to stand for 14 minutes at room temperature then centrifuged at a minimum of 4000 rpm for 15 minutes. 2- In a spectrophotometer cuvette 20 μl of the sample supernatant was mixed thoroughly with 1 ml reagent B and then incubated for 10 minutes at 37ºC. 3- The absorbance was read at 500 nm against a blank containing 20 μl distilled water instead of sample. The same procedure was repeated using 20 μl of cholesterol standard reagent C. Calculation Cholesterol in supernatant mg/dl Absorbance of sample X 200 X 1.5 The LDL-cholesterol concentration in the sample is calculated as follows: LDL-cholesterol mg/dl Total cholesterol – cholesterol in the supernatant 7-Determination of serum free fatty acids 246 Reaction principle FFAs were determined according to Shimizus’s method. FFAs are converted by acyl-CoA synthetase Acyl CS into acyl-coenzyme A acylCoA which reacts with oxygen in the presence of acyl-CoA oxidase ACOD to form 23-enoyl-coenzyme A enoyl-CoA. The resulting hydrogen peroxide converts 244-tribromo-3-hydroxy-benzoic acid TBHB and 4-aminoantipyrine to a red dye in the presence of peroxidase.

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80 Free fatty acids + CoA+ ATP ⎯ Acyl ⎯⎯ ⎯ CS →Acyl-CoA + AMP + pyrophosphate Acyl-CoA + O 2 ⎯⎯ ACOD ⎯⎯→Enoyl-CoA + H 2 O 2 H 2 O 2 + 4-aminoantipyrine + TBHB ⎯⎯ Peroxidase ⎯⎯→ Red dye + 2H 2 O + HBr Reagents • Reaction mixture A - ATP coenzyme A acyl-CoA-synthetase peroxidase ascorbate oxidase 4-aminoantipyrine dissolved in potassium phosphate buffer pH 7.8. - Tribromohydroxy-benzoic acid magnesium chloride and stabilizers. • Reaction mixture B: Acyl-CoA-oxidase ACOD and stabilizers in dilute solution. • N-ethyl-maleinimide solution with stabilizers. Method In a spectrophotometer cuvette 50 μl of sample and 1 ml of reaction mixture A were mixed kept at 25ºC for approximately 10 minutes then 0.05 ml of N-ethyl-maleinimide solution was added. The reagents were mixed again and the absorbance A 1 was read at wavelength of 546 nm. This was followed by the addition of 50 μl of reaction mixture B and the absorbance A 2 was read again after 15 minutes. The same procedure was repeated for the blank using 0.05 ml of distilled water instead of the sample. The absorbance differences A 2 -A 1 was calculated for both blank and sample. Calculation 1.15 Concentration of serum free fatty acids mmol/L X A 19.3×1×0.05

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81 1.192 X A Where: - A absorbance difference of sample - absorbance difference of blank - 1.15 final volume in ml - 19.3 absorption coefficient of dyestuff at 546 nm - 1 light path in cm - 0.05 sample volume 8- Determination of serum monocyte chemoattractant protein-1 247 248 Serum MCP-1 was determined by an in-vitro enzyme-linked immunosorbent assay using RayBio ® rat MCP-1 ELISA kit. Principle of the assay This assay is based on the immobilization of an antibodies specific for rat MCP-1 as the solid phase. MCP-1 present in the serum sample becomes bound to the wells by the immobilized antibodies then reacts with biotinylated anti-rat MCP-1 antibodies. On adding horseradish peroxidase enzyme HRP conjugated streptavidin and 33’55’ tetramethylbenzidine TMB a blue color is formed which then changes to yellow color after addition of a stop solution. The intensity of the color generated is directly proportional to the amount of MCP-1 in the sample. Reagents 1. 96 well MCP-1 microplate coated with anti-rat MCP-1. 2. Wash buffer concentrate 20X: diluted with 19 parts distilled water. 3. Standards: recombinant rat MCP-1 reconstituted with diluents A to prepare serial dilutions of MCP-1 standards. 4. Assay diluent A: 0.09 sodium azide as preservative and used as a diluent for standard/sample. 5. Assay diluent B 5X: diluted with 4 parts distilled water.

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82 6. Detection biotinylated anti-rat MCP-1 antibodies: reconstituted with 100 μl of 1X assay diluents B and then diluted 65 fold with 1X diluent B. 7. Horseradish peroxidase enzyme HRP-conjugated streptavidin: diluted 5000 fold with 1X diluent B. 8. TMB substrate 33’55’ tetramethylbenzidine. 9. Stop solution: 2 M sulfuric acid. Method: All reagents and samples were brought to room temperature 18-25°C before use. 1. 100 l of each standard and sample were added into appropriate wells. The microplate was covered well and incubated for 2.5 hours at room temperature or overnight at 4°C with gentle shaking. 2. The solution was discarded and washed 4 times with 1X wash solution using an autowasher. After the last wash any remaining wash buffer was removed. 3. 100 l of 1x prepared biotinylated antibody was added to each well. The microplate was incubated again for 1 hour at room temperature with gentle shaking. 4. The solution was discarded and the wash was repeated. 5. 100 l of prepared streptavidin solution was added to each well. The microplate was incubated again for 45 minutes at room temperature with gentle shaking after which the solution was discarded and the wash was repeated. 6. 100 l of TMB one-step substrate reagent was added to each well and the microplate was incubated again for 30 minutes at room temperature in the dark with gentle shaking. 7. 50 l of stop solution was added to each well and the absorbance was read at 450 nm immediately. Calculation The concentration of serum MCP-1 in the samples was determined in pg/ml from a calibration curve which was constructed from the standards.

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83 The zero standard was used as a blank with its average value subtracted from each well. The calibration curve of MCP-1 is illustrated in Figure 16. Concentration pg/ml Figure 16: Standard curve of monocyte chemoattractant protein-1 MCP- 1 9- Determination of serum C-reactive protein 249 Reaction principle 0 0. 2 0. 4 0. 6 0. 8 1 1. 2 1. 4 1. 6 1. 8 2 0 25 0 50 0 75 0 100 0 125 0 150 0 175 0 200 0 225 0

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84 The serum level of CRP was measured by turbidimetric immunoassay according to Otsuji’s method using an N-Assay TIA CRP-S kit Nittobo Medical Tokyo. Serum C-reactive protein causes agglutination of the latex particles coated with anti-rat C-reactive protein. The agglutination of the latex particles is proportional to the CRP concentration and can be measured by turbidimetry. Reagents Reagent A: Glycine buffer 0.1 mol/L sodium azide 0.95 gm/L pH 8.6 Reagent B: Suspension of the latex particles coated with anti-rat CRP antibodies sodium azide 0.95 gm/L Reagent C: CRP standard C-reactive protein 65.6 mg/L Working reagent: A mixture of reagent A and B Method In a spectrophotometer cuvette the following reagents were pipetted - 1 ml working reagent - 7 μl sample The contents were mixed then the absorbance was read at 540 nm against a blank containing 7 μl distilled water after 10 seconds A 1 and after 2 minutes A 2 The same procedure was repeated using 7 μl of the standard reagent C instead of the sample. Calculation A2 - A1 sample The concentration of serum CRP mg/L X 65.6 A2 - A1 standard

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85 10- Determination of serum nitric oxide end products nitrite NO2 - and nitrate NO3- 250 Principle The nitrite and nitrate concentration was determined by simple Griess reaction. Because the nitric oxide NO has a short half-life 2-30 sec it is preferable to determine nitrite the stable product of NO which may be further oxidized to nitrate. So the Griess reaction was supplemented with the reduction of nitrate to nitrite by NADPH-dependent nitrate reductase. Reagents - Nitrate reductase from Aspergillus niger 3 U/ml in phosphate buffer saline - 0.32 mM NADPH in 20 mM Tris buffer pH 7.6. - Methanol: diethyl ether 3:1 v/v - 6.5 M HCl. - 37.5 mM sulphanilic acid. - 12.5 mM N-1-naphthyl ethylenediamine NED - Standard sodium nitrite solution from 0 to 100 μmol/L Procedure 1 -Nitrate reduction and deproteinization An aliquot of 100 μl of serum was incubated with 50 μl nitrate reductase and 50 μl of NADPH for 30 minutes at room temperature. After the reduction 100 μl of the mixture was incubated overnight with 900 μl methanol: diethyl ether 3:1 v/v. After the overnight incubation the sample was centrifuged at 10000 rpm for 10 min at 40°C and the supernatant was used for determination of nitrite. 2 -Nitrite determination Aliquots of 600 μl of the deproteinized sample 150 μl of 6.5 M HCl and 150 μl of 37.5 mM sulphanilic acid were added to a centrifuge tube and then incubated at 40°C for 30 min. After incubation 150 μl of 12.5 mM NED

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86 was added and the sample was centrifuged at 10000 rpm for 10 min at 40°C. The absorbance of the clear supernatant was measured at 540 nm. Calculation The concentration of NO in the samples was determined in nmol/ml from a calibration curve which was constructed by preparing serial concentrations of sodium nitrite Figure 17. Concentration nmol/ml Figure 17: Standard curve of nitric oxide 0. 0 0. 1 0. 2 0. 3 0. 4 0. 5 0. 6 0. 7 0. 8 0 2 0 4 0 6 0 8 0 10 0 12 0

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87 II- Oxidative stress parameters 1-Determination of hepatic malondialdehyde MDA by thiobarbituric acid reaction 251 Reaction principle Hepatic malondialdehyde was determined by the thiobarbituric acid TBA method which was described by Ohkawa for the assay of lipid peroxides in various animal studies. This thiobarbituric acid TBA test is most frequently used as an index of lipid peroxidation. It depends on the fact that when polyunsaturated fatty acids PUFAs or esters containing 3 or more double bonds undergo auto-oxidation a secondary product of lipid peroxidation which is referred to as malondialdehyde is produced. In this assay one molecule of malondialdehyde the most abundant aldehyde product of lipid peroxidation reacts with two molecules of thiobarbituric acid TBA at pH 3.5 to yield a pink chromagen that can be detected spectrophotometerically at 532 nm. Reagents - 1.15 potassium chloride. - 0.8 thiobarbituric acid TBA in distilled water - 8.1 sodium dodecyl sulphate SDS in distilled water - 20 acetic acid pH 3.5 was adjusted with 1 N NaOH - N-butanol - 11 33-tetramethoxypropane TMP Method 1- A small part of the liver was cut weighed and homogenized in 9 volumes of 1.15 KCl to prepare 10 homogenate w/v.

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88 2- An aliquot of 0.1 ml liver homogenate was added to 0.2 ml of SDS. This was followed by the addition of 1.5 ml of acetic acid and 1.5 ml of aqueous solution of TBA. This mixture was finally made up to 4 ml with distilled water vortexed and then heated in a water bath at 95 °C for 60 minutes. 3- After cooling to room temperature 1 ml of distilled water and 5 ml of nbutanol were added followed by the vigorous shaking. After centrifugation at 4000 rpm for 10 minutes the absorbance of the organic layer was read at 532 nm using Jenway 6305 spectrophotometer against a blank containing 0.1 ml distilled water instead of the sample and treated exactly like the sample. Calculation The level of MDA in the samples in nmol/gm tissue was determined from a standard curve made by preparing serial dilutions of 1133tetramethoxypropane TMP Sigma Chemical Co-USA in 97 ethanol and treating them like the samples. The standard curve of MDA is shown in Figure 18.

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89 Concentration nmol/ml Figure 18: Standard curve of MDA 2-Determination of hepatic reduced glutathione GSH 252 Principle The GSH was measured according to Murphy’s method. This method is based on the reductive cleavage of Ellman’s reagent 55’-dithiobis- 0. 0 0. 1 0. 2 0. 3 0. 4 0. 5 0. 6 0 1 0 2 0 3 0 4 0 5 0

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90 2nitrobenzoic acid DTNB by SH group of glutathione to yield a yellow color with a maximum absorbance at 412 nm. 2GSH + DTNB GSSG + 2 TNB yellow colored chromophore Reagents 1. Trichloroacetic acid TCA and disodium salt of ethylene diamine tetraacetic acid EDTA: disodium salt of EDTA 372 mg and TCA 50 gm were dissolved in one liter of distilled water. 2. Solution A: 0.2 M prepared by dissolving 27.8 gm monobasic sodium phosphate Merck Co Germany in one liter of distilled water. 3. Solution B: 0.2 M prepared by dissolving 53.65 gm dibasic sodium phosphate Merck Co Germany in one liter of distilled water. 4. 0.1 M sodium phosphate buffer pH 8: prepared by adding 26.5 ml of solution A to 473.5 ml of solution B and the volume was adjusted to one liter with distilled water. 5. 0.1 M sodium phosphate buffer pH 7: prepared by adding 195 ml of solution A to 305 ml of solution B and the volume was adjusted to one liter with distilled water. 6. 0.01 M DTNB: prepared by dissolving 39.5 mg DTNB in 200 ml of 0.1 M sodium phosphate buffer pH 7. Procedure 1. The sample was prepared by the addition of an aliquot of 200 μl of the clear supernatant of the 20 liver homogenate in TCA to 4.7 ml of 0.1 M sodium phosphate buffer pH 8 followed by 100 μl of 0.01 M DTNB and vortexed immediately for few seconds. 2. The absorbance of the resultant yellow color was measured spectrophotometerically at 412 nm using Jenway 6305 spectrophotometer within 25 minutes of addition of DTNB against a blank containing 200 μl distilled water instead of the sample.

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91 Calculation The concentration of reduced glutathione in the liver samples was determined from a standard curve made by preparing serial dilutions of standard reduced glutathione in phosphate buffer pH 8 and treating them as samples Figure 19. Results were subsequently expressed as μg reduced glutathione/mg protein by dividing the concentration of glutathione in the sample by the protein concentration in the same sample.

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92 Concentration μg/ml Figure 19: Standard curve of reduced glutathione 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 400 800 1200 1600 2000 2400 2800

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93 3-Determination of total protein 253 Reaction Principle Lowrys method was used for the protein determination. The color produced is thought to be due to a complex between the alkaline copperphenol reagent and tyrosine and tryptophan residues of the protein in the sample. The protein concentration of each sample was estimated by referring to the standard curve constructed using bovine serum albumin fraction V Sigma Chemical Co Poole England. Reagents - Sodium hydroxide 0.1 M - Sodium carbonate anhydrous 2 in 0.1 M NaOH - K + /Na + tartarate 2 - Copper sulphate 1 - Lowry C reagent: prepared immediately before use by mixing volumes of sodium carbonate K + /Na + tartarate and copper sulphate reagent in a ratio 100:1:1 - Folin-Ciocalteau reagent Sigma Chemical Co Poole England. The working reagent was prepared by diluting the stock reagent 1:1 v/v with distilled water immediately before use. Method The sample was diluted in distilled water 1:10. Aliquots of 10 μl of diluted samples were mixed with 2.5 ml of Lowry C reagent. After incubation 10 minutes at room temperature 0.25 ml of working FolinCiocalteau reagent was added. The tubes were then mixed and incubated in a dark place for one hour at room temperature after which the absorbance was read at 695 nm using Jenway 6305 spectrophotometer. A blank containing distilled water instead of the sample was treated similarly. Calculation

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94 The total protein amount was calculated in mg/gm tissue with reference to the protein standard curve which was constructed by preparing serial concentrations of bovine serum albumin fraction V Figure 20. Concentration mg/ml Figure 20: Standard curve of protein 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 9 0 1 2 3 4 5 6 7 8

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95 IV- Conventional pathological assesment of excised pancreas by Haematoxylin and Eosin HE staining Cure Diabetes in 21 Days Excised pancreas were cut into 1-2 cm sections and subjected to the following treatment: 1- Fixation Phase: Formalin 10 was used as a fixative for 12-24 hours. Fixative prevents autolysis of cells by lysosomal enzymes and protects cells from damage during dehydration and mounting. Fixative also stabilizes the protein skeleton of the cells giving the cells some structural support to resist deformation and crushing. 2- Dehydration step: it was done by ascending grades of alcohol 3- Embedding step: dehydrated tissues were embedded in a paraffin blocks. 4- Haematoxylin and Eosin HE staining Phase: Thin sections 3-4 m were stained by conventional haematoxylin and eosin HE stain. a. Haematoxylin staining for 4 min: Haematoxylin Genzyme England is a salt that dissociates in water into positive and negative ions. Its positive ion basic-alkaline readily combines with negatively charged regions of cellular macromolecules especially phosphate groups of nucleic acids coloring them blue to purple or black. Any substance cell or tissue that tends to be stained in this way by hematoxylin is said to be basophilic. Because of their high nucleic acid DNA and RNA content the nuclei of cells are usually basophilic. However the cytoplasm of cells is generally not basophilic unless it contains unusually high amounts of RNA or another macromolecule with appropriate negative charge regions as for example when it is very active in protein synthesis. b. Washing with tap water

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96 c. Eosin staining for 2 min: Eosin water soluble Chematec U.K is also a salt that dissociates in water into ions. Its negative ion which is acidic in nature readily combines with positively charged regions of cellular macromolecules especially positively charged regions of cytoplasmic proteins coloring them a variety of colors ranging from pink to red or orange. Any substance cell or tissue that tends to be stained in this way by eosin is said to be acidophilic or eosinophilic. 5- Dehydration Phase: Samples were passed in ascending series 70 80 95 absolute ethanol for 1 min. 6- Clearing and Dealcoholization Phase: Clearing was performed in equal parts of absolute ethanol and xylene mixture then followed by two changes of xylene. 7- Mounting Phase: One drop of DPX Chematec U.K was used for mounting. The slides were examined by microscope ZEISS Axioskop2 Germany stained sections were examined in all slides and then photographed by camera.

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97 4- Statistical analysis of the data 254 255 To Cure Diabetes Permanently Click Here All values are reported as means S.E.M. Statistical comparisons for differences between groups were performed by analysis of variance ANOVA.Data handling were processed using the microcomputer program statistical package for social science SPSS version 18.0 SPSS Chicago IL USA 256 . Statistically significant differences were assumed at p value less than 0.05. 1- Arithmetic mean X This represents the value around which the sum of the deviations of the individual variations have taken plus and minus cancel out i.e. a measure of central tendency X X n Where: - X Arithmetic mean - X sum of values recorded - n number of observations 2- The standard deviation S.D. This is used as a measure of dispersion. It was calculated as follows 2 X 2 − X ± S.D n n −1

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98 Where: - X 2 Sum of squared values - X 2 Square of the sum of values 3- Standard error of mean S.E.M. This is used as a measure of precision and statistical reliability of the mean i.e. it is the correction of the standard deviation in relation to the number of observations. It was calculated as follows: ± S.D. ±S.E.M. 4- Comparison between more than two groups means ANOVA test F-test analysis of variance This test of significance is used for comparison between more than two groups of variance according to the following equation: BetweenGroupMeansSquaresB.M.S F WithinGroupMeanSquaresW.M.S Where: BetweenGroupSumSquaresB.S.S B. M. S. Groups −1 B. S. S. X2 − X2 K n n

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99 Where : - K Number of each group. - n Number of samples in all group. WithinGroupSumSquaresW.S.S W.M.S n −numberof Groups W. S. S X 2 − X K 5- Least significant difference L.S.D. L.S.D. is used only when F-value was significant to detect the presence of significance between each group. L.S.D t0.05 S2p n11 +n12 Where: - t 0.05 The critical value from t-table 2.101. - n 1 Number of the first group. - n 2 Number of the second group. - S 2 P Pooled variance.

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100 RESULTS To Cure Diabetes Permanently Click Here In the present study the effects of streptozotocin-induced type 2 diabetes in adult male albino rats on different serum biochemical parameters and hepatic oxidative stress markers are discussed. A summary of the effects obtained after treating diabetic rats with metformin and Lcysteine alone or in combination for 2 weeks on these parameters are also presented. All results are expressed as mean ± S.E.M. I- Effect of streptozotocin-induced type 2 diabetes mellitus on the studied parameters in male albino rats 1- Biochemical metabolic parameters 1.1. Fasting serum glucose FSG FSG level was significantly increased in STZ-induced type 2 diabetic rats as compared to normal rats. The mean value of the normal control group was 89.90±2.58 mg/dl while that of the STZ-rats was 194.40±2.17 mg/dl at p 0.001. Table 1 and Figure 21 1.2. Fasting serum insulin FSI Type 2 diabetic rats had shown significantly higher values of FSI than the normal control rats at p 0.001. The FSI mean was 4.01±0.09 ng/ml for the untreated diabetic rats as compared to 1.37±0.04 ng/ml for the normal rats. Table 1 and Figure 22 1.3. Homeostasis model assessment of insulin resistance HOMA-IR The calculated HOMA-IR mean was 48.13±1.20 in the untreated diabetic group which showed a statistically significant increase when compared to 7.61±0.29 in the normal control group at p 0.001. Table 1 and Figure 23

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101 2- Lipid profile The deleterious effects of experimentally-induced type 2 diabetes on lipid profile are shown in table 2 and figure 24A-24G. 2.1. Serum triglycerides TGs Mean value of serum triglycerides was significantly increased in the diabetic untreated rats in comparison to the normal non-diabetic controls at p 0.001. The mean value of triglycerides for normal controls was 39.10±2.03 mg/dl while that of the untreated diabetic rats was 162.80±1.81 mg/dl. Table 2 and Figure 24a 2.2. Serum total cholesterol TC The present study revealed that there was a significant increase in total cholesterol level in STZ-diabetic rats with a mean value 126.10±1.70 mg/dl as compared to the normal group in which the mean value was 79.70±1.41 mg/dl at p 0.001. Table 2 and Figure 24b 2.3. Serum high-density lipoprotein cholesterol HDL-C There was a significant decrease in the mean value of HDL-C of the untreated diabetic group 28.0±0.77 mg/dl when compared to the mean of the normal controls 58.0±1.15 mg/dl at p 0.001. Table 2 and Figure 24c 2.4. Serum low-density lipoproteins cholesterol LDL-C Serum LDL-C was significantly increased in STZ-diabetic untreated rats with a mean value of 65.54±0.87 mg/dl as compared to the mean value 13.88 ±0.82 mg/dl for the normal controls at p 0.001. Table 2 and Figure 24d 2.5. Serum free fatty acids FFAs The mean value of FFAs in the untreated diabetic rats was significantly higher than that in the normal group at p 0.001. The FFAs mean value was

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102 1.32±0.03 mmol/L for the untreated diabetic rats as compared to 0.27±0.03 mmol/L for the normal non-diabetic rats. Table 2 and Figure 24e 2.6. Non-HDL-cholesterol Experimental STZ-induced diabetes resulted in significantly elevated values of the calculated non-HDL-C at p 0.001. Non-HDL-C mean value increased significantly from 21.70±1.03 mg/dl in normal rats to 98.10±0.92 mg/dl in diabetic rats. Table 2 and Figure 24f 2.7. Triglycerides to HDL-cholesterol ratio TGs/HDL The untreated STZ-diabetic rats had shown higher mean value of TGs/HDL ratio 5.85±0.06 as compared to the mean value 0.68±0.15 in the normal group at p 0.001. Table 2 and Figure 24g 3- Hepatic oxidative stress parameters 3.1. Hepatic malondialdehyde MDA As shown in Table 3 and Figure 25 STZ-induced type 2 diabetes resulted in a pro-oxidant state manifested by a significant increase in lipid peroxidation products estimated as hepatic MDA 24.76±0.57 nmol/gm wet tissue as compared to normal non-diabetic control rats with a mean value of 9.12±0.37 nmol/gm wet tissue at p 0.001. 3.2. Hepatic reduced glutathione GSH The hepatic GSH content in the untreated diabetic rats expressed in ratio to total protein content was significantly lower than normal control rats 135.31±1.64 μg/mg protein versus 250.10±2.31 μg/mg protein at p 0.001. Table 3 and Figure 26 4- Serum inflammatory parameters 4.1. Monocyte chemoattractant protein-1 MCP-1

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103 Experimentally-induced type 2 diabetic rats showed a significantly increased level of serum monocyte chemoattractant protein-1 when compared to the normal control rats at p 0.001. The mean value of MCP-1 for the normal non-diabetic group was 157.84±2.60 pg/ml while that of the untreated diabetic group was 670.42±9.01 pg/ml. Table 4 and Figure 27 4.2. Serum C-reactive protein CRP Mean value of C-reactive protein was significantly increased in the untreated diabetic rats as compared to the normal non-diabetic controls with mean values 3.25±0.08 mg/L and 0.40±0.03 mg/L respectively at p 0.001. Table 4 and Figure 28 4.3. Serum nitric oxide NO Untreated diabetic rats had shown significantly higher values of serum nitric oxide than the normal control rats at p 0.001. The NO mean was 65.95±2.07 nmol/ml for the untreated diabetic rats as compared to 18.77±0.62 nmol/ml for the normal non-diabetic control rats. Table 4 and Figure 29

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104

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105 Effect of STZ-induced type 2 diabetes on fasting serum glucose fasting serum insulin and HOMA-IR in male albino rats Figure 21: Effect of STZ-induced type 2 Figure 22: Effect of STZ-induced type 2 diabetes on fasting serum glucose diabetes on fasting serum insulin in in male albino rats male albino rats

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106

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107 Figure 23: Effect of STZ-induced type 2 diabetes on HOMA-IR in male albino rats : Significant in comparison to normal rats

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108 Effect of STZ-induced type 2 diabetes on lipid profile in male albino

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109

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110 rats Figure 24a: Effect of STZ-induced type 2 diabetes on serum triglycerides in male albino rats

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111 Figure 24c: Effect of STZ-induced type 2 diabetes on serum HDL-C in male albino rats Figure 24b: Effect of STZ- induced type 2 diabetes on serum total cholesterol in male albino rats Figure 24d: Effect of STZ-induced type 2 diabetes on serum LDL-C in male albino rats Figure 24e: Effect of STZ-induced type 2 diabetes on serum free fatty acids in male albino rats

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112 Figure 24f: Effect of STZ-induced type 2 diabetes on non-HDL-C in male albino rats Figure 24g: Effect of STZ-induced type 2 diabetes on TGs/HDL ratio in male albino rats : Significant in comparison to normal rats

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113 Effect of STZ-induced type 2 diabetes on oxidative stress parameters in male albino rats

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114

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115 Figure 25: Effect of STZ-induced type 2 Figure 26: Effect of STZ-induced type 2 diabetes on hepatic diabetes on hepatic reduced malondialdehyde in male albino glutathione in male albino rats rats : Significant in comparison to normal rats

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116

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117

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118

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119 Effect of STZ-induced type 2 diabetes on inflammatory parameters in male albino rats

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120 Figure 27: Effect of STZ-induced type 2 diabetes on serum monocyte chemoattractant protein-1 in male albino rats Figure 28: Effect of STZ-induced type 2 diabetes on serum C- reactive protein in male albino rats Figure 29: Effect of STZ-induced type 2 diabetes on serum nitric oxide in male albino rats : Significant in comparison to normal rats

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121 II- Effect of oral administration of metformin Lcysteine and their combination for 2 weeks on the studied parameters in STZ-induced male albino rats 1- Biochemical metabolic parameters 1.1. Fasting serum glucose FSG • Metformin treated group Treatment of STZ-induced type 2 diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally was associated with significant decrease in FSG level at p 0.001. The mean values of the FSG were decreased from 194.40±2.17 mg/dl in the untreated diabetic group to 143.00±1.57 mg/dl after metformin treatment LSD ≥ 7.542. Table 5 and Figure 30 • L-cysteine treated group When L-cysteine was administrated orally in a dose of 300 mg/kg/day for 2 weeks to the STZ-diabetic rats it resulted in a significant decrease in the mean value of FSG from 194.40±2.17 mg/dl to 167.20±2.36 mg/dl at p 0.001 LSD ≥ 7.542. Table 5 and Figure 30 • Combination treated group Our study revealed that the combination treatment of STZ-induced type 2 diabetes in male albino rats by oral metformin 300 mg/kg/day and oral L- cysteine 300 mg/kg/day for 2 weeks caused a significant reduction in the mean value of FSG from 194.40±2.17 mg/dl to 127.20±1.42 mg/dl at p 0.001 LSD ≥ 7.542. Table 5 and Figure 30 Thus both metformin and L-cysteine alone or in combination significantly decreased FSG level as compared to untreated diabetic rats at p 0.001. However the glucose lowering effect of metformin was more evident than that of L-cysteine as the average values of FSG in the two groups were 26.4 and 14 below the untreated diabetic level respectively. When both drugs were given in combination greater decreases in the FSG to about 34.6 below the STZ-diabetic values were observed.

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122

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123

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124 Figure 30: Effect of treatment with the studied drugs for 2 weeks on fasting serum glucose in male albino rats

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125 : Significant in comparison to normal rats 1.2. Fasting serum insulin FSI • Metformin treated group Metformin treatment to the STZ-diabetic rats in a dose of 300 mg/kg/day for 2 weeks orally caused a significant decrease in the FSI reaching a value of 2.64±0.05 ng/ml as compared to the untreated diabetic group in which the mean value of FSI was 4.01±0.09 ng/ml at p 0.001 LSD ≥ 0.205. Table 6 and Figure 31 •L-cysteine treated group Treating STZ-diabetic rats with oral L-cysteine in a dose of 300 mg/kg/day for 2 weeks significantly reduced the FSI when compared to the untreated group at p 0.001. The mean value of FSI was 3.24±0.04 ng/ml in the L-cysteine treated group while that of the diabetic group was 4.01±0.09 ng/ml LSD ≥ 0.205. Table 6 and Figure 31 • Combination treated group Concurrent administration of both metformin 300 mg/kg/day and Lcysteine 300 mg/kg/day for 2 weeks orally to the STZ-induced diabetic rats reduced that FSI significantly as compared to the untreated diabetic rats

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126 with mean values of 2.19±0.03 ng/ml and 4.01±0.09 ng/ml respectively p 0.001 LSD ≥ 0.205. Table 6 and Figure 31 The effects of both drugs given alone and in combination on FSI were qualitatively similar to their effect on FSG. Treatment with either metformin or L-cysteine alone for 2 weeks resulted in significant decreases in FSI to about 34.2 and 19.2 below the STZ-diabetic values respectively p 0.001. The reduction of FSI was more evident in the combination group than either drug alone reaching values 45.4 below the untreated diabetic rats.

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127

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128

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129 Figure 31: Effect of treatment with the studied drugs for 2 weeks on fasting serum insulin in male albino rats : Significant in comparison to normal rats

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130 1.3. Insulin resistance HOMA-IR • Metformin treated group Treatment with oral metformin in a dose of 300 mg/kg/day for 2 weeks significantly decreased the HOMA-IR mean value to 23.31±0.40 as compared to the mean value of untreated diabetic rats 48.13±1.20 at p 0.001 LSD ≥ 2.460. Table 7 and Figure 32 • L-cysteine treated group Treatment with L-cysteine 300 mg/kg/day for 2 weeks orally caused a significant reduction in the HOMA-IR levels 33.41±0.70 in comparison to the STZ-diabetic group 48.13±1.20 at p 0.001 LSD ≥ 2.460. Table 7 and Figure 32 • Combination treated group The study also revealed that the combination oral of metformin 300 mg/kg/day and oral L-cysteine 300 mg/kg/day for 2 weeks was associated with a significant decrease in the mean value of HOMA-IR 17.21±0.31 when compared to the untreated diabetic group 48.13±1.20 at p 0.001 LSD ≥ 2.460. Table 7 and Figure 32 As presented here treatments with either metformin or L-cysteine alone or in combination for 2 weeks resulted in significant decreases in HOMA-IR. The mean values of HOMA-IR in the metformin and Lcysteine groups decreased significantly by about 51.6 and 30.6 from the untreated diabetic values respectively reaching greater significant decreases in the combination treated group 64.2 below the untreated diabetic rats. All decreases carried a statistical significance at p 0.001.

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131

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133 Figure 32: Effect of treatment with the studied drugs for 2 weeks on HOMA- IR in male albino rats : Significant in comparison to normal rat

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134 2- Lipid profile 2.1. Serum triglycerides TGs • Metformin treated group In the metformin treated group given metformin orally in a dose of 300 mg/kg/day for 2 weeks the mean value of the serum TGs was 122.60±1.065 mg/dl which was significantly less than that of the untreated diabetic rats 162.80±1.81 mg/dl at p 0.001 LSD ≥ 5.732. Table 8 and Figure 33a • L-cysteine treated group Treatment of diabetic rats with L-cysteine in a dose of 300 mg/kg/day for 2 weeks orally resulted in a non-significant decrease in serum TGs as compared to the untreated diabetic rats. The mean values of both groups were 157.50±1.06 mg/dl and 162.80±1.81 mg/dl respectively at p 0.05 LSD 5.732. Table 8 and Figure 33a • Combination treated group Treatment of STZ-diabetic rats with both metformin 300 mg/kg/day and L-cysteine 300 mg/kg/day for 2 weeks orally was associated with a significant reduction in the mean value of serum TGs 118.50±1.07 mg/dl when compared to the untreated diabetic rats with mean value of 162.80±1.81 mg/dl at p 0.001 LSD ≥ 5.732. Table 8 and Figure 33a Thus metformin treatment alone caused a statistically significant reduction in serum triglycerides reaching values of 24.7 below STZdiabetic rats p 0.001. This decrease in the combination treated group was quantitatively similar to the metformin group as the mean value of serum triglycerides was 27.2 below diabetic values without significant differences between the two groups. In the L-cysteine group the level of serum triglycerides was not significantly affected as they decreased by only 3.3 from untreated diabetic values.

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135

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136

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137 Figure 33a: Effect of treatment with the studied drugs for 2 weeks on serum triglycerides in male albino rats : Significant in comparison to normal rats

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138 2.2. Serum total cholesterol TC • Metformin treated group Treating STZ-diabetic rats with oral metformin in a dose of 300 mg/kg/day for 2 weeks caused a significant decrease in the mean value of total cholesterol to 99.60±1.12 mg/dl when compared to the untreated diabetic rats 126.10±1.70 mg/dl at p 0.001 LSD ≥ 4.940. Table 9 and Figure 33b • L-cysteine treated group A non-significant decrease in the mean value of total cholesterol resulted by L-cysteine treatment given orally in a dose of 300 mg/kg/day for 2 weeks as compared to the untreated STZ-diabetic rats at p 0.05. The mean value of L-cysteine treated group was 121.20±0.88 mg/dl while that of the STZ-diabetic group was 126.10±1.70 mg/dl LSD 4.940. Table 9 and Figure 33b • Combination treated group Combination treatment of metformin in a dose of 300 mg/kg/day and L- cysteine in a dose of 300 mg/kg/day given orally for 2 weeks to STZdiabetic rats showed a significant decrease in the mean level of total cholesterol from a value of 126.10±1.70 mg/dl in the untreated diabetic group to reach a value of 96.50±1.02 mg/dl after treatment at p 0.001 LSD ≥ 4.940. Table 9 and Figure 33b As resulted in serum triglycerides metformin treated group and combination treated group showed a quantitatively similar decreases in the serum total cholesterol level without statistically significant differences between the two groups. The mean values of total cholesterol in both groups were 21 and 23.5 below the untreated diabetic value respectively. While L-cysteine treated group showed a non-significant decrease in the serum total cholesterol mean value as it only caused 3.9 decrease from the untreated diabetic value.

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141 Figure 33b: Effect of treatment with the studied drugs for 2 weeks on serum total cholesterol in male albino rats : Significant in comparison to normal rats 2.3. Serum high-density lipoprotein cholesterol HDL-C • Metformin treated group Oral metformin treatment 300 mg/kg/day for 2 weeks significantly increased the mean level of serum HDL-C 40.20±0.77 mg/dl as compared to the mean value of the untreated diabetic rats which was 28.0±0.77 mg/dl at p 0.001 LSD ≥ 3.069. Table 10 and Figure 33c • L-cysteine treated group Oral L-cysteine in a dose 300 mg/kg/day for 2 weeks produced a significant increase in the mean value of serum HDL-C in comparison to the untreated diabetic rats with mean values of 34.10±0.80 mg/dl and 28.0±0.77 mg/dl respectively at p 0.001 LSD ≥ 3.069. Table 10 and Figure 33c • Combination treated group Treatment of STZ-rats with oral metformin 300 mg/kg/day in combination with oral L-cysteine 300 mg/kg/day for 2 weeks caused a significant increase in serum HDL-C mean value 45.90±0.64 mg/dl when compared to 28.0±0.77 mg/dl in the diabetic group at p 0.001 LSD ≥ 3.069. Table 10 and Figure 33c Thus the serum HDL-C mean value was greatly increased in the metformin treated group to about 143.6 that of untreated diabetic rats p 0.001. L-cysteine treatment caused less significant increases in serum HDL- C as compared to the metformin treated group reaching an average of 121.8 of diabetic value p 0.001. Moreover the increase in serum HDLC was even

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142 higher in the combination treated group increasing up to 163.9 of STZ- diabetic value.

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143 Figure 33c: Effect of treatment with the studied drugs for 2 weeks on serum high-density lipoprotein cholesterol in male albino rats : Significant in comparison to normal rats

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144 2.4. Serum low-density lipoprotein cholesterol LDL-C • Metformin treated group A dose of 300 mg/kg/day of oral metformin given to the STZ- diabetic rats for 2 weeks resulted in a significant reduction in the LDL-C mean value to reach 34.88±0.85 mg/dl in comparison to the untreated diabetic group mean value which was 65.54±0.87 mg/dl LSD ≥ at p 0.001 2.887. Table 11 and Figure 33d • L-cysteine treated group Treatment with oral L-cysteine 300 mg/kg/day for 2 weeks was associated with a significant decrease in the mean value of LDL-C

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145 55.60±0.90 mg/dl as compared to 65.54±0.87 mg/dl in the untreated diabetic rats at p 0.001 LSD ≥ 2.887. Table 11 and Figure 33d • Combination treated group The treatment of diabetic rats with a combination of metformin 300 mg/kg/day and L-cysteine 300 mg/kg/day orally for 2 weeks decreased significantly the mean level of LDL-C to 26.90±0.43 mg/dl when compared to the untreated diabetic rats 65.54±0.87 mg/dl at p 0.001 LSD ≥ 2.887. Table 11 and Figure 33d Therefore metformin treatment for 2 weeks caused a significant decrease in serum LDL-C averaging 46.8 below the untreated diabetic values p 0.001. In the L-cysteine group less but significant reduction in LDL-C was observed reaching value of about 15.2 below STZ-diabetic value p 0.001. The combined effect of both drugs on serum LDL-C was more profound than either drug alone as its level significantly decreased by about 59 from untreated diabetic rats p 0.001.

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146

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147 Figure 33d: Effect of treatment with the studied drugs for 2 weeks on serum low-density lipoprotein cholesterol in male albino rats

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148 : Significant in comparison to normal rats 2.5. Serum Free fatty acids FFAs • Metformin treated group When metformin administrated orally to the STZ-diabetic rats in a dose 300 mg/kg/day for 2 weeks it caused a significant decrease in the mean value of FFAs 0.66±0.02 mmol/L as compared to 1.32±0.03 mmol/L in the untreated diabetic rats at p 0.001 LSD ≥ 0.087. Table 12 and Figure 33e • L-cysteine treated group The oral administration of L-cysteine in a dose of 300 mg/kg/day for 2 weeks significantly decreased the FFAs mean value to reach 0.89±0.03 mmol/L in comparison to the mean value in untreated diabetic group which was 1.32±0.03 mmol/L at p 0.001 LSD ≥ 0.087. Table 12 and Figure 33e • Combination treated group Concurrent treatment with both metformin 300 mg/kg/ day and Lcysteine 300 mg/kg/day for 2 weeks orally was associated with a significant reduction in the mean value of FFAs when compared to the untreated STZ-diabetic rats at p 0.001. The FFAs mean values in both groups were 0.50±0.01 mmol/L and 1.32±0.03 mmol/L respectively LSD ≥ 0.087. Table 12 and Figure 33e As shown treatment with either metformin or L-cysteine alone for 2 weeks caused significant decreases in the mean value of FFAs to reach 50 and 32.6 below the values of the untreated diabetic rats respectively p 0.001. Meanwhile the combination between both drugs resulted in a more evident reduction in FFAs level reaching about 62.1 below STZ-diabetic value p 0.001.

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151 group diabetic group treated group treated group treated group Figure 33e: Effect of treatment with the studied drugs for 2 weeks on serum free fatty acids in male albino rats : Significant in comparison to normal rats 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Normal control Untreated Metformin L-Cysteine Combination

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152 2.6. Non-HDL-cholesterol • Metformin treated group Metformin treatment given in a dose of 300 mg/kg/day for 2 weeks orally resulted in a significant reduction in the non-HDL-C mean values 59.40±0.95 mg/dl when compared to 98.1±0.92 mg/dl in the untreated STZ-diabetic rats at p 0.001 LSD ≥ 3.243. Table 13 and Figure 33f • L-cysteine treated group Treatment of STZ-diabetic rats with L-cysteine in a dose of 300 mg/kg/day for 2 weeks orally caused a significant decrease in the mean value of non-HDL-C when compared to the untreated diabetic rats at p 0.001. The mean values of the both groups were 87.10±0.48 mg/ dl and 98.10±0.92 mg/ dl respectively LSD ≥ 3.243. Table 13 and Figure 33f • Combination treated group The current study revealed that the combination between metformin and L-cysteine both given orally in a dose of 300 mg/kg/day for 2 weeks was associated with a significant reduction in the mean value of the calculated non-HDL-C reaching a value of 50.60±0.97 mg/dl as compared to the untreated diabetic mean value which was 98.10±0.92 mg/dl at p 0.001 LSD ≥ 3.243. Table 13 and Figure 33f Therefore metformin treatment alone resulted in a more significant decrease in the mean value of non-HDL-C as compared to the L-cysteine group p 0.001. Non-HDL-C mean values in both groups were 39.4 and

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153 11.2 below the untreated diabetic value respectively. Such decreases were more significant in the combination treated group reaching average value of 48.4 below the untreated diabetic value.

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155 group diabetic group treated group treated group treated group Figure 33f: Effect of treatment with the studied drugs for 2 weeks on non- HDL-cholesterol in male albino rats : Significant in comparison to normal rats 0 20 40 60 80 100 120 Normal control Untreated Metformin L-Cysteine Combination

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156 2.7. Triglycerides to HDL- cholesterol ratio TGs/HDL • Metformin treated group There was a significant decrease in TGs/HDL ratio in rats treated with metformin in a dose of 300 mg/kg/day for 2 weeks orally to reach a mean value of 3.06±0.13 in comparison with the untreated diabetic rats with a mean value of 5.85±0.06 at LSD ≥ p 0.001 0.342. Table 14 and Figure 33g • L- cysteine treated group It was also observed that treatment of diabetic rats with L- cysteine in the previously mentioned dose was associated with a significant decrease in TGs/HDL ratio when compared to the STZ-diabetic rats at p 0.001. The mean values of both groups were 4.65±0.03 and 5.85±0.06 respectively

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157 LSD ≥ 0.342. Table 14 and Figure 33g • Combination treated group: The results revealed that the combination treatment between metformin in a dose of 300 mg/kg/day and L-cysteine in a dose of 300 mg/kg/day for 2 weeks orally reduced significantly the mean value of TGs/HDL ratio to 2.58±0.04 as compared to 5.85±0.06 in the untreated diabetic rats at p 0.001 LSD ≥ 0.342. Table 14 and Figure 33g Thus it was concluded that metformin treatment caused a more significant reduction in the level of TGs/HDL ratio in comparison to the Lcysteine treated group reaching values about 47.7 and 20.5 below the untreated diabetic value respectively at p 0.001. Such decrease in the level of TGs/HDL ratio was more significant in the combination treated group reaching average value of 55.9 below the STZ-diabetic value. .

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160 Figure 33g: Effect of treatment with the studied drugs for 2 weeks on triglycerides to HDL-cholesterol ratio in male albino rats : Significant in comparison to normal rats 3. Hepatic oxidative stress parameters To Cure Diabetes Permanently Click Here

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161 4. 3.1. Hepatic malondialdehyde MDA • Metformin treated group The hepatic MDA in STZ-diabetic rats treated with metformin in a dose of 300 mg/kg/day orally for 2 weeks was significantly lower than that of the untreated diabetic rats at p 0.001. The mean values of both groups were 18.69±0.37 nmol/gm wet tissue and 24.76±0.57 nmol/gm wet tissue respectively LSD ≥ 1.403. Table 15 and Figure 34 • L-cysteine treated group Treating STZ-diabetic rats with oral L-cysteine in a dose of 300 mg/kg/day for 2 weeks significantly reduced the increased hepatic MDA levels seen in the untreated diabetic rats at p 0.001. The hepatic MDA mean value was decreased from 24.76±0.57 nmol/gm wet tissue in the untreated diabetic group to 15.44±0.20 nmol/gm wet tissue after Lcysteine treatment LSD ≥ 1.403. Table 15 and Figure 34 • Combination treated group According to the data presented in Table 15 and Figure 34 oral metformin 300 mg/kg/day and oral L-cysteine 300 mg/kg/day given together for 2 weeks significantly decreased the hepatic MDA levels to 12.78±0.33 nmol/gm wet tissue as compared to the MDA mean value 24.76±0.57 nmol/gm wet tissue in the untreated diabetic group at p 0.001 LSD ≥ 1.403. Thus it was observed that the concentration of hepatic MDA in the metformin treated group decreased significantly to approximately 76 of untreated diabetic values. The reduction in MDA level was more evident in the L-cysteine group reaching an average of 62.4 of diabetic values. However treatment of diabetic rats with metformin and L-cysteine in combination decreased MDA levels more significantly than either drug alone to reach an average MDA value about 51 that of the untreated diabetic rats. All decreases carried a statistical significance at p 0.001.

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165 Figure 34: Effect of treatment with the studied drugs for 2 weeks on hepatic malondialdehyde in male albino rats : Significant in comparison to normal rats 3.2. Hepatic reduced glutathione GSH • Metformin treated group Treatment with metformin in a dose of 300 mg/kg/day for 2 weeks orally increased significantly the hepatic GSH mean level to reach a value of 171.35±1.56 μg/mg protein versus 135.31±1.64 μg/mg protein in the

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166 untreated diabetic rats at p 0.001 LSD ≥ 5.718. Table 16 and Figure 35 • L-cysteine treated group There was a significant increase in the hepatic GSH in the group treated with L-cysteine in a dose of 300 mg/kg/day for 2 weeks orally in comparison to the untreated diabetic rats at p 0.001. The mean values of hepatic GSH for the two groups were 199.24±1.09 μg/mg protein and 135.31±1.64 μg/mg protein respectively LSD ≥ 5.718. Table 16 and Figure 35 • Combination treated group The study revealed that the combination treatment of metformin 300 mg/kg/day and L-cysteine 300 mg/kg/day orally for 2 weeks was associated with a significant increase in the hepatic GSH levels to reach a mean value 228.01±0.82 μg/mg protein in comparison to the untreated diabetic group in which the mean value of hepatic GSH was 135.31±1.64 μg/mg protein at p 0.001 LSD ≥ 5.718. Table 16 and Figure 35 Thus treatment with metformin significantly increased the GSH values by 26.6 above the untreated diabetic values. The GSH levels in the L- cysteine treated group reached significantly higher values about 47.2 above that of untreated diabetic group. Moreover in the combination treatment group the GSH content was more greatly increased as compared to the metformin and the L-cysteine treated groups reaching a concentration about 68.5 that of untreated diabetic rats.

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169 Figure 35: Effect of treatment with the studied drugs for 2 weeks on hepatic reduced glutathione in male albino rats : Significant in comparison to normal rats 4. Inflammatory parameters

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170 4.1. Serum monocyte chemoattractant protein-1 MCP-1 • Metformin treated group As could be seen in Table 17 and Figure 36 treatment of diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally caused a significant decrease in serum level of MCP-1 498.33±5.97 pg/ml as compared to the mean value of untreated STZ-diabetic rats 670.42±9.01 pg/ ml at p 0.001 LSD ≥ 20.552. • L-cysteine treated group There was a significant decrease in serum MCP-1 in diabetic rats treated with L-cysteine in the aforementioned dose in comparison to the untreated diabetic rats at p 0.001. The mean values for both groups were 369.70±4.87 pg/ml and 670.42±9.01 pg/ml respectively LSD ≥ 20.552. Table 17 and Figure 36 • Combination treated group It was also observed that the diabetic rats received metformin 300 mg/kg/day and L-cysteine 300 mg/kg/day orally in combination for 2 weeks showed a significant decrease in MCP-1 level in comparison to the untreated diabetic rats with mean values of 249.97±3.44 pg/ml and 670.42±9.01 pg/ml respectively at p 0.001 LSD ≥ 20.525. Table 17 and Figure 36 Therefore treatment with metformin for 2 weeks significantly decreased the MCP-1 values to an average of 25.7 below diabetic values. Oral L-cysteine treatment gave significantly lower levels of MCP-1 as compared to the metformin treated group p 0.001 reaching values lower than control diabetic values by about 44.9. Combining both drugs together caused further significant reduction in the mean MCP-1 values to about 62.7 from STZ-diabetic levels.

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172 Figure 36: Effect of treatment with the studied drugs for 2 weeks on serum monocyte chemoattractant protein-1 in male albino rats : Significant in comparison to normal rats 4.2. Serum C-reactive protein CRP • Metformin treated group Treatment of STZ-diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally caused a significant reduction in the mean value

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173 of CRP to reach a value of 2.27±0.07 mg/L when compared to the untreated diabetic rats with mean value 3.25±0.08 mg/L at p 0.001 LSD ≥ 0.229. Table 18 and Figure 37 • L-cysteine treated group A significant decrease in serum CRP was observed in the diabetic rats treated with L- cysteine in a dose of 300 mg/kg/day given orally for 2 weeks in comparison to the untreated diabetic rats at p 0.001. The mean values of serum CRP were 1.54±0.06 mg/L and 3.25±0.08 mg/L LSD ≥ respectively 0.229. Table 18 and Figure 37 • Combination treated group

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174 Treating STZ- diabetic rats with both oral metformin in a dose of 300 mg/kg/day and oral L-cysteine in a dose of 300 mg/kg/day for 2 weeks resulted in a significant decrease in serum CRP as compared to the untreated diabetic rats at p 0.001. The mean value of serum CRP was decreased from 3.25±0.08 mg/L in the untreated STZ-diabetic group to 0.88±0.06 mg/L after treatment with both drugs in combination LSD ≥ 0.229 Table 18 and Figure 37. Similarly the effect of L-cysteine was more substantial than that of metformin on the serum CRP level. Both metformin and L-cysteine caused significant decreases in CRP levels reaching values about 30.2 and 52.6 lower than the untreated diabetic values. In the combination treatment group the effect on CRP was more profound as the level went down by approximately 73 from the untreated diabetic values. All decreases carried a statistical significance of p 0.001.

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177 Figure 37: Effect of treatment with the studied drugs for 2 weeks on serum C- reactive protein in male albino rats : Significant in comparison to normal rats

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178 4.3. Serum nitric oxide NO • Metformin treated group STZ-diabetic rats treated with metformin in a dose of 300 mg/kg/day for 2 weeks orally showed a significant decrease in the serum NO level to reach a mean value of 37.04±0.94 nmol/ml in comparison to the mean value of the untreated diabetic rats which was 65.95±2.07 nmol/ml at p 0.001 LSD ≥ 4.032. Table 19 and Figure 38 • L-cysteine treated group There was a significant reduction in serum NO from its mean value in the untreated STZ-diabetic group 65.95±2.07 nmol/ml to reach a value of 46.51±0.54 nmol/ml in the group treated with L-cysteine in a dose of 300 mg/kg/day for 2 week orally at p 0.001 LSD ≥ 4.032. Table 19 and Figure 38 • Combination treated group A significant decrease was resulted in the mean value of serum NO in STZ-diabetic rats treated with combination between both metformin and Lcysteine in the aforementioned dose for 2 weeks orally as compared to the untreated diabetic rats at p 0.001. The mean values for the two groups were 27.01±0.51 nmol/ml and 65.95±2.07 nmol/ml respectively LSD ≥ 4.032. Table 19 and Figure 38

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179 In contrast to L-cysteine’s stronger effect on MCP-1 and CRP it caused a smaller reduction in NO levels as compared to the metformin treated group. Nitric oxide values averaged 70.5 and 56.2 of the untreated diabetic values in the L-cysteine and metformin treated groups respectively p 0.001. Greater reductions in nitric oxide levels were observed in the combination treatment group reaching values about 59 below the STZ-diabetic levels p 0.001.

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182 Figure 38: Effect of treatment with the studied drugs for 2 weeks on serum nitric oxide in male albino rats

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183 : Significant in comparison to normal rats 5. Histopathological examination of pancreas To Cure Diabetes Permanently Click Here Pancreatic islet cells from control rats exhibited normal architecture namely normal size and shape with eosinophilic cytoplasm and centrally placed nuclei Fig. 39A. Degenerated islets with irregular contour cytoplasmic vacuolization and apoptotic cells were observed in pancreatic tissue from untreated diabetic rats Fig. 39B. Treatment of diabetic rats with either L-cysteine Fig. 39C or metformin Fig. 39D resulted in moderate regeneration of pancreatic islets. Well formed regenerating islets were observed in rats treated with both drugs in combination Fig. 39E indicating more protection against pancreatic tissue damage.

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184 Figure 39: Histopathological evaluation of pancreatic sections stained with hematoxylin and eosin HE stain X 10. A Section of normal pancreas showing pancreatic islets of normal size and shape with centrally placed nuclei. B Pancreatic section of diabetic rats showing degenerated islet of irregular shape with cytoplasmic vacuolization and apoptotic cells indicated by arrow. C D Pancreatic sections of L-cysteine-treated and metformin-treated rats respectively showing moderately regenerating islets. E Section of the pancreas of the combined therapy group showing well formed regenerated islet.

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188 DISCUSSION To Cure Diabetes Permanently Click Here Diabetes mellitus is a multifactorial metabolic syndrome affecting at least 150 million people worldwide and causing many serious socio- economic problems. It is characterized by absolute or relative deficiency of insulin and pancreatic polypeptide secretion and imperfection of insulin receptor or post- receptor events with derangement in carbohydrate protein and lipid metabolism. Insulin resistance is the core pathophysiological feature in type 2 diabetes mellitus leading to increased hepatic glucose production and decreased glucose uptake and disposal. This is followed by decreased insulin secretion as a result of progressive pancreatic -cell dysfunction. These dual defects eventually result in chronic hyperglycemia a clinical hallmark of diabetes mellitus 257-259 . Indeed chronic hyperglycemia is contemplated to produce a notable decline in the level of intracellular antioxidants and to generate proxidants through diverse pathways. These pathways include mutilation of the redox equilibrium augmentation of advanced glycation products glycosylation of antioxidative enzymes activation of protein kinase C or overproduction of mitochondrial superoxides. These finally lead to oxidative stress in various tissues 257 particularly in pancreatic -cells which are highly susceptible to damage by ROS due to low antioxidant enzymes expression 260 . In type 2 diabetes ROS activate -cell apoptotic pathways impair insulin synthesis and also contribute to insulin resistance and diabetic complications 261 . Nowadays increasing evidence from human population studies and animal research has established correlative as well as causative links between chronic inflammation and insulin resistance which leads finally to type 2 diabetes mellitus 262 . There is now a wealth of evidence indicating close ties between metabolic and immune systems from an emerging paradigm that metabolic imbalance with starvation and immunosuppression on one end of the spectrum and obesity and inflammatory diseases on the other end 263 .

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189 Obesity particularly visceral obesity and its associated hyperlipidemia are hallmarks and major risk factors of type 2 diabetes mellitus. Adipose tissue plays an important role in the development of inflammation insulin resistance and type 2 diabetes mellitus. Resistance of dysfunctional fat cells to the antilipolytic effects of insulin leads to chronic elevation in plasma FFAs levels. These cells in turn produce excessive amounts of cytokines such as TNF- IL-6 and resistin that further increase insulin resistance inflammation and atherosclerosis as well as these cells secrete reduced amounts of insulin sensitizing cytokines such as adiponectin 264 . Moreover elevated circulating levels of FFAs which are derived from adipocytes contribute to insulin resistance by inhibiting glucose uptake glycogen synthesis and glycolysis as well as by increasing hepatic glucose production 265 . FFAs were reported to stimulate expression of gluconeogenic enzymes including glucose-6-phosphatase 266 . Although the antidiabetic drugs may be effective for glycemic control in type 2 diabetes mellitus at least in the early stages they do not appear to be effective in entirely preventing the progression of ROS-mediated organ damage 261 . The pathophysiological changes in type 2 diabetes mellitus sets the scene for considering antioxidant and anti-inflammatory therapy together with reduction in visceral adiposity and tissue lipid content as an adjunct to the commonly used oral antidiabetics in the management of this disease 1 . There are several available antioxidants that hold promise as new approaches for the treatment of insulin resistance and type 2 diabetes mellitus including N-acetyl cysteine and -lipoic acid 149 . Therefore the aim of the present study was to investigate the metabolic derangements dyslipidemia oxidative stress and inflammation associated with experimentally-induced type 2 diabetes mellitus. Moreover it was conducted to evaluate the metabolic and lipid profile improvements as well as the antioxidant and anti-inflammatory effects of the amino acid L-cysteine in the management of STZ-induced rat model of type 2 DM. The study was also designed to assess any possible additive effect of Lcysteine with the well-established gold star antidiabetic drug metformin on the aforementioned parameters in the STZ-induced type 2 diabetes in rats.

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190 For induction of a rat model of type 2 diabetes rats were initially fed with high fat diet which was used to produce insulin resistance an early feature of type 2 diabetes. This is followed by injecting rats with a low dose of streptozotocin which has been known to induce a mild impairment of insulin secretion. Therefore this rat model closely mimics the natural history of the disease events which progresses from insulin resistance to cells dysfunction as well as resembles metabolic characteristics of type 2 diabetes in human 267 . The results of the present study revealed that injecting rats with a low dose of streptozotocin after their maintenance on high fat diet for 2 months was associated with hyperglycemia hyperinsulinemia and insulin resistance which was presented by calculating HOMA-IR a mathematical model that relates fasting glucose and insulin levels to insulin resistance. These findings were in accordance with the results previously reported by Zhang et al in 2003 237 and Abdin et al in 2010 39 . High fat diet HFD has been shown to induce insulin resistance by different mechanisms but considered mainly through Randle or glucosefatty acid cycle. Briefly the presence of high level of triglycerides due to excess fat intake could constitute a source of increased fatty acid availability and oxidation. The preferential use of increased fatty acids for oxidation blunts the insulin-mediated reduction of hepatic glucose output and reduces the glucose uptake and utilization in skeletal muscle leading to compensatory hyperinsulinemia a common feature of insulin resistance 268 . This explains the hyperinsulinemia observed in our animal model. Another potential mechanism of high fat diet-induced insulin resistance could involve the activation of PKC and inhibitor of NF-B kinase- IKK by the elevated FFAs level. These serine kinases are mediators of inflammation resulting in the inhibition of insulin-stimulated glucose transport 149 . FFAs also affect insulin-signaling pathway where their elevated levels impair IRS- 1 phosphorylation and PI3K activation following insulin stimulation 269 .

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191 Conversion of this insulin resistant state to overt type 2 diabetes with frank hyperglycemia was achieved by the low dose streptozotocin. STZ induces -cell death through DNA alkylation and increasing the -cells oxidative stress thus establishing a state of relative insulin deficiency and hyperglycemia 267 . Elevation of blood glucose may be attributed to reduced entry of glucose to peripheral tissues muscle and adipose tissue increased glycogen breakdown and increased gluconeogenesis 270 . Moreover the results of the present study revealed that STZinduced diabetic rats showed significant increases in serum triglycerides total cholesterol LDL-C free fatty acids calculated nonHDL-cholesterol and triglycerides to HDL-C ratio in addition to a significant decrease in serum HDL-C level. In agreement to our results a previous study reported that the levels of triglycerides and total cholesterol were further accentuated after STZ injection to high fat diet-fed rats 268 . Sahin et al in 2007 271 also showed that high fat diet and STZ induced type 2 diabetic rats were hypertriglyceridemic and hypercholesterolemic. Another recent study reported that type 2 diabetes in rats induced by HFD and low dose STZ was associated with significant increases in serum levels of triglycerides total cholesterol LDL-C and FFAs as well as a significant decrease in HDL-C 272 . In addition many studies in the literature showed the characteristic atherogenic dyslipidemia associated with diabetes in both diabetic animals and humans which was seen as increased levels of triglycerides total cholesterol LDL-C non-HDL-cholesterol and free fatty acids as well as reduced HDL-C 273-275 . Hypertriglyceridemia may be caused by the increased hepatic TGs production or decreased TGs removal or both. In insulin resistant states lipolysis is stimulated in adipose tissues increasing the delivery of FFAs from adipose tissues to liver and consequently increases the hepatic lipid content and circulating TGs-rich VLDL production. Moreover hyperinsulinemia enhances the expression of hepatic sterol regulatory element binding protein SREBP a key transcription factor. SREBP can activate a cascade of enzymes involved in fatty acid and cholesterol

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192 biosynthesis such as fatty acid synthase FAS and HMG-CoA reductase 276 . Since liver TGs production is mainly determined by the free fatty acid synthesis rate which is controlled at the level of transcription by SREBP thus induction of SREBP expression will finally result in a rise in triglycerides 277 . Hypertriglyceridemia may be also due to a defect in lipoprotein lipase which hydrolyzes circulating TGs causing impaired plasma triglyceride removal. In addition it was reported that STZ-diabetic rats exhibited VLDL receptor deficiency in skeletal muscle heart and adipose tissue thus decreasing VLDL clearance 278 . Therefore the hypertriglyceridemia observed in our rat model of type 2 diabetes may be due to increased absorption and formation of triglycerides in the form of chylomicrons following exogenous consumption of diet rich in fat or through increased endogenous production of TGs-enriched hepatic very low-density lipoprotein VLDL and decreased TGs uptake in peripheral tissue 268 . The increase in total cholesterol and LDL-C demonstrated in our results was in accordance to a previous study which showed that serum total cholesterol and LDL-C levels were elevated in a model of STZinduced type 2 diabetes in rats 279 . It was also showed in another recent study that hyperglycemia was accompanied with a marked increase in total cholesterol LDL-C and triglycerides as well as a reduction in HDL-C in HFD/STZ diabetic rats 270 . Serum cholesterol levels are mainly incorporated in LDL fraction indicating harmful effects. This frank hypercholesterolemia found in our rat model of type 2 diabetes mellitus may be attributed to increased dietary cholesterol absorption from the small intestine following the intake of high fat diet 268 or may be due to increased rate of cholesterol synthesis which is stimulated 2-3 fold in the gut of STZ-induced diabetic rats 280 . Furthermore increases in VLDL secretion can lead to chain reactions in other lipoprotein and lipids leading to the increased levels of LDL-C and total cholesterol 276 . It was also reported that the status of hyperglycemia can accelerate LDL modifications with subsequent AGEs formation 281 . Moreover insulin resistant and diabetic states are associated with a reduction in HDL-C which is one of the main defenses against

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193 atherosclerosis. Beside its role in reverse cholesterol transport HDL has an array of antioxidant mechanisms which may prevent the formation and promote removal of lipid peroxides from cell membranes and from other lipoproteins such as the proatherogenic LDL 282 . Our study revealed a decrease in serum HDL-C. This was in accordance to a previous study which showed dyslipidemia with a significant decrease in HDL-C level in STZ-induced diabetic rats 283 . This decrease in circulating HDL seen in insulin resistant states is intimately linked to the overproduction of TGs-rich lipoproteins such as VLDL and chylomicrons. Although the mechanisms are not entirely clear available data implicate TGs-enrichment of HDL particles leading to particle instability and degradation. The interaction between HDL that is TGs enriched and hepatic lipase action has been suggested to play an important role in the enhanced catabolism of HDL in insulin resistant hypertriglyceridemic states 276 . Our results also showed an elevated fasting serum FFAs. This was in accordance to previous studies which showed a significant elevation in the concentration of FFAs in high fat diet/STZ diabetic rats 39 271 . The net plasma concentration of FFAs results from a delicate equilibrium between enzyme-regulated lipolysis of plasma triglycerides-rich lipoproteins such as VLDL lipolysis of TGs stored in adipose tissue and FFAs uptake by peripheral tissues. Under normal conditions insulin stimulates postprandial uptake of glucose as well as FFAs esterification and storage. Insulin and glucose are also believed to regulate lipoprotein lipase LPL activity which provides an essential first step in the delivery of FFAs to adipose tissue for storage as well as plasma TGs removal. In the insulin resistant state there is an increase in release of FFAs from adipose tissue. This may result in an increased influx of FFAs into the liver and the muscle. This “vicious cycle” may result in attenuation in insulin signaling in these tissues and may exacerbate insulin resistance 276 . Thus increased free fatty acids flux from adipose tissue to non-adipose tissues resulting from abnormalities in fat metabolism either in storage or lipolysis is both a consequence of insulin resistance and aggravating factor participating in and amplifying many of the fundamental metabolic derangements that are characteristic to insulin resistance and type 2 diabetes 284 .

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194 In our research a significant elevation in the levels of the calculated atherogenic indices non-HDL-cholesterol and triglycerides to HDL-C ratio was observed. These two indices are particularly useful in predicting cardiovascular disease risk in patients with diabetes 285 286 . High TGs/HDL- C ratio was also reported to correlate with the small dense LDL and insulin resistance 286 . Furthermore the results of the present study revealed a significant increase in hepatic malondialdehyde concentration and a significant decrease in hepatic reduced glutathione content in the high fat diet/STZ induced type 2 diabetes in rats. These results indicate the presence of oxidative stress with subsequent cellular damage in the liver of STZ-induced rat model of type 2 diabetes mellitus. Oxidative stress with excessive generation of free radicals and depleted levels of free radical scavenging enzymes have been demonstrated in both experimental animal models of diabetes and human diabetic subjects. Oxygen radicals are the major causative agents for distant organ damage as increased production of OH • O 2 •- and H 2 O 2 have detrimental effects on various tissues. Our findings were in accordance to previous studies which showed that high fat diet/STZ type 2 diabetic rats had a significant elevation in hepatic MDA concentrations 271 287 . An earlier study also reported a significant increase in the MDA level which was due to decreased activity of most of the antioxidant enzymes 288 . This increase in MDA was correlated with hyperglycemia in diabetic patients 289 . Furthermore previous studies showed a significant reduction in the level of reduced glutathione in liver and serum of HFD/STZ diabetic rats 287 290 . In addition many studies showed a marked decreased level of reduced glutathione in the plasma of diabetic patients 79 291 292. Increased oxidant production and subsequent antioxidants consumption in diabetic and insulin-resistant states can originate from the metabolism of both glucose and FFAs 293 . Abundant evidences demonstrate that chronic exposure to high circulating glucose or FFAs increases ROS production that in turn causes lipid peroxidation and membrane damage as well as decreases insulin content and glucosestimulated insulin secretion of -cells 68 . Lipid

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195 peroxidation provides useful information for prognosis of diabetes. It is frequently used as an index of tissue oxidative stress in which oxygen interacts with polyunsaturated fatty acids and leads to the formation of toxic products such as MDA that plays an important role in the pathogenesis and progression of diabetes and its complications 290 . Increased ROS production results from hyperglycemia-induced increase in the proton gradient across the inner mitochondrial membrane. When the gradient exceeds a threshold complex III electron transfer is blocked leading to leakage of electrons with formation of superoxide 294 . This increased superoxide production is the central and major mediator of diabetes tissue damage. 295 . It is hypothesized that excess ROS inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. This inhibition in turn mediates the activation of the proposed mechanisms of hyperglycemia-associated tissue damage 294 and leads to direct inactivation of eNOS as well as results in decreases in insulin promoter activity insulin gene transcription and expression insulin secretion induction of -cells apoptosis and impairment in energy production contributing to the development and progression of diabetes mellitus 57 . Hyperglycemia also results in increased flux through the polyol pathway causing NADPH depletion impaired glutathione reductase activity and a decrease in the GSH:GSSG ratio. In addition increased AGEs formation and their binding to RAGE receptors result in generating more ROS increasing GPx activity and depleting of glutathione which may further enhance lipid peroxidation 296 . Moreover elevated FFAs have numerous adverse effects on mitochondrial function including the uncoupling of oxidative phosphorylation and generation of more ROS including superoxide thus impairing endogenous antioxidant defenses by reducing intracellular glutathione 297 . This depletion of GSH leads to hamper the stabilizing effects of GSH on NO. Thus in diabetes excessive formation of superoxide anion O 2 •– resulted which in turn by a direct reaction with nitric oxide NO results in the formation of peroxynitrite ONOO a potent oxidant. ONOO was found to be strongly correlated with oxidative stress and apoptosis. However under physiological conditions O 2 •– is predominantly removed by SOD and the formation of ONOO will be minimal 298 .

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196 The results of the present study revealed that high fat diet/STZ diabetic rats showed a significant increase in the serum levels of the inflammatory markers monocyte chemoattractant protein-1 MCP-1 C-reactive protein CRP and nitric oxide NO. Our results were in accordance to a previous study which showed significant increased levels of TNF- IL-6 and CRP which were all in significant positive correlation with insulin resistance HOMA-IR in the diabetic experimental rat model 39 . It was also reported that the inflammatory marker CRP a non-specific acute phase reactant is commonly elevated in human insulin resistant states 299 . Moreover it was reported that animal models of obesity have shown significant increases in circulating proinflammatory mediators including CRP MCP-1 and IL-6 300 . In addition a recent study showed a significant increase in the level of MCP-1 in an animal model of type 2 diabetes Goto-Kakizaki rats fed with high fat diet 301 . A previous study showed also a significant increase in the serum nitric oxide level in STZ diabetic rats fed with fat containing diet 302 . However another study reported a decrease in the serum level of NO in the HFD/STZ type 2 diabetic rats 303 . The reasons for these discrepancies between our results and them are not clear but may be related to the different animal models different STZ dose used severity and time course of hyperglycemia. Inflammation is strongly suggested as a primary cause of obesitylinked insulin resistance hyperglycemia hyperlipidemia and atherosclerosis rather than merely a consequence 263 . Nowadays the circulating inflammatory markers and the acute phase reactants are considered strong predictors for the development of type 2 diabetes and its possible associated cardiovascular complications 304 . The inhibition of signaling downstream of the insulin receptor is a primary mechanism through which inflammatory signaling leads to insulin resistance. This was assured by exposure of cells to TNF- which stimulates inhibitory phosphorylation of serine residues of IRS-1. This phosphorylation reduces both tyrosine phosphorylation of IRS-1 and its ability to associate with the insulin receptor and thereby inhibits downstream signaling and insulin action decreasing insulin sensitivity 70 263 . This might in turn interfere with the anti-inflammatory effect of insulin promoting further inflammation. It was reported that a low dose infusion of

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197 insulin suppresses intranuclear NF- κB binding and reduces plasma intercellular adhesion molecule-1 ICAM-1 plasma tissue factor PAI-1 and monocyte chemotactic protein-1 MCP-1 concentrations. An interruption of insulin signal transduction would prevent the antiinflammatory effect of insulin from being exerted 305 . Moreover it was found that the expression of multiple genes in vascular cells including redox-sensitive transcription factors such as NFB could be activated by oxidative stress. Overexpression of these genes stimulates the secretion of many proinflammatory cytokines such as TNF IL-6 and MCP-1. The increasing level of these markers is known to decrease insulin sensitivity and increase vascular inflammation. Thus oxidative stress plays a key role in the regulatory pathway that progresses from elevated glucose to monocyte and endothelial cell activation in the enhanced vascular inflammation of diabetes 75 . It was reported that in hyperglycemic conditions the increased glucose uptake by endothelial cells causes excess production of ROS in mitochondria which inflicts oxidative damage and activates inflammatory signaling cascades resulting in endothelial injury that in turn might attract inflammatory cells such as macrophages and further exacerbate the local inflammation 70 . This can be explained by the findings of a previously published study showed that transient exposure of cultured human aortic endothelial to hyperglycemia induces persistent epigenetic changes in the promoter of the NF-B leading to a sustained increase in the expression of the NF-B responsive proatherogenic genes MCP-1 and VCAM-1 as well as the proinflammatory genes IL-6 and iNOS 306 . The plasma level of C-reactive protein was found to be increased in both T1DM and T2DM. It plays a significant role in inflammation and atherogenesis. CRP causes numerous proinflammatory and proatherogenic effects in endothelial cells such as decreased endothelial NO and prostacyclin and increased cell adhesion molecules MCP-1 IL-8 and PAI1 307 . In monocytes/macrophages CRP increases ROS and proinflammatory cytokine release promotes monocyte chemotaxis and adhesion and increases oxidized LDL uptake. Moreover in vascular smooth muscle cells CRP has been shown to increase inducible NO production and increase NF- B activity resulting in increased oxidative stress and vascular smooth muscle cell proliferation 308 .

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198 Monocyte chemoattractant protein-1 MCP-1 also known as chemokine ligand 2 Ccl2 and its cognate receptor chemokine receptor 2 Ccr2 are also major components of insulin resistance 299 . MCP-1 plays important roles in the vascular inflammatory process through its multiple actions which include recruitment of neutrophils and T lymphocytes into the subendothelial space monocyte adhesion to endothelium and migration of vascular smooth muscle cells 73 . Many studies on the MCP-1have shown that its expression is increased in obese mice suggesting that changes in its level promote the recruitment of macrophages to adipose tissue and cause inhibition of tyrosine phosphorylation in liver and skeletal muscle resulting in inflammation and insulin resistance 73 74 299 . A very important mediator synthesized by endothelial cells is nitric oxide NO because of its vasodilatory antiplatelet antiproliferative antiinflammatory and antioxidant properties. NO inhibits adhesion of leucocytes as well as cytokine-induced expression of vascular cell adhesion molecule-1 VCAM-1 and monocyte chemotactic protein-1 MCP-1 probably through the inhibition of the transcription factor nuclear factorB 307 . NO is rapidly inactivated by O 2 • ¯ a major potential pathway of NO reactivity. Oxidative breakdown of NO gives rise to radicals such as peroxynitrite and peroxynitrous acid not only decreasing NO levels but increasing oxidative stress 115 . The endothelium of diabetic subjects is a great producer of superoxide instead of NO 309 resulting in a decreased NO/ROS ratio. This undesirable effect may be due to endothelial nitric oxide synthase eNOS uncoupling which results from many factors. These include the oxidative stress as eNOS contains a free cysteine SH group at its catalytic site making it susceptible to inactivation by ROS and aldehydes 115 as well as the increased production of nitroarginine a competitive inhibitor for Larginine the substrate for generating NO 309 . Several inducers of insulin resistance including FFAs proinflammatory cytokines and oxidative stress activate the expression of NOS-2 the gene that encodes iNOS a mediator of non-specific tissue damage leading to excessive nitric oxide production 299 310 . It was reported that iNOS induction is associated with impaired insulin action in skeletal

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199 muscle indicating that iNOS may be involved in the pathogenesis of chronic metabolic disorders such as atherosclerosis insulin resistance and obesity- linked type 2 diabetes in which an inflammatory condition is believed to play a pathologic role 310 . In the insulin signaling pathway NO can reduce Akt activity by causing snitrosylation of a specific cysteine residue. Increased iNOS activity also results in the degradation of IRS-1 in cultured skeletal muscle cells 299 . Moreover overexpression of iNOS mRNA in the pancreatic islets of Zucker diabetic rats suggested that high NO production in this tissue might cause impaired insulin secretion 310 . On the other hand eNOS expression decreases in the insulin resistance syndrome triggering endothelial dysfunction 310 as it was showed that its level is partially regulated by insulin 115 . Nitric oxide and NO donors have been demonstrated to have an autoinhibition capacity of NOS via specific binding sites on the enzyme however a relatively marked lower concentration of NO is required for inhibition of constitutive NOS eNOS and nNOS than for iNOS. Therefore it is possible that in metabolic syndrome insulin resistance and type 2 diabetes mellitus the associated state of proatherogenesis and low-grade inflammation increases cytokines such as TNF- and IL-1 levels causing iNOS induction producing a substantial concentrations of NO that could autoinhibit the cNOS in-vivo resulting in a condition in which iNOS is overexpressed while cNOS is concurrently inhibited 310 . In our study serum NO levels were higher in diabetic rats supporting the hypothesis that NO overproduction in acute hyperglycemia may be associated with eNOS inhibition and iNOS overexpression . As mentioned type 2 diabetes is a complex progressive disorder that is difficult to treat effectively in the long-term. The majority of patients is obese at diagnosis and will be unable to achieve or sustain near normoglycemia without oral antidiabetic agent 183 . Metformin is the gold star in treatment of diabetes whose history dates to medieval times. Metformin is currently the drug of first choice for the treatment of type 2 diabetes being prescribed to at least 120 million people worldwide. Furthermore metformin has been used against the development of type 2 DM in high-risk persons with a 31 reduction in incidence 311

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200 In our present study treatment of STZ-diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally resulted in a significant decrease in the levels of fasting serum glucose insulin and HOMA-IR as compared to the untreated diabetic rats. These findings are in accordance to metformin’s well known antihyperglycemic effects already described in both animals and human studies 312 313 . However we also showed in our research that the metformin treated group exhibited better metabolic control than L-cysteine group. This was also assured by HOMA-IR which indicated that reduction in insulin resistance by metformin is significantly better than L-cysteine. This is probably secondary to the increased insulin sensitivity and concomitant decrease in hyperglycemia. Metformin exerts an antihyperglycemic effect primarily by suppressing basal hepatic glucose production mainly through inhibiting gluconeogenesis and by increasing glucose disposal in skeletal muscle 167 . This preferential action of metformin in hepatocytes is due to the predominant expression of organic cation transporter-1 OCT1 which has been shown to facilitate cellular uptake of metformin 314 . Metformin also increases insulin sensitivity by 20–25 mainly by promoting weight loss 149 . Although this effect on weight varies between patient populations it is preferred for obese patients 167 . A recent study suggests that increased OCT1 gene expression in adipose tissue of obese subjects might contribute to the increased metformin action in these subjects 315 . A previous study reported that metformin treatment caused an increase in insulin sensitivity resulted from significant increases in visfatin and adiponectin 316 . Although the function of visfatin is not currently understood visfatin may have a dual role an autocrine/paracrine function that facilitates differentiation and fat deposition on visceral adipose tissue and an endocrine role that modulate insulin sensitivity in peripheral organs 317 . The results of the present study also showed that treatment of STZ- diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally was associated with significant improvements in lipid profile as compared to the untreated diabetic rats. This was revealed by significant decreases in serum triglycerides total cholesterol LDLC

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201 free fatty acids calculated non-HDL-cholesterol and TGs to HDL ratio as well as by a significant increase in HDL-C. Metformin not only lowers blood glucose concentration but also inhibits adipose tissue lipolysis reduces circulating levels of FFAs improves lipid profiles and reduces the rate of formation of advanced glycation end products 167 311 . The literature showed discrepant results about the influence of metformin on lipid profile 318 . Some studies reported reduction only in TC levels 318 319 while others reported reduction of TC and TGs with an increase of HDL-C 320 321 . Still other studies showed no changes in lipid profile 322 323 . Another investigation showed an association of metformin with an improvement in the lipid profile even in non-diabetic patients 324 . The reasons for these conflicts are not clear but may be related to the different animal species and models used or different patients’ disease profile as well as different dose and duration of metformin treatment. In accordance to our results a previous study showed that metformin caused a significant reduction of fasting serum triglycerides cholesterol and total lipids in STZ diabetic rats 313 . In addition randomized controlled trials in humans treated with metformin showed a significant reduction in triglycerides and LDL-C as well as an increase in HDL-C 312 . Another study reported that metformin decreased total cholesterol and LDL-C in type 2 diabetic patients intensively treated with insulin. However nonsignificant changes in HDL-C and triglycerides were resulted 325 . It was also previously reported that metformin lowered fasting plasma levels of triglycerides free fatty acids total cholesterol LDL and non-HDL cholesterol in patients with type 2 diabetes 326 . In contrast to our work a previous study showed no effect of metformin on LDL or HDL cholesterol inspite of the decrease in serum free fatty acids and triglycerides levels in diabetic patients treated with metformin 327 . Furthermore metformin therapy of type 2 diabetic patients increased LDL particle size and decreased plasma concentrations of remnant lipoprotein cholesterol which reflects increased atherogenicity. Metformin

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202 also decreased the plasma concentrations of methylglyoxal in diabetic patients as well as oxidative stress and related oxidation of LDL 328 . Despite the large number of studies that have established its mode of action the molecular target of metformin action was elusive for several years until Zhou et al demonstrated in 2001 329 that metformin treatment activates the energy sensor AMP-activated protein kinase AMPK a major cellular regulator of lipid and glucose metabolism in rat hepatocytes and thereafter it was confirmed that metformin treatment stimulates AMPK in tissues in both humans and rodents 314 . AMPK has been identified as a key regulator of cellular energy status 330 and has been implicated in the control of hepatic glucose and lipid homeostasis by many effects on both gene and short term regulation of specific enzymes 331 . It works as an intracellular fuel gauge which becomes activated in response to a variety of metabolic stresses that typically change the cellular AMP/ATP ratio caused by increasing ATP consumption or reducing ATP production 314 . Thus AMPK activation results in the stimulation of glucose uptake in muscle fatty acid oxidation in muscle and liver and the inhibition of hepatic glucose production cholesterol and triglyceride synthesis and lipogenesis 330 . Chronic activation of AMPK may also induce the expression of muscle glucose transporter GLUT4 329 . Therefore the increased phosphorylation and activation of AMPK by metformin lead to its beneficial effects on glucose and lipid metabolism. Phosphorylation and inactivation of acetyl-CoA carboxylase ACC as a result of AMPK activation serves to inhibit the rate-limiting step of lipogenesis. Reduced synthesis of the ACC product malonyl-CoA is also predicted to increase fatty acid oxidation 329 . Moreover the synthesis of lipogenic enzymes along with sterol regulatory elementbinding protein 1 SREBP-1 a key lipogenic transcription factor is suppressed 149 . These effects are likely to contribute to metformin’s invivo ability to lower triglycerides and VLDL thus improving dyslipidemia associated with diabetes 329 . The net metformin effect is thus to decrease glucose and lipid synthesis and to increase fat oxidation. The reduced glucose output from the liver and

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203 the decrease in ectopic fat accumulation in hepatocytes augment hepatic sensitivity to insulin 332 . The improvement in lipid profile may be not only through the correction of abnormal glucose metabolism in diabetic condition but also due to an intrinsic potential of metformin to improve lipid abnormalities. Metformin also was reported to cause inhibition of lipid peroxidation and lipoproteins oxidation as well as improvement in antioxidants level in hyperlipidemic rats. All accumulatively indicate a protective effect of metformin against oxidative stress mediated complications 333 . Perhaps this protective effect of metformin may be the cause of the amelioration of lipid profile and oxidative stress parameters seen in our results. Moreover our study insured the beneficial effect of metformin treatment on the cardiovascular complications of diabetes by a significant reduction in triglycerides to HDL-cholesterol ratio. This was in accordance to another study which showed a significant reduction in this ratio after metformin treatment 334 . The present study showed that treatment of STZ-diabetic rats with metformin in a dose of 300 mg/kg/day for 2 weeks orally was associated with a significant reduction in lipid peroxidation reflected by the decrease in hepatic malondialdehyde level as well as it caused a significant increase in the antioxidant capacity revealed by the increase in the hepatic reduced glutathione level as compared to the untreated diabetic rats. This may be due to its antioxidant action and protective effects on the liver. Previous studies showed that metformin exerts antioxidant activity in streptozotocin-induced diabetic rats 335 and decreases erythrocyte susceptibility to oxidative stress in type 2 diabetic patients 336 . Additionally when metformin therapy was given to high fructose dietinduced type 2 diabetic rats it lowered the levels of TBARS and lipid hydroperoxides decreasing lipid peroxidation as well as it significantly enhanced or maintained the activities of antioxidant enzymes SOD CAT GPx vitamin E and vitamin C in the liver of the rats 337 . In another study metformin treatment resulted in a significant increase in hepatic and kidney GSH content and activities of antioxidant enzymes SOD CAT GST with significant decrease in TBARS levels in liver and

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204 kidney when compared to untreated diabetic rats 338 . Additionally it was reported that metformin increased the expression of the antioxidant thioredoxin Trx which could mediate some of the metformins effect on ROS reduction 339 . The drug is known to inhibit oxidative phosphorylation. Although the mechanisms by which this effect is achieved are still not fully known it was suggested that metformin can enter the mitochondria accumulate within these organelles and inhibit mildly and specifically the enzymatic activity of complex-1 of the mitochondrial respiratory chain. When islet cells are exposed to metformin a lower amount of ROS of mitochondrial origin is likely to be produced which restores a sort of vicious circle leading to reduced oxidative stress 340 . This is considered an AMPKindependent mechanism through which metformin exerts a direct antioxidant effect 331 . Therefore metformin through mitochondrial complex-1 inhibition can prevent the consequences of oxidative stress on apoptosis and can diminish the mitochondria-related toxicity of hyperglycemia 341 . Thus beyond its glucose-lowering effects metformin exhibits antioxidant properties that contribute to the vasculoprotective effects 342 . Furthermore metformin was reported to possess a direct scavenging effect against oxygenated free radicals generated in-vitro and to decrease intracellular production of ROS in aortic endothelial cells through the reduction of both NADPH oxidase and/or the mitochondrial respiratory chain pathways 343 . The increase of NADPH formation by metformin can be used in regeneration of reduced glutathione by GSH reductase 341 thus reducing intracellular ROS by increasing the activity of the antioxidative glutathione system 339 . A previous study showed that metformin increased glucose- 6phosphate dehydrogenase activity and enhanced the pentose phosphate pathway related formation of NADPH 344 . One of the unique findings in the present study is the antiinflammatory effect of metformin. The results revealed that treatment of STZ-diabetic rats with metformin was associated with significant decreases in the serum levels of the inflammatory markers CRP MCP-1 and NO in comparison to the untreated diabetic rats. It was reported that metformin might prevent microvascular and macrovascular complications of diabetes mellitus by exhibiting vascular

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205 anti-inflammatory and anti-atherogenic activities. Metformin improves vascular endothelial functions inhibits IL-1-induced release of the proinflammatory cytokines improves diabetic dyslipidemia and reduces plasminogen activator inhibitor-1 resulting in subsequent improvement of capillary flow 333 . It was also demonstrated that metformin attenuated the proinflammatory responses in human vascular wall cells and macrophages 345 . However the detailed molecular mechanisms underlying these antiinflammatory effects are not fully understood. One study suggested that metformin can exert this anti-inflammatory effect through NF-B inhibition 346 . Another study showed that metformin attenuated the cytokine-induced expression of proinflammatory and adhesion molecule genes by inhibiting the activation of NF-B via AMPK activation. In addition this study showed that metformin inhibited the NF-B–dependent gene expression of various inflammatory and cell adhesion molecules including VCAM-1 ICAM-1 and MCP-1 347 . Recently it was shown that metformin significantly inhibited TNF production in isolated human monocytes attenuating the inflammatory responses 345 . Moreover obese subjects treated with metformin displayed a significant reduction in the inflammatory marker MCP-1 supporting the hypothesis that metformin can improve the low-grade inflammatory state observed in diabetes 348 . In addition a previous study showed that in overweight type 2 diabetic patients metformin reduced the concentration of CRP and regulated it specifically 349 . However another report showed modest CRP reduction after one year treatment with metformin 350 . In contrast to the results reported in patients with type 2 DM metformin had no effect on CRP and TNF- concentrations in patients with impaired glucose tolerance 351 suggesting that metformin likely reduces levels of inflammatory markers by reducing hyperglycemia improving insulin sensitivity and/or promoting weight loss in patients with T2DM 349 . Furthermore the anti-inflammatory effects of metformin were demonstrated by iNOS inhibition resulting in a decrease in serum NO level. Metformin could inhibit iNOS through its well-known mechanism the AMPK activation which is believed to contribute to its insulinsensitizing

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206 actions in diabetic subjects. However inhibition of iNOS induction and reduced NO formation represent a novel mechanism by which metformin improves insulin action in muscle 352 . Previous studies demonstrated the beneficial effects of metformin on the endothelium which appear to be mediated through its effects to improve insulin resistance. Although it was suggested that metformin ameliorates endothelial function independently of glycemia. Nevertheless it is not clear whether these effects are due to the release of endothelial NO or improvement in its signaling as metformin was found to increase endothelial NO levels through phosphorylation of eNOS in cultured endothelium cells 167 . However several studies have suggested that the vasculoprotective effects of metformin are mainly due to improved NO signaling 298 311. Thus the AMPK activation via metformin inhibits iNOS which results in decrease in the elevated NO associated with acute hyperglycemia. On the other hand it improves the endothelial dysfunction associated with chronic hyperglycemia by increasing endothelial NO probably via phosphorylation of eNOS. Eventually it is intriguing to suggest that metformin does not only improve the metabolic defects in diabetes by increasing glucose uptake and decreasing gluconeogenesis but additionally protects the vasculature by reducing the oxidative stress and inflammation 341 346 . Therefore the unique efficacy of metformin in the treatment of hyperglycemia and insulin resistance dependant complications is presumably not only an expression of one mechanism alone but of the diverse and manifold properties of this old but useful drug 301 341 . Interestingly in our present research L-cysteine treatment of the STZ-diabetic rats in a dose of 300 mg/kg/day for 2 weeks orally alone decreased significantly the levels of fasting serum glucose insulin and HOMA-IR. In accordance to our work a previous study demonstrated that Lcysteine supplementation significantly lowered the circulating levels of plasma glucose and glycated hemoglobin and the HOMA index of insulin resistance in ZDF rats a model of type 2 diabetes 75 . In addition previous

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207 studies reported lower glucose intolerance and insulin level in high fructose fed rats or in hyperinsulinemic subjects following thiol or sulfur amino acids supplementation 140 141 . Interestingly dietary cysteine was also shown to improve glucose control and alleviate sucrose-induced insulin resistance 353 . The resulted improvement in glycemic control and insulin resistance associated with cysteine treatment may be due to a decrease in AGEs formation. It is known that insulin resistance and impaired glucose metabolism can result in the accumulation of reactive aldehydes including methylglyoxal and glyoxal. These aldehydes react with free SH and amino groups of proteins and DNA to form advanced glycation end products AGEs. Thiols such as cysteine also bind these aldehydes allowing them to be excreted in bile and urine. This results in a reduction in the development of diabetic complications 115 . It was demonstrated that AGERAGE interaction increased intracellular oxidative stress causing a decrease in glucose uptake in cultured adipocytes. This effect was completely reversed by cysteine supply 136 . In addition another study showed that methylglyoxal decreased insulin-induced IRS-1 tyrosine phosphorylation and decreased kinase activity of PI3K impairing insulin signaling in cultured adipocytes. This impairment was prevented by the addition of N- acetyl cysteine to the cell culture 137 . One of our novel findings is the beneficial effect of L-cysteine treatment on lipid profile as compared to the untreated diabetic rats. L- cysteine treatment of the STZ-diabetic rats in a dose of 300 mg/kg/day for 2 weeks orally resulted in significant reductions in the levels of LDL- C free fatty acids calculated non-HDL-cholesterol and triglycerides to HDL-C ratio as well as it significantly increased the serum level of HDL- C. However L-cysteine did not show any significant reductions in the level of serum triglycerides and total cholesterol. Our results were in accordance to Diniz et al in 2006 354 who showed that cysteine supplementation prevented the elevation of oxidized LDL induced by high sucrose intake. They also reported that it had beneficial effects on enhancing HDL/triacylglycerol reducing cholesterol/HDL and normalizing effectively serum triacylglycerol and VLDL. It is evident that these beneficial effects on serum lipids were related to cysteine’s antioxidant property in both liver and serum as cysteine

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208 promotes the maintenance of protein structure against oxidation by ROS facilitating the lipoprotein receptors functions and improving the cellular uptake of serum lipids from the blood 354 . Another earlier study showed that HDL-C was increased by cysteine supplementation suggesting the possibility that a decrease in serum HDL-cholesterol may be related to changes of the plasma thiol level and/or the thiol/disulfide redox status 355 . However the molecular mechanisms by which L-cysteine supplementation improved lipid profile are not fully known. These improvements may reflect a tendency towards an overall improvement in general health and tissue metabolic status 356 . In our present study we also found that elevated malondialdehyde level in the liver of the STZ-diabetic rats was more decreased by Lcysteine supplementation in a dose of 300 mg/kg/day for 2 weeks orally than by metformin indicating better reduction of lipid peroxidation. Moreover L-cysteine caused a more significant increase in hepatic reduced glutathione than metformin suggesting more improvements in oxidative stress. Previously Blouet et al in 2007 139 provided the first demonstration that under sucrose-induced oxidative stress increasing cysteine intake markedly prevented postprandial deterioration of the redox status reported as being a key determinant of the detrimental effects of nutrients in the initiation of diabetes and atherosclerosis. Moreover previous studies have reported a benecial effect of cysteine supplementation in the prevention of post-exercise oxidative stress in human subjects 353 357 . It was also showed that the addition of N-acetylcysteine to drinking water supplied to high sucrose-fed Wistar rats resulted in increased SOD and GSH levels glutathione peroxidase activity and the GSH/GSSG ratio 354 . Moreover Oral NAC given to type 2 diabetic patients increased intraerythrocytic GSH levels and the GSH/GSSG ratio 115 . Using cysteine derivatives or cysteine-rich dietary proteins has been shown to increase blood and intracellular glutathione which may affect the thiol redox status 353 . Moreover there is evidence which may support the idea that the effects of cysteine on glucose and lipid homeostasis are mediated by an increase in hepatic GSH output which in turn positively affects body redox status and early events of the insulin signaling

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209 pathway 139 . As it was shown that the cysteine supply determines decisively the intracellular glutathione concentration of lymphocytes and macrophages thus prevents the LDL oxidation and cellular apoptosis mediated by the decrease in the intracellular GSH concentration 355 . A previous study also showed that cysteine supplementation resulted in the enhancement of the endogenous antioxidant capacity which was revealed by the decrease in thiobarbituric acid reactive substances TBARS 358 . Moreover it enhanced GSH reductase activity thus shifting NADPH to reduce GSSG 359 . Another study also showed that increasing dietary cysteine content dose dependently increased hepatic GSH. This indicates that hepatic GSH delivery and turnover were increased in rats. This study also showed significant correlations between muscle intracellular redox status and insulin signaling suggesting that the beneficial effects of cysteine-rich diets on glucose homeostasis may be mediated by their ability to maintain the peripheral GSH status 139 . Cysteine not only provides the cells with the rate-limiting ingredient of GSH but also helps to maintain other antioxidative molecules in their reduced form. Moreover it was reported that cysteine reacted directly with H 2 O 2 producing a concentration-dependent protective effect against H 2 O 2 induced oxidative stress in insulinoma cells. Therefore these effects were proposed to contribute at least partly to the mechanisms by which cysteine improves and maintains -cell viability and function as well as the integrity of nuclear DNA 295 . In addition to L-cysteine’s ability to improve glycemic control lipid profile and oxidative stress it caused a significant decrease in CRP MCP-1 and NO levels when it was given in a dose of 300 mg/kg/day for 2 weeks orally in comparison to the untreated diabetic group. This reflects L-cysteine’s anti-inflammatory effect. This finding was in accordance to a recent study which showed that L-cysteine supplementation lowered significantly blood levels of CRP and MCP-1 in ZDF rats 75 . It was postulated that the inhibitory effect of L-cysteine on the release of proinflammatory cytokines might be mediated partly by inhibiting oxidative stress pathways. Cysteine could have prevented NF-B activation which is a primary inducer of the inflammatory pathway possibly by inhibiting the glucose-mediated production of ROS thus reducing MCP-1 and CRP levels. This reduction is likely to increase insulin sensitivity and

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210 thereby improving glucose metabolism 75 . This can also explain the observed lowering of blood glucose insulin and HOMAIR in L-cysteine supplemented diabetic rats as observed in our results. The significant reduction in NO concentration by L-cysteine supplementation seen in our study may be due to the inhibition of iNOS activation. It was reported that cysteine supplementation inhibits the proinflammatory cytokines TNF- and IL-1 blocks the activation of NF-B and increases the anti-inflammatory cytokine IL-10. The induction of IL-1 and activation of NF-B were shown to precede the induction of iNOS. Thus through this mechanism cysteine can inhibit iNOS activation leading to decrease in the elevated NO concentration associated with the inflammatory conditions 360 . It was reported that chronic cysteine supplementation in alloxan induced diabetic mice reduced the activation of NF-B in the pancreas with the potential of reducing the production of proinflammatory cytokines such as interleukins and iNOS 361 . A previous study showed that cysteine supplementation for 3 weeks to diabetic rats reduced the immunostaining of iNOS as well as nitrotyrosine which indicated the inhibitions of iNOS activation and peroxynitrite- mediated cytotoxicity. They suggested that the improvement of the diabetic rats by cysteine supply is due to the suppression of oxidative stress which results in the preservation of proteins such as receptors enzymes transport proteins and structural proteins as well as reduction of cellular damage through lipid peroxidation. This finding provides evidence that antioxidant therapy offers protection against cellular damage in various animal models of diabetes as well as in diabetic patients and also reveals the strong antioxidant effect of L-cysteine 358 . Nevertheless as a major endogenous antioxidant cysteine can improve the endothelial function as it protects endothelial NO eNOS and signaling pathways from the oxidative effects of ROS thereby preserving NO bioavailability. A previous study showed that NAC increased eNOS expression and activity in both bovine and human endothelial cells in culture 362 . Moreover in type 1 diabetic rats NAC supplied in drinking water for eight weeks normalized endothelium-dependent vasodilatation in the aorta 363 .

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211 It should be noted that in our study the antioxidant and antiinflammatory effects of L-cysteine exceeded that of metformin except on nitric oxide. The effect of metformin on nitric oxide was higher. This may be attributed to the ability of metformin to inhibit the activated iNOS via AMPK activation. However controversy data suggest that L-cysteine has no or mild inhibitory effect on iNOS after its activation. The results of the present study also showed that the combination between metformin and L-cysteine each given in a dose of 300 mg/kg/day for 2 weeks orally was associated with better significant improvements in all studied parameters than either drug alone as compared to the untreated diabetic rats. This was evidenced by significant decreases in the levels of fasting serum glucose insulin calculated HOMA-IR serum triglycerides total cholesterol LDL-C free fatty acids calculated non-HDL-cholesterol and triglycerides to HDL-C ratio MCP-1 nitric oxide CRP and hepatic MDA. In addition there were significant increases in serum HDL-C and hepatic GSH. On the other side results of combination treatment were similar to metformin treatment in lowering triglycerides and total cholesterol suggesting that this lowering is due to the effect of metformin alone. However the combination treatment was significantly better for all other parameters than the metformin group. In comparison to L-cysteine treated group the combination treatment was far better in all measured parameters. The improvements in oxidative stress and anti-inflammatory parameters seen in the combination group may be due to combing between two drugs both have antioxidant and anti-inflammatory activities resulting in a synergistic effect. This gave better results than either drug used alone. The augmented results seen in combination of metformin and L-cysteine in terms of further improvement in glycemic control lipid profile oxidative stress and inflammation could be in part explained by the double inhibiting of NF-B activation. However research into the molecular mechanism for the augmented hypoglycemic antioxidant and anti-inflammatory effects of the combination between metformin and Lcysteine is needed to be further investigated and clinical trials on diabetic patients are needed. In fact medical science still has many unknown regions to explore. Therefore further research will be always needed to help in the evaluation and integration of many remedies and search strategies and to offer a

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212 framework that helps in guiding clinical decision making for suitable choice of different medication in different situation for world prosperity. CONCLUSION AND RECOMMENDATIONS To Cure Diabetes Permanently Click Here From the results of the present study it could be concluded that: - High fat diet/STZ induced experimental diabetes is a reliable mean for creating an animal model which mimics type 2 diabetes mellitus in humans. This model represents a good example of type 2 diabetes mellitus simulating the metabolic derangements oxidative stress and inflammation that occur in humans. This pattern indicates a harmful effect of STZ which should be taken into consideration in patients under STZ treatment as an antitumor agent. Besides it gives a convincing clue that dietary imbalance is an essential factor in both initation and development of type 2 diabetes mellitus. - Environmental factors especially the visceral obesity are implicated in the pathogenesis of type 2 diabetes mellitus. - Type 2 experimental diabetes is associated with chronic hyperglycemia and elevated free fatty acids that resulted in elevated oxidants and lipid peroxides as well as elevated inflammatory markers. Therefore the amelioration of oxidative stress and inflammation besides the glycemic and lipid control in diabetic patients must be taken into consideration in the new therapeutic approaches. - Diabetes mellitus is associated with characteristic lipid profile referred to as dyslipidemia triad which is usually an increase in triglycerides and LDL-cholesterol with a concomitant decrease in HDL-cholesterol. - Metformin is the gold star of the antidiabetic drugs. The results of the present study show that metformin improves insulin sensitivity and lipid profile as well as it exhibits antioxidant and anti-inflammatory effects besides its glycemic control property.

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213 - L-cysteine is a promising therapeutic agent which can correct some of the derangements occurring in type 2 diabetes mellitus especially the oxidative stress and vascular inflammation. The antioxidant and the antiinflammatory effects of L-cysteine are more evident than that of metformin in the improvement of oxidative stress markers as the reduction of hepatic MDA level and the increase of hepatic GSH level as well as in the reduction of the inflammatory markers serum MCP-1 and CRP. Besides L-cysteine possesses anti-hyperglycemic effects. These findings are novel and need to be explored in the diabetic patients. If it works as well as this research hints then cysteine supplementation could be used as an adjuvant therapy for better management of diabetes mellitus. - The combination of metformin and L-cysteine in the aforementioned doses was associated with utmost glycemic control as well as improvement of lipid profile oxidative stress and inflammation. This combination was more effective than either drug alone in decreasing fasting serum glucose insulin atherogenic lipoprotein LDL-C free fatty acids MCP-1 CRP NO and hepatic MDA as well as in raising HDL-C and hepatic GSH. This reveals synergistic effect of this drug combination. After the present study was completed we recommend that: - Nutrient management of the type 2 diabetic patients should be applied especially using the balanced diet and weight reduction since obesity especially resulting from high fat diet leads to increased visceral fat resulting in insulin resistance and subsequently type 2 diabetes mellitus. - Metformin is still a mysterious drug with a lot to explore about its mechanism and effects. It has a very unique effect on diabetic dyslipidemia. It ameliorates the cardiovascular disease risk that is known to be an important secondary complication of diabetes mellitus. It also exerts antioxidant and anti-inflammatory effects that contribute to better management of diabetes. - The antioxidants should be used as an add-on therapy to the commonly used oral antidiabetics is of great beneficial effect for scavenging free radicals improving antioxidant status and ameliorating the inflammatory condition associated with diabetes. Moreover as a result of improving the

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214 overall body status and health antioxidants can exert additional improvement in the glycemic control of the diabetic patients. - In view of synergistic effects of metformin and L-cysteine further research should be carried out to adjust the optimum doses of both drugs to be used in combination aiming at achieving maximum glycemic control target lipid profile and amelioration of both oxidative stress and inflammation in diabetic patients. - Thus the present study may be considered an addition to the present knowledge about the new mechanism of actions for both metformin and L-cysteine as antioxidants and anti-inflammatory drugs. - Still a lot to be known about diabetes its pathophysiology and its management which represent challenges for researchers to discover.

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215 SUMMARY Want to Cure Diabetes Click Here Type 2 diabetes mellitus is now a worldwide epidemic disease affecting at least 150 million people and is projected to increase to 439 million in 2030. Complex genetic predisposition interplays with sedentary life style to induce a state of insulin resistance which progresses to glucose intolerance hyperglycemia and overt type 2 diabetes when the pancreatic β- cells are unable to maintain previously high rate of insulin secretion. Great evidences demonstrate that chronic hyperglycemia and elevated free fatty acids seen in diabetic patients underlie oxidative stress and inflammation which are known to be implicated in the pathogenesis of diabetes mellitus. This state does not emerge from a single dominant route but through multiple pathways that provide several targets for therapeutic intervention. Hence a new potential approach which is the use of antioxidants and anti-inflammatory agents may signify a useful pharmacologic overture to the management of diabetes. This approach capitalizes on previous data obtained from both human and animal studies implicating lipid oversupply oxidative stress and chronic inflammation as root causes in the development and exacerbation of insulin resistance and diabetes mellitus. In view of these facts the present investigation was undertaken to assess the extent of oxidative stress the antioxidant defense and inflammatory states in high fat diet/STZ induced diabetic rats and the benefits resulting from the combination of the well-established antihyperglycemic drug metformin and the antioxidant amino acid Lcysteine. This study was also designed to reveal the effects of metformin and/or L-cysteine on the glycemic control and lipid profile as well as their ability to improve the oxidative stress and inflammation seen in the high fat diet /STZ model of type 2 diabetes mellitus. Fifty male albino rats weighing between170-200 gram were used in the current study. Rats were divided into 5 groups 10 rats in each. In all groups except group V rats were kept on a high fat diet for 2 months after which rendered diabetic by intravenous injection of 15 mg/kg streptozotocin into

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216 the caudal vein. After diabetes was confirmed drug treatment was carried out for 2 weeks as follows: Group I: Diabetic rats received distilled water orally and served as untreated diabetic control. Group II: Diabetic rats treated orally with metformin HCl in a dose of 300mg/kg/day dissolved in distilled water. Group III: Diabetic rats treated orally with L-cysteine in a dose of 300mg/kg/day dissolved in distilled water. Group IV: Diabetic rats treated orally with both metformin HCl in a dose of 300mg/kg/day and L-cysteine in a dose of 300mg/kg/day. Group V: Fed conventional rat chow and served as normal non-diabetic control. After 2 weeks of treatment 12 hours fasted rats were sacrificed by decapitation. Blood was collected and serum was separated for the determination of the following parameters: - Fasting glucose level. - Insulin. - Triglycerides. - Total cholesterol. - HDL-cholesterol. - LDL-cholesterol. - Free fatty acids. - Monocyte chemoattractant protein MCP-1. - C-reactive protein CRP. - Nitric oxide NO. - HOMA-IR non-HDL-cholesterol and triglycerides to HDL-cholesterol ratio were then calculated using their corresponding equations. Immediately after collection of blood livers were excised washed with ice-cold saline and preserved for the assessment of:

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217 - Malondialdehyde MDA. - Reduced glutathione GSH. - Protein content. Results of the present study paralleled the published data on the hyperglycemia dyslipidemia and the increasingly oxidative stress and inflammatory conditions seen in experimentally induced diabetes. Our study showed that experimentally induced diabetes mellitus was associated with a statistically significant elevated fasting serum insulin confirming the presence of insulin resistant state which was accompanied by a significant elevated fasting serum glucose level. This state mimics the natural progressive history of type 2 diabetes mellitus in humans. The levels of triglycerides total cholesterol LDL-cholesterol and free fatty acids as well as the calculated non-HDL-cholesterol and TGs to HDL ratio were also elevated while HDL-cholesterol level was decreased significantly. These findings suggest the deleterious effects of hyperglycemia and insulin resistance in the adipose tissue seen in the diabetic state. The results also revealed that type 2 diabetes resulted in a pro-oxidant state manifested by an increase in hepatic MDA generated by lipid peroxidation and this is coupled with reduction of GSH which is a protective thiol antioxidant. Since GSH represents the largest source of cellular reducing equivalents its decrease could significantly affect the overall cellular redox environment. Moreover the significantly elevated levels of the inflammatory markers MCP-1 C-reactive protein and nitric oxide indicate that inflammation plays an important key role in the pathogenesis of insulin resistance and diabetes mellitus. Metformin treated group of rats showed a statistically significant decrease in fasting serum glucose insulin and significant improvement in insulin resistance. Moreover it caused significant decreases in triglycerides total cholesterol free fatty acids LDL-C non- HDLcholesterol and triglycerides to HDL-C ratio as well as a significant increase in HDL- cholesterol. This study also demonstrates the antioxidant and the antiinflammatory effects of metformin which were suggested by the ability of metformin to

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218 decrease hepatic MDA level and to increase hepatic GSH concentration which reflects its ability to ameliorate the hazardous effects of both lipid peroxidation and oxidative stress states accompanied with diabetes mellitus. As well as its ability to decrease significantly serum MCP-1 CRP and NO which are known to reflect the overall inflammatory status. However L-cysteine treated group of rats showed a more statistically significant protection from oxidative stress and inflammation than metformin. This was in terms of the decrease in hepatic MDA level and the increase in hepatic reduced GSH concentration as well as in the decrease in MCP-1 and CRP. It also showed some improvements in other biochemical parameters include fasting serum glucose insulin LDL-C HDL-C and free fatty acids. However the effect of metformin on these parameters was more statistically significant than L-cysteine group. Interestingly the effects of metformin and L-cysteine combination for 2 weeks on all measured or calculated parameters were more superior as compared to the effects of either drug when used alone suggesting their synergism. However the decreases in serum triglycerides and total cholesterol were totally attributable to metformin as L-cysteine showed non- significant effect on both parameters. Taken together our results indicate that fasting hyperglycemia hyperinsulinemia unfavorable lipid profile as well as the oxidative stress and inflammatory states caused by the diabetic state were improved in a statistically significant way to a great extent on giving combination treatment than either drug when used alone. Thus our results obtained from the combination of metformin and Lcysteine confirm the beneficial and protective effects that can be resulted from the use of an antioxidant drug with anti-inflammatory effect as an add-on therapy to the well-known antidiabetic drugs used for management and treatment of type 2 diabetes mellitus. This study is considered as a new addition to the current knowledge about the derangements occurring in type 2 diabetes mellitus and its evaluation. This study also reveals a new mechanism of action for metformin as an anti-inflammatory drug in addition to its well-known antihyperglycemic effect and its ability to improve dyslipidemia and oxidative stress in type 2 diabetes mellitus.

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219 The study shows that the antioxidant L-cysteine also has antiinflammatory and antihyperglycemic effects as well as an ability to improve some of the disturbances of the lipid profile present in type 2 diabetes mellitus. This is novel action and needs to be explored in type 1 and type 2 diabetic patients as it can be used as an adjuvant therapy for the reduction of oxidative stress vascular inflammation and cardiovascular diseases in diabetic patients.

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