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Effect of exercise on conduit and resistance artery function in the db/db model of type 2 diabetes Moien Afshari, Farzad 2009

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EFFECT OF EXERCISE ON CONDUIT AND RESISTANCE ARTERY FUNCTION IN THE db/db MODEL OF TYPE 2 DIABETES    by    Farzad Moien Afshari    MSc University of British Columbia 2002 MD Mashad University of Medical Sciences 1996    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    Doctor of Philosophy   in   The Faculty of Graduate Studies    (Pharmacology and Therapeutics)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    December 2008   © Farzad Moien Afshari, 2008 ABSTRACT Vasculopathy is the major cause of cardiovascular complications leading to mortality in diabetes mellitus. The earliest stage for diabetic vasculopathy is endothelial dysfunction, which is related to increased oxidative stress. Another abnormality in diabetic vasculature is an increased contractile response to α-adrenergic stimulation, which may lead to hypertension and accelerated atherosclerosis. Exercise decreases diabetic vascular complications with poorly identified mechanisms. The tested hypothesis was that exercise, independent of weight loss and glycemic control, prevents endothelial dysfunction in type 2 diabetes mellitus via a decrease in oxidative stress due to increased antioxidant defense mechanisms. In this study, the mechanisms whereby low- and moderate-intensity aerobic exercise i) improve endothelial function and ii) alter vasoconstrictive response to adrenergic stimulation were assessed in db/db mice. Moderate-intensity exercise lowered body weight, increased mitochondrial SOD (Mn-SOD) and both total and phosphorylated (Ser1177) endothelial nitric oxide synthase (eNOS) protein production; it also decreased plasma (8-isoprostane) and aortic oxidative stress (nitrotyrosine and protein carbonyl levels). Low-intensity exercise did not alter body weight; however, it upregulated cytosolic Cu,Zn-SOD instead of Mn-SOD, and still demonstrated all the above benefits in the db/db aortae. Importantly, both exercise protocols improved endothelial function without altering hyperglycaemic status in db/db mice. In coronary arteries, smooth muscle contractile responses to U-46619 (a thromboxane agonist) or to increases in transmural pressure were not altered in diabetes mellitus. Moderate-exercise restored endothelium-dependent vasodilatation in coronary arteries of db/db mice, accompanied by increased expression of Mn-SOD and decreased nitrotyrosine levels in the hearts. The maximal force generated by phenylephrine was greater in db/db aortae and exercise did not attenuate the response. PKC inhibition normalized the augmented phenylephrine-induced vasoconstriction in db/db to that observed in WT mice. PKC activation, independent of exercise  ii produced greater vasoconstrictor responses in db/db compared to WT mice. In conclusion exercise reversed diabetic endothelial dysfunction independently of improvements in body weight or hyperglycaemia in association with upregulation of eNOS and specific SOD isoforms. Vascular benefits of moderate exercise were independent of changes in myogenic tone. The augmented adrenergic-mediated vasoconstriction in db/db mice was likely due to increased PKC activity that was not affected by exercise.  iii TABLE OF CONTENTS Abstract ....................................................................................................................................................ii Table of Contents....................................................................................................................................iv List of Tables ..........................................................................................................................................ix List of Figures ..........................................................................................................................................x List of Abbreviations .............................................................................................................................xii Acknowlegements.................................................................................................................................xiv Dedication ..............................................................................................................................................xv Co-authorship Statement.......................................................................................................................xvi  1. INTRODUCTION............................................................................................................................1 1.1 Diabetes mellitus ........................................................................................................................1 1.2 Diabetes mellitus and cardiovascular disease.............................................................................3    1.2.1  Polyol pathway.................................................................................................................4   1.2.2  Increased production of advanced glycation end products (AGEs).................................4   1.2.3  PKC activation .................................................................................................................5   1.2.4  Increased activity of the hexosamine pathway ................................................................6   1.2.5  The missing link...............................................................................................................6 1.3 Endothelial function and diabetes mellitus.................................................................................8 1.4 Superoxide dismutase and the vasculature .................................................................................9 1.5 Exercise, diabetes and vascular antioxidant defense ................................................................11 1.6 Obese mouse models of type 2 diabetes ...................................................................................12 1.7 db/db mouse and endothelial dysfunction ................................................................................14 1.8 Hypothesis ................................................................................................................................15  iv 1.8.1 Objectives ......................................................................................................................16 1.9 Project road map .......................................................................................................................16 1.10 Bibliography ............................................................................................................................21  2. EXERCISE RESTORES AORTIC ENDOTHELIAL FUNCTION INDEPENDENT OF WEIGHT LOSS OR HYPERGLYCAEMIC STATUS IN db/db MICE ..................................29 2.1 Introduction...............................................................................................................................30 2.2 Methods ....................................................................................................................................31  2.2.1  Animals and exercise regimen .......................................................................................31  2.2.2  Estimation of body fat using nuclear magnetic resonance (NMR)...............................  31  2.2.3  Collection of blood and tissue samples..........................................................................32  2.2.4  Measurement of plasma parameters...............................................................................33  2.2.5  Oral glucose tolerance test (OGTT)..............................................................................  33  2.2.6  Isometric force measurement .........................................................................................33  2.2.7  8-isoprostane enzyme immunoassay..............................................................................34  2.2.8  Spectrophotometric quantification of tissue nitrite........................................................34  2.2.9  Western Blot ..................................................................................................................35  2.2.10 Protein Carbonyl levels..................................................................................................35  2.2.11 Statistical analysis and calculations ...............................................................................36 2.3 Results.......................................................................................................................................36 2.3.1 Influence of exercise on body weight and body fat .......................................................36 2.3.2 Influence of exercise on plasma lipid profile ...............................................................37 2.3.3 Influence of exercise on glycemic status ......................................................................37 2.3.4 Endothelium-dependent and -independent vasodilation following exercise ................37 2.3.5 Aortic nitric oxide bioavailability following exercise ..................................................38  v 2.3.6 Changes in eNOS production and tissue nitrite levels following exercise ...................38 2.3.7 Differential regulation of intracellular antioxidants following exercise.......................39 2.3.8 Influence of exercise on whole body and tissue-specific oxidative stress....................39 2.4  Discussion .................................................................................................................................40 2.5  Bibliography .............................................................................................................................58 ............................................................................................................................................................. 3. EXERCISE RESTORES CORONARY VASCULAR FUNCTION INDEPENDENT OF MYOGENIC TONE OR HYPERGLYCEMIC STATUS IN db/db MICE ..............................62 3.1  Introduction...............................................................................................................................63 3.2  Materials and methods ..............................................................................................................64 3.2.1 Animals ..........................................................................................................................64 3.2.2 Exercise protocol ...........................................................................................................64 3.2.3 Blood and tissue samples ...............................................................................................64 3.2.4 Oral glucose tolerance test (OGTT)...............................................................................65 3.2.5 Citrate synthase (CS) enzyme assay ..............................................................................65 3.2.6 Western Blot analysis ....................................................................................................66 3.2.7 Immunofluorescence......................................................................................................66 3.2.8 Resistance artery preparation.........................................................................................67 3.2.9 Glucose and Insulin........................................................................................................68 3.2.10 Chemicals and solutions ................................................................................................68 3.2.11 Statistical analysis and calculations ...............................................................................69 3.3  Results.......................................................................................................................................69 3.3.1 Exercise, body weight, heart weight and plasma parameters ........................................69 3.3.2 Exercise decreases whole body and tissue oxidative stress ...........................................70 3.3.3 Exercise and coronary arteriolar tone ............................................................................71  vi 3.3.4 Exercise and endothelium dependent arteriolar relaxation............................................71 3.3.5 Exercise and endothelial NO bioavailability .................................................................72 3.4  Discussion .................................................................................................................................72 3.5  Bibliography .............................................................................................................................87  4. EFFECT OF EXERCISE ON AUGMENTED AORTIC VASOCONSTRICTION IN THE db/db MOUSE MODEL OF TYPE 2 DIABETES ...................................................................92 4.1 Introduction...............................................................................................................................93 4.2 Methods.....................................................................................................................................93 4.2.1 Animal groups...............................................................................................................93 4.2.2 Exercise training program.............................................................................................94 4.2.3 Isometric force measurement ........................................................................................94 4.2.4 Measurement of plasma parameters..............................................................................95 4.2.5 Citrate synthase assay ...................................................................................................95 4.2.6 Drugs and chemicals .....................................................................................................96 4.2.7 Statistical analysis and calculations ..............................................................................96 4.3  Results......................................................................................................................................96 4.3.1 Body weight ...................................................................................................................96 4.3.2 Plasma Parameters .........................................................................................................96 4.3.3 Efficacy of exercise training program............................................................................97 4.3.4 Endothelium-dependent and -independent vasodilation................................................97 4.3.5 Aortic contractile responses...........................................................................................97 4.3.6 Effect of cyclooxygenase inhibitor (indomethacin)......................................................  98 4.3.7 Effect of endothelin-1 receptor antagonist (bosentan), and Rho-kinase inhibitor (Y- 27632) ............................................................................................................................ 98  vii 4.3.8 Effect of NOS blocker (L-NAME) ................................................................................ 98 4.3.9 Effect of PKC inhibitor (Calphostin-C)......................................................................... 98 4.3.10 PKC activator (indolactam) concentration-response curve ........................................... 99 4.4 Discussion ................................................................................................................................. 99 4.5 Bibliography ........................................................................................................................... 110  5. CONCLUSIONS .......................................................................................................................... 114 5.1  Future Directions ................................................................................................................... 114 5.2  Conclusions ............................................................................................................................ 115 5.3  Bibliography ........................................................................................................................... 118  6. APPENDIX A- MEASUREMENT OF WATER AND FOOD CONSUMPTION IN SEDENTARY AND EXERCISING db/db AND WILD-TYPE MICE ................................... 119  6.1 Methods.................................................................................................................................... 119  6.2 Results...................................................................................................................................... 119  6.3 Discussion ................................................................................................................................ 120  7. APPENDIX B- EFFECT OF LOW AND MODERATE INTENSITY EXERCISE ON AORTIC FUNCTION IN WT MICE ........................................................................................ 122  8. APPENDIX C- EFFECT OF LOW-INTENSITY EXERCISE ON CORONARY ENDOTHELIAL FUNCTION IN db/db MICE ........................................................................ 123   viii LIST OF TABLES 2-1   Plasma parameters of diabetic (db/db) and wild type (WT) mice ................................................ 45 2-2   Emax and EC50 values for ACh and SNP concentration-response curves ...................................... 46 2-3   Emax and EC50 values for ACh concentration-response curves before and after SOD and L-Arg plus BH4 incubation ........................................................................................................................ 47 3-1a General Characteristics of Animals .............................................................................................. 78 3-1b Oral Glucose Tolerance Test performed in mice that were exercised for five weeks .................. 78 3-2   Vasomotor responses of isolated coronary arteries ...................................................................... 79 4-1 Exercise training protocol for mice............................................................................................. 102 4-2 Serum parameters at the end of study in all experimental groups .............................................. 103  ix LIST OF FIGURES 1-1 Mechanisms of vasculopathy in DM ............................................................................................ 17 1-2 Schematic model for NO degradation by oxygen free radicals (O2•) in diabetes and the suggested preventive role of exercise ............................................................................................................ 18 1-3 The antioxidant defense mechanism pathways............................................................................. 19 1-4 Algorithm indicating animal groups used in this study ................................................................ 20 2-1 Age- and exercise-related changes in body weights of mice and OGTT values .......................... 48 2-2 Traces illustrating tension (mN) of aortic rings in differennt mouse groups................................ 50 2-3 ACh and SNP concentration-response curves from preconstricted aortic rings........................... 52 2-4 Effect of SOD and L-Arg plus BH4 incubation on ACh-induced vasodilation ............................ 53 2-5 Endothelial nitric oxide synthase (eNOS) protein expression and aortic nitrite levels ................ 55 2-6 Western Blot analysis of antioxidant protein expression.............................................................. 56 2-7 Oxidative stress related parameters .............................................................................................. 57 3-1 Protein expression of antioxidants and eNOS in the whole heart................................................. 80 3-2 Intracardiac localization of Mn-SOD by immunofluorescence .................................................... 82 3-3 Myogenic tone and passive distensibility in coronary septal arteries........................................... 83 3-4 Endothelium-dependent and -independent response traces in coronary septal arteries................ 84 3-5 Concentration response curves for endothelium-dependent and endothelium-independent vasorelaxation in coronary arteries ............................................................................................... 85 3-6 Effect of L-Arg + BH4 and SOD incubation on ACh mediated vasorelaxation in coronary septal arteries of sedentary and exercised db/db mice ............................................................................86 4-1 Age- and exercise-related changes in body weight of WT and db/db mice ...............................104 4-2 The maximum response to ACh in aortic rings of WT, db/db and exercised db/db mice..........105 4-3 Traces illustrating tension (mN) of aortic rings from the following mouse groups ...................106  x 4-4 PE-induced constriction in aortae of WT, db/db, and exercised db/db mice..............................107 4-5 PE concentration-response curves in the presence and absence of calphostin-C .......................108 4-6 Indolactam concentration-response curve...................................................................................109 6-1 Food and water consumption in mice .........................................................................................121 7-1 Ach, SNP and PE dose response curves in aortae of sedentary WT mice and WT mice exercised with low and moderate intensity ..........................................................................................................122 8-1 Endothelium-dependent and endothelium-independent vasorelaxation in coronary arteries of WT mice vs. db/db mice vs. db/db mice exercised with low-intensity....................................................... 123 8-2 U46619-induced constriction curves in the presence or absence of L-NAME in coronary arteries of sedentary and exercised WT and db/db mice ..................................................................................124  xi LIST OF ABBREVIATIONS ACh: Acetylcholine ADA: American Diabetes Association AGES: Advanced Glycation End Products ADMA: Asymmetric Dimethylmethylarginine ANOVA: Analysis of Varience AP-1: Activator Protein-1 L-Arg:  L-Arginine AsA: ascorbate ATP: Adenosine Triphosphate BH :  4 tetrahydrobiopterin BHT: Butylated Hydroxy Toluene BSA: Bovine Serum Albumin CAD: Coronary Artery Disease CoA: Coenzyme-A CPMG: Carr Purcell Meiboom Gill CYP-450: Cytochrome P-450 DAG: Diacylglycerol DAPI: 4'-6-Diamidino-2-Phenylindole DAsA: Dehydroascorbate DCA: DM: Diabetes Mellitus DTNB: 5, 5'-Dithiobis-(2-Nitrobenzoate) EC: Endothelial Cell EC50:  Concentration causing 50% of the maximum effect EDHF: Endothelium Derived Hyperpolarizing Factor EDTA: Ethylene-Diamine-Tetra-Acetic acid EGTA: Ethylene Glycol Tetraacetic Acid E max:  Maximal response that can be produced by the drug eNOS: Endothelial Nitric Oxide Synthase EPCs: Endothelial Projenitor Cells ET-1: Endothelin-1 FITC: Fluorescein isothiocyanate GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase GSH: L-Glutathione GSSG: Oxidized Glutathione HDL: High Density Lipoprotein HLA: Human Leukocyte Antigen LDL: Low Density Lipoprotein MDA: Malondialdehyde Mn-SOD: Manganese Superoxide Dismutase NMR: Nuclear Magnetic Resonance NAD: Nicotinamide Adenine Dinucleotide NADH: Nicotinamide Adenine Dinucleotide-oxidase NADP: Nicotinamide Adenine Dinucleotide Phosphate NADPH: Nicotinamide Adenine Dinucleotide Phosphate-oxidase NO: Nitric Oxide  xii OGTT: Oral Glucose Tolerance Test PAI-1: Plasminogen Activator Inhibitor-1 PKB: Protein Kinase B PKC: Protein Kinase C PSS: Physiological Salt Solution RAGEs: Receptor for Advanced Glycation End Products RNA: Ribonucleic Acid ROS: Reactive Oxygen Species SDS: Sodium Dodecyl Sulfate SNP: Sodium Nitroprusside SOD: Superoxide Dismutase T 2:  Time 2 TBS-T Tris-buffered saline containing 0.1% Tween-20 TCA: Tricarboxylic Acid TGF: Transforming Growth Factor TNFα: Tumor Necrosis Factor-α TRIS: Tris (hydroxymethyl) aminomethane VEGF: Vascular Endothelial Growth Factor WT: Wild Type  xiii ACKOWLEGEMENTS  I would like to express my deepest gratitude to my supervisor, Professor Ismail Laher, Ph.D.; his extensive knowledge, encouragement and guidance have provided a strong basis for the present thesis. I would also like to thank the faculty and staff of the Department of Anesthesia, Pharmacology and Therapeutics at the University of British Columbia for their friendly support throughout my studies at UBC. I owe my loving thanks to my wife, Baharak Farrokhnia. It would have been impossible for me to finish this work without her encouragement and understanding. Special thanks are owed to my parents, who have constantly supported and persuaded me all through my years of education. Scholarships of the University of British Columbia throughout my graduate studies are gratefully appreciated.   xiv    To my wife and my parents whose love and support have made everything possible.  xv CO-AUTHORSHIP STATEMENT In chapter 2,3, and 4 that are published, the initial idea of looking at the effect of exercise on vascular function in db/db mouse model of type 2 diabetes belongs to Dr I. Laher. All experiments in these three chapters were carried out by myself, except for the following: Dr M. Khazaei assisted with developing the exercise protocol and vascular function experiments (chapters 2 and 4), M.M. Rahman and N. Sallam assisted with 8-isoprostane measurement (chapter  2 and 3), Dr. S. Ghosh assisted with molecular biology and immunohistochemistry experiments (Chapter 2 and 3).  Citrate synthase assay was performed in Dr. R.W. Brownsey’s laboratory by myself with assistance of M.M. Rahman. Oral glucose tolerance test was performed with equipments and assistance of Dr. T.J. Kieffer’s laboratory and S. Elmi (chapter 2 and 3). S. Elmi and N. Sallam also helped with spectrophotometric quantification of tissue nitrite (chapter 2).    xvi 1 Introduction 1.1  Diabetes mellitus The Greek word “diabetes” literally means “flowing through”, and is used to describe diseases of excessive urination. The Latin adjective for “sweet” specifies the disease in which the excessive urine produced has a high concentration of glucose, diabetes mellitus (DM).  Diabetes mellitus is characterized clinically by polydipsia (excessive thirst), polyuria (frequent urination), and polyphagia (excessive eating); high fasting and random blood glucose levels are diagnostic of the disease. In 2006, the World Health Organization estimated that over 180 million people had diabetes. By 2030, they estimate that this figure will be at least 360 million (World Health Organization, 2006). The earliest historical record of diabetes mellitus comes from a 3rd Dynasty Egyptian papyrus, circa 1552 B.C.E.   The physician Hesy-Ra described polyuria as a symptom (Canadian Diabetes Association, 2007a) and it was noted that ants were attracted to the urine of people with a mysterious emaciating disease (MacCracken & Hoel, 1997). Over 3000 years later, a German medical student Paul Langerhans (1869) announced in a dissertation that the pancreas contained nine different cell types, two of which had not been reported before. One type was a spindle-shaped centroacinar glandular cell that secreted digestive enzymes and the second cell type had a polygonal structure that was found together in large numbers and was suggested to be lymph nodes by Langerhans. The latter cells later named the “Islets of Langerhans” had no known function at that time. In 1921, Dr. Banting, a physiologist from the University of Toronto, presented a paper to the American Physiological Society at Yale University describing the beneficial effect of some pancreatic extracts on diabetes. The next year, insulin extracts were first tested on a human being, a 14-year-old boy named Leonard Thompson; the treatment was considered a success. In 1923, Dr. Banting and Dr. Macleod shared the Nobel Prize in physiology for the discovery of insulin. The Eli Lilly and Company and the University of Toronto  1 subsequently started the mass production of insulin in North America (Canadian Diabetes Association, 2007a). Further systematic research indicated that DM is a heterogeneous group of disorders attributable either to insulin resistance, insulin deficiency, or both. In 1980, the World Health Organization (WHO) certified the classification developed by the National Diabetes Data Group (NDDG). According to this classification, two major types of DM were identified: insulin dependent DM (IDDM) and non-insulin dependent DM (NIDDM) (WHO, NDDG 1980). However in 1997, the American Diabetes Association (ADA) proposed that the terms IDDM and NIDDM be replaced respectively by Type 1 and Type 2 diabetes mellitus (Report of Expert Committee on the Diagnosis and Classification of DM 1997). Type 1 DM: includes 5-10% of all cases of diabetes and occurs following immune-mediated (90%) or idiopathic (10%) destruction of pancreatic β-cells (Harris, 2004). It usually affects children and adolescents and is often called juvenile-onset DM. It occasionally affects adults older than 30.  The immune-mediated form results from a cell-mediated autoimmune destruction of pancreatic β-cells. In 85-90% of cases, one or more autoantibodies against islet cells, insulin, glutamic acid decarboxylase, or tyrosine phosphatases can be detected.  Genetic factors, including specific HLA sub-types (DR3, DR4), increase the risk of autoimmune Type 1 DM. There is also an association with environmental factors, such as viral infections (Report of Expert Committee of the Diagnosis and Classification of DM 2003). Type 2 DM: The majority of patients with this type of diabetes are obese, or have a high percentage of their body fat distributed in the abdominal area.  Obesity and abdominal fat have been shown to cause insulin resistance.  Type 2 diabetics have lower serum insulin levels than would be expected given the degree of hyperglycemia, indicating the presence of a defect in insulin secretion as well.  The risk of developing type 2 DM increases with age, obesity, and decreased physical activity. Individuals with type 2 DM are at increased risk of developing macrovascular and microvascular complications. The  2 genetic predisposition to type 2 DM is often strong, more so than in the autoimmune form of type 1 DM. However, the genetics of type 2 DM are multifactorial and not clearly defined (2003).  1.2  Diabetes mellitus and cardiovascular disease Both type 1 and type 2 DM are independent risk factors for developing coronary, cerebrovascular, and peripheral arterial disease. It has even been said that "diabetes is a cardiovascular disease" (Grundy et al, 1999). Approximately 80% of people with diabetes will die as a result of arterial disease leading to myocardial infarction or stroke (Canadian Diabetes Association, 2007b). Compared to the rest of the population of patients with cardiovascular disease, the mortality rate is higher in diabetic patients with cardiovascular pathology (Nesto, 2004). Diabetic patients have a two- to four-fold increased risk of developing microvascular disease (affecting the retina, kidney, and peripheral nerves) and macrovascular disease (affecting the coronary, cerebral and peripheral arteries) (Soro-Paavonen & Forbes, 2006).  Clinical studies have shown that tight control of blood glucose can prevent the development and progression of microvascular complications (UKPDS 1998). Most cells are able to reduce glucose import in a hyperglycemic environment, so that their internal glucose concentration remains constant. In contrast, cells damaged by hyperglycemia are unable to do this efficiently (Heilig et al, 1995; Kaiser et al, 1993). Thus, DM selectively damages cells such as endothelial and mesangial cells, whose glucose transport rate does not decline rapidly as a result of hyperglycemia, leading to high glucose concentrations inside the cell. This is an important observation suggesting that the cause of hyperglycemic complications is likely to be a change in the normal mechanisms inside these cells, rather than those outside. Four main hypotheses have been proposed for hyperglycemia-induced vascular complications. The four hypotheses are: increased polyol pathway activity; increased advanced glycation end products (AGEs) formation; activation of protein kinase C (PKC) isoforms; and increased hexosamine pathway  3 activity. Until recently there was no unifying hypothesis linking these four mechanisms (Brownlee, 2001).  1.2.1 Polyol pathway In the polyol pathway, glucose is reduced to sorbitol by aldose reductase, a reaction coupled to the oxidation of NADPH to NADP.  Sorbitol is then oxidized to fructose by sorbitol dehydrogenase, a reaction coupled with the reduction of NAD to NADH. Several mechanisms have been proposed to explain the potential harmful effects of hyperglycemia- induced increases in polyol pathway activity.  An initial hypothesis was that increased sorbitol levels caused osmotic stress.  But sorbitol concentrations measured in diabetic vessels and nerves were found to be too low to induce osmotic damage.  Decreased (Na+-K+) ATPase activity has been associated with sorbitol, but in hyperglycemia this has more convincingly been attributed to PKC-related increases in the production of arachidonate and PGE2, two inhibitors of the (Na+-K+) ATPase pump. Later, it was proposed that NADPH would be depleted in hyperglycemia, consumed in the co-reaction with the reduction of glucose to sorbitol. Since NADPH is required for regenerating reduced glutathione (GSH), this could induce or exacerbate intracellular oxidative stress. This was in fact supported by findings in diabetic transgenic mice overexpressing aldose reductase, who had decreased lens GSH (Lee & Chung, 1999) and in knock-out mice for aldose reductase, who had no decrease in nerve GSH content and conduction velocity (Forstermann & Munzel, 2006).  1.2.2 Increased production of advanced glycation end products (AGEs) AGEs are produced by the non-enzymatic, spontaneous reaction between reducing sugars and amino groups on proteins, lipids and nucleic acids. They were originally thought to arise from reactions between extracellular proteins and glucose. However, it now seems likely that intracellular  4 hyperglycemia is the primary inciting event in the production of both intracellular and extracellular AGEs (Brownlee, 2001). AGEs produce a cytotoxic effect by three common mechanisms. First, modification of intracellular proteins by AGEs alters their function. Second, modification of the extracellular matrix machinery by AGEs leads to their abnormal interaction with other matrix components and with the receptors for matrix proteins (integrins) located on the cell membrane. Third, plasma proteins modified by AGEs bind to receptors on a variety of cells including endothelial cells and increase receptor-mediated production of reactive oxygen species (Brownlee, 2001). Various studies in diabetic animal models using aminoguanidine (Hammes et al, 1991; Soulis-Liparota et al, 1991) and OPB-9195 (Nakamura et al, 1997), two structurally unrelated inhibitors of AGEs production, have shown prevention of diabetic microvascular disease in the form of retinopathy, nephropathy and neuropathy. This shows the potential importance of the role of AGEs in the pathogenesis of diabetes complications.  1.2.3 PKC activation Incubation of blood vessels with elevated concentrations of glucose enhances the intracellular concentration of Diacylglycerol (DAG), which will ultimately activate PKC. Among the isoforms of PKC, it is mainly the β- and δ-isoforms that are activated; however the levels of other isoforms may also increase. Activation of PKC leads to the pathologic alteration of a variety of vasoactive compounds (eNOS, ET-1, VEGF) and increases oxidative stress (NADPH oxidase activation), which eventually leads to endothelial and vascular pathology. In animal models, treatment with an inhibitor specific for PKC-β significantly reduced diabetic retinopathy and nephropathy (Brownlee, 2001).    5 1.2.4 Increased activity of the hexosamine pathway High levels of intracellular glucose cause shunting of glucose metabolism into the hexosamine pathway, which may lead to a number of diabetic complications. The hexosamine pathway turns fructose-6-phosphate away from glycolysis in order to provide substrates for reactions dependent on UDP-N-acetylglucosamine, such as proteoglycan synthesis and the synthesis of O-linked glycoproteins.  The pathway also generates as byproducts inflammatory mediators that have been linked to diabetic complications, including TGF-α, TGF-β1 (Kolm-Litty et al, 1998) and PAI-1 (Du et al, 2000).  The mechanism by which the hexosamine shunt mediates hyperglycemia-induced increases in gene expression of the inflammatory mediators is not known. But inhibition of the rate-limiting enzyme in the hexosamine pathway (glutamine: fructose-6-phosphate amidotransferase) blocks hyperglycemia-induced increases in the transcription of the above-mentioned inflammatory mediators. The hexosamine pathway also has an important role in insulin resistance mediated by hyperglycemia and hyperlipidemia (Hawkins et al, 1997; Marshall et al, 1991).  1.2.5 The missing link As discussed above, specific inhibitors of the aldose reductase pathway, AGEs formation, PKC activation and the hexosamine shunt each improve different diabetes-induced pathologies in vitro and in vivo (Brownlee, 2001).  The possibility of a common factor linking these four mechanisms of hyperglycemia-induced damage was initially a matter of some debate (Engerman et al, 1994; Faraci & Didion, 2004; Hammes et al, 1991; Lee & Chung, 1999; Nakamura et al, 1997). The question has now been resolved by the recent finding that the four hyperglycemia-induced pathogenic processes have a final common pathway: increased generation of superoxide by the mitochondrial electron-transport chain (Du et al, 2000; Nishikawa et al, 2000). When the electrochemical potential difference generated by the proton gradient across the inner mitochondrial membrane is high, the lifetime of superoxide generating electron-transport intermediates is prolonged. It seems that there is a threshold level above  6 which superoxide generation is significantly increased (Korshunov et al, 1997). It has been found that hyperglycemia raises the proton gradient above this threshold level due to overproduction of electron donors by the tricarboxylic acid (TCA) cycle (Du et al, 2000). This subsequently causes a significant increase in the generation of superoxide by vascular endothelial cells. Overexpression of manganese superoxide dismutase (Mn-SOD), the mitochondrial form of superoxide dismutase, eliminated the signal produced by reactive oxygen species. Mitochondrial superoxide production induced by hyperglycemia activates the four damaging pathways (of hyperglycemia-induced damage)  by inhibiting glyceraldehyde phospho dehydrogenase (GAPDH) (Du et al, 2003). Inhibition of GAPDH activity by hyperglycemia does not occur when mitochondrial overproduction of superoxide is reversed by antioxidants. When GAPDH activity is inhibited, levels of all glycolytic intermediates that are upstream of GAPDH including glyceraldehyde-3-phosphate increase, which directly activates two of the four pathways (AGEs, and PKC). Also further upstream, levels of the glycolytic metabolite fructose-6 phosphate increases, which enhances flux through the hexosamine pathway. Finally, inhibition of GAPDH increases intracellular levels of the first glycolytic metabolite, glucose and therefore increases flux through the polyol pathway. Inhibition of GAPDH activity using antisense DNA increases the activity of each of the four pathways at normal glucose levels to the same extent as that induced by hyperglycemia. These findings rule out the possibility that other hyperglycemia-induced metabolic changes account for these observations (Brownlee, 2005; Du et al, 2003). Inhibition of GAPDH by hyperglycemia occurs via activation of poly(ADP-ribose) polymerase. Superoxide generated secondary to hyperglycemia generates polymers of ADP-ribose which inhibits GAPDH activity in vivo. Inhibition of mitochondrial superoxide production either with antioxidants, or by inhibition of poly(ADP-ribose) polymerase (PARP) both prevented reduction of GAPDH activity by ADP-ribose in hyperglycemia (Brownlee, 2005; Du et al, 2003). It is important to note that although the unifying theory helps to simplify the mechanisms of vasculopathy in DM, this is still a complex issue that involves many different pathways as is shown in Fig 1-1.  7 1.3  Endothelial function and diabetes mellitus Endothelial dysfunction is a hallmark of diabetes mellitus, and is directly associated with an increased risk of cardiovascular complications (Halcox et al, 2002). In fact endothelial dysfunction occurs in prediabetes (such as impaired glucose tolerance) (Hsueh & Quinones, 2003). Endothelium-dependent vasodilators such as acetylcholine (ACh) can be applied to pre-constricted isolated blood vessels mounted in a wire- or pressure myograph to assess endothelial function (Mulvany & Halpern, 1977; Lui et al, 2000).  ACh dilates blood vessels by increasing nitric oxide release from the endothelium. ACh works on endothelial muscarinic receptors to increase cytoplasmic calcium concentration leading to activation of endothelial nitric oxide synthase (eNOS). eNOS produces NO by converting L-arginine to L-citrulline in a reaction that requires calcium, BH4, and NADPH as co-factors (Marletta, 1993). Reduced NO availability in diabetes is not due to decreased eNOS protein expression (Okon et al, 2003). In fact, there is even the possibility that diabetes upregulates eNOS protein expression in endothelial cells (Cosentino et al, 1997). Although eNOS is present, it is possible that the activity (Wang et al, 2002) and regulation of this enzyme may be negatively altered in diabetes mellitus (Jensen et al, 1989). Moreover, decreased availability of the eNOS substrate, L-Arg, or co-factors such as BH4, also occurs in diabetes (Pannirselvam et al, 2003a). It has been shown that plasma and vascular tissue levels of L-Arg and BH4 are significantly lower in diabetic subjects (Pannirselvam et al, 2003a; Pieper et al, 1996; Pieper & Dondlinger, 1997). The cofactor BH4 can be degraded by peroxynitrite (Milstien & Katusic, 1999), which is produced when excess oxygen free radicals in diabetic vessels react with NO. BH4 deficiency and toxic peroxynitrite are able to uncouple eNOS (Katusic, 2001). Uncoupled eNOS consumes L-Arg to generate more oxygen free radicals instead of NO, leading to a relative L- Arg deficiency (Fig 1-2). Endothelial dysfunction in diabetes mellitus may be related to increased degradation of nitric oxide due to increased oxidative stress, and may be preventable by exercise. Increased generation of reactive  8 oxygen species and / or decreased antioxidant mechanisms leads to increased oxidative stress in diabetes. In support of this are the increased superoxide levels in arteries of STZ-induced type 1 diabetic rats (Hink et al, 2001). Reactive oxygen species inhibit endothelium-dependent relaxation by inactivation of NO (Andrews et al, 2005; Bagi et al, 2003; Gryglewski et al, 1986; Maejima et al, 2001; Nishikawa et al, 2000; Pieper, 1998; Tuck, 2003) and the concurrent production of peroxynitrite, which degrades BH4 (indirect uncoupling) (Pannirselvam et al, 2003a) and uncouples the enzyme (Zou et al, 2002). In the presence of SOD, scavenging of oxygen free radicals prevents direct NO degradation while at the same time decreasing the formation of peroxynitrite (Fig 1-2). The net result of these effects is that SOD prevents the uncoupling of eNOS and also the depletion of the eNOS cofactor BH4 and substrate L-Arg (Fig 1-2). In the diabetic state, the SOD enzyme or its activity is decreased. This has been supported by the demonstration that mitochondrial SOD (Mn-SOD) mRNA expression is markedly decreased in the diabetic rat aorta (Kamata & Kobayashi, 1996). SOD activity is also decreased in the plasma (Abou-Seif & Youssef, 2004;Flekac et al, 2008) and white blood cells (Uchimura et al, 1999) of diabetic patients.  1.4  Superoxide dismutase and the vasculature Superoxide anions (O2•) are generated during oxidative phosphorylation in the mitochondria of all animal cells. Mitochondria are the major site of excessive production of reactive oxygen species in diabetes (Nishikawa et al, 2000). Antioxidant defence mechanisms, including SOD, protect cells against the toxic effects induced by free radicals (Fig 1-3). Three isoforms of SOD exist in the blood vessels of mammals. Each of these isoforms has a distinct gene, however, they all catalyze the same reaction (Faraci & Didion, 2004). Two of these SOD isoforms are intracellular -- cytoplasmic (Cu,Zn- SOD) and mitochondrial (Mn-SOD) -- and the third is extracellular (EC-SOD), which is also a Cu,Zn- SOD (Faraci & Didion, 2004). SODs are important components of antioxidant mechanisms since they  9 are the primary defence mechanism against oxygen free radicals, converting them to hydrogen peroxide and oxygen. Hydrogen peroxide is further degraded to water and oxygen by catalase and other antioxidant pathways (Fig 1-3). In the normal mouse aorta, the activity of Cu,Zn-SOD accounts for 50% to 80% of total SOD activity. Mn-SOD accounts for approximately 2% to 12% of total vascular SOD, and EC-SOD accounts for the remainder. A similar pattern of expression was observed in human arteries (Faraci & Didion, 2004). EC-SOD can be measured in extracellular fluids (Adachi et al, 1994; Marklund, 1982; Marklund et al, 1986) and is secreted into the extracellular fluid by cells such as fibroblasts, endothelial cells, smooth muscle cells, and some immune cells and then EC-SOD binds to sulfated polysaccharides such as heparin and heparan sulfate on endothelial cells (Carlsson et al, 1995; Marklund, 1984; Tan et al, 2006). It is likely that EC-SOD has a major role is determination of NO bioavailability in blood vessels (Fukai et al, 2002), so that deficiency in EC-SOD and CuZn-SOD produces similar dysfunction of endothelial cells (Jung et al, 2003). Thus to protect NO over its entire diffusion pathway (from endothelial cell where it is produced to its target in vascular muscle), normal expression of both CuZn-SOD and EC-SOD is essential. These observations may explain how incubation of blood vessels with cell impermeable Cu,Zn-SOD improves endothelial fuction. The beneficial effects of SOD activity are as follows. First, SOD is protective against superoxide- induced cytotoxicity, such as the inactivation of mitochondrial proteins containing iron–sulfur (Fe–S) centers and damage to these complexes resulting in the release of free iron leading to the formation of hydroxyl radicals (highly reactive ROS). A second major function of SOD is to protect NO and NO- mediated signaling. NO reacts with superoxide at a rate three times faster than the dismutation of superoxide by SOD (Beckman & Koppenol, 1996; Darley-Usmar et al, 1995). Because of the efficiency of the reaction, the local concentration of SOD is a key determinant of bioactivity of NO. Finally, SOD converts free oxygen radicals to hydrogen peroxide, which is a signaling molecule and regulator of gene expression that may act as a trophic mediator of vascular muscle, function as an endothelium-derived hyperpolarizing factor (EDHF), and has also been suggested to be an  10 endothelium-derived relaxing factor (EDRF) without functioning as an EDHF (Blanc et al, 2003; Chaytor et al, 2003; Matoba & Shimokawa, 2003). Local concentrations of hydrogen peroxide and its mediated effects are modified by other antioxidant enzymes such as glutathione peroxidases or catalase (Fig 1-3). Deficiency in Cu,Zn-SOD results in greater levels of vascular superoxide and peroxynitrite, augmented myogenic tone, increased vasoconstrictor responses, and impaired endothelium-dependent (NO-mediated) relaxation in both large arteries and microvessels.  1.5  Exercise, diabetes and vascular antioxidant defense The ability of exercise to benefit cardiovascular function and decrease mortality is generally accepted (Jolliffe et al, 2001; Myers et al, 2002; Paffenbarger, Jr. et al, 1986; Sesso et al, 2000). Long-term exercise improves vascular function in animal models of diabetes (Minami et al, 2002) as well as in humans with CAD (Hambrecht et al, 2000) and type 1 DM (Fuchsjager-Mayrl et al, 2002). However, the mechanisms by which exercise promotes improved coronary artery function are incompletely understood. Early studies suggested that exercise improves cardiovascular function by decreasing risk factors, including plasma lipids, blood pressure, blood glucose, and body mass index (Kingwell et al, 1996). However, recent findings indicate that although long-term vigorous exercise decreases some cardiovascular risk factors, exercise can improve vascular function even without significantly reducing other known risk factors (Green et al, 2003; McGavock et al, 2004).  The direct beneficial effect of exercise on endothelial function in diabetes may result from decreased oxidative stress by a reduction in oxygen free radical generation, from tighter glucose control, or from increased free radical scavenging systems such as SOD (Chang et al, 2004). Two major possibilities exist for the upregulation of SOD by exercise: (1) the effect of shear stress (Inoue et al, 1996b; Dimmeler et al, 1999), and (2) the stimulating effect of bouts of oxidative stress induced by exercise. Shear stress may directly activate antioxidant mechanisms. For example, Cu,Zn-  11 SOD (cytoplasmic SOD) is known to be a shear-stress-sensitive gene product (Inoue et al, 1996a). Thus, increases in Cu,Zn-SOD may contribute to mechanisms by which exercise exerts protective effects within the vasculature. Under normal conditions, the mitochondrial electron transport chain is the main source of superoxide, converting up to 5% of molecular O2 to superoxide (Faraci & Didion, 2004). Because of its mitochondrial localization, Mn-SOD is considered to be a first line of defense against oxidative stress. To fit this role, Mn-SOD is particularly sensitive to the local generation of reactive oxygen species (ROS) and can be upregulated by them, as occurs in vascular cells during bouts of exercise. Exercise-induced increase in oxidative stress triggers the cellular events leading to increased Mn-SOD expression. The redox sensitive transcription factors including activator protein-1 (AP-1) and nuclear factor-κB may be involved in a sequence of events leading to the observed antioxidant adaptations (Arrigo, 1999). Therefore, exercise increases NO bioavailability by augmenting the SOD antioxidant defence and protecting NO as it diffuses from endothelium to its major target, soluble guanylate cyclase, in vascular muscle (Fig 1-2).  1.6  Obese mouse models of type 2 diabetes Monogenic animal models of obesity and type 2 DM include the ob and db mouse.  Genetically obese mice with inactivating mutations in the gene encoding leptin (ob gene) or the gene encoding the leptin receptor (db gene) have been known for many years. Leptin is synthesized primarily by adipose tissue and secreted into the circulatory system, and is a satiety factor with receptors in the hypothalamus. Human leptin is a relatively small protein (16 kDa) that shares extensive homology with mice (84%) and rats (83%). Lack of leptin signaling leads to early onset obesity, hyperphagia, and hypothalamic hypogonadism. Unlike leptin-deficient mice, humans deficient in leptin do not always suffer from hyperinsulinemia, hyperglycemia, hypercorticism, or hypothermia, however there are examples of leptin-deficient patients that exhibit fasting hyperglycemia (Montague et al, 1997; Farooqi et al, 1999;  12 Licinio et al, 2004). Both ob and db are autosomal recessive with full penetrance. Homozygous mutants of either gene are infertile and are bred by heterozygous mating. The obese (ob) phenotype shows several characteristics of type 2 diabetes including insulin resistance, glucose intolerance, and mild hyperglycemia. Even though they have severe insulin resistance, ob/ob mice are not always severely diabetic although colonies of mice in some centers manifest more severe diabetes (Nobe et al, 2004; Herberg & Coleman, 1977). In contrast, the db mice are consistently extremely hyperglycemic and diabetic. The db/db mouse is a genetic model of non-insulin dependent type 2 DM (Coleman, 1982; Leibel et al, 1997), and is characterized by a defect in leptin receptors (Tartaglia et al, 1995). The defect in leptin receptors in db/db mice leads to impairments of leptin regulation of food intake and body weight (Chua, Jr. et al, 1996). This defect results in the expression of diabetes with preceding hyperinsulinemia, hyperglycemia, and extreme obesity (Coleman, 1978). The db/db mouse was identified in 1966 as an obese mouse that was hyperphagic soon after weaning (Hummel et al, 1972). The diabetic gene (db) is transmitted as an autosomal recessive trait. The db gene encodes for a G-to-T point mutation of the leptin receptor, leading to abnormal splicing and defective signaling of the adipocyte-derived hormone leptin (Chen et al, 1996; Lee et al, 1996). The lack of leptin signaling in the hypothalamus leads to persistent hyperphagia and obesity with consequently high leptin and insulin levels. In this animal, hyperinsulinemia is noted by 10 days of age and blood glucose levels are slightly elevated at 1 month of age (Chen et al, 1996). After 1 month of age, db/db mice are distinguishable from WT and heterozygous mice by the presence of increased fat deposition in the inguinal and axillary region. The db/db mouse develops frank hyperglycemia with glucose values of 9.7 ± 1.6 mM by 8 weeks of age and 15.7 ± 4.3 mM at 10 weeks of age (Chen et al, 1996). Progressive hyperglycemia is noted with mean levels of glucose of 28.6 ± 13.2 mM and peak levels reaching as high as 44mM at 16 weeks of age (Hummel et al, 1972; Kobayashi et al, 2000). After 5-6 months of age, body weight and insulin levels begin to fall in association with pancreatic islet cell degeneration  13 (Hummel et al, 1972; Kobayashi et al, 2000). Thus, the db/db mouse is a useful model of type 2 DM, in that they are obese, hyperglycemic, and insulin-resistant (Chen et al, 1996; Kobayashi et al, 2000). However, after about 16 weeks of age, they progress to more frank diabetes, sometimes resembling insulin-dependent type 1 DM with a complete loss of pancreatic beta cell function. For a comprehensive review of all non-insulin-dependent animal models of DM, including the db/db mouse model, see reference (McIntosh & Pederson, 1999). Compared to the db/db mouse that is severely diabetic at 8 weeks of age, other models of obesity and type 2 diabetes have a much milder insulin resistance and full-blown diabetes does not occur until much later during the animals’ lifespan (McIntosh & Pederson, 1999).  1.7  db/db mouse and endothelial dysfunction It is important to note that endothelial dysfunction in the db/db mouse is not genetically mediated and instead occurs due to hyperglycemia (Miike et al, 2008b). Previous studies have investigated several aspects of endothelial dysfunction in db/db mouse as summarized below.  1) Increased oxidative stress in db/db mice is related to endothelial dysfunction. For example, a link between AGEs / RAGEs, TNFα, increased oxidative stress and endothelial dysfunction in db/db mice has been explained (Gao et al, 2008; Su et al, 2008; Zhang et al, 2008; Gao et al, 2007). Also CYP-450 monooxygenase enzymes may play a role in generation of excessive free oxygen radicals in db/db mice leading to EC dysfunction (Elmi et al, 2008).  2) eNOS protein in db/db mice could be deficient secondary to oxidative stress (Srinivasan et al, 2004) or a deficiency of the eNOS cofactor BH4 could be the cause of endothelial dysfunction in db/db mice (Pannirselvam et al, 2003b; Pannirselvam et al, 2002).  14  3) Dysfunction of ACh receptors and/or receptor G-protein coupling probably leads to impaired endothelium-dependent relaxation in db/db mice arteries (Miike et al, 2008a).  4) Caveolae deformity related to an enhanced expression of caveolin-1 mRNA and protein may impair endothelium-dependent relaxation in db/db mice (Lam et al, 2006).  In light of the significance of prior investigations in db/db mouse, endothelial dysfunction is well established in this model of type 2 diabetes. As discussed earlier, Nishikawa et al. showed that hyperglycemia is the cornerstone of endothelial dysfunction in diabetes (Nishikawa et al, 2000). At the same time, in db/db mice compared with other animal models of type 2 diabetes hyperglycemia is more advanced and occurs much earlier (McIntosh & Pederson, 1999). Therefore, it is expected that this model (compared to other animal models of type 2 diabetes) has a more advanced endothelial dysfunction at an earlier stage of life. If exercise can reverse endothelial dysfunction in this advanced model of diabetes with severe endothelial dysfunction, it will most likely also be beneficial in other models of diabetes. Therefore, the db/db mouse was used in this study to assess the effect of exercise on endothelial dysfunction in type 2 diabetes.  1.8  HYPOTHESIS Exercise, independent of weight loss and glycemic control, prevents endothelial dysfunction in type 2 diabetes via a decrease in oxidative stress and increased antioxidant defense mechanisms.     15 1.8.1 Objectives 1) To assess the effect of exercise with and without significant weight loss on prevention of endothelial dysfunction in aortae of db/db mice and in particular the effect of exercise on NO bioavailability, oxidative stress and SOD subtype expression/ activity in the aortae.  2) To explore the effect of exercise on coronary artery myogenic tone and endothelial dysfunction in db/db mice and in particular to assess the effect of exercise on NO bioavailability, oxidative stress and antioxidant defense in coronary arteries.  3) To investigate the mechanisms behind over-constriction in response to adrenergic stimulation in db/db aortae and to evaluate the effect of exercise on the constriction response.  1.9  Project road map For this study we used 5-week-old db/db and WT mice that were divided into subgroups of sedentary or exercised for 7 weeks and were euthanized at 12 weeks of age as seen in Fig 1-4. After euthanization, we assessed endothelial cell function, in aorta and coronary arteries. We also evaluated oxidative stress and antioxidant defense in arteries and plasma of mice. Aortic smooth muscle response to adrenergic stimulation and coronary myogenic tone were also assessed.  16  Figure 1-1: Mechanisms of vasculopathy in DM. In DM, increased oxidative stress is the cornerstone of ETC dysfunction that decreases NO and endothelium derived hyperpolarizing factor (EDHF) and eventually leads to vascular dysfunction. A variety of mechanisms contribute to increased oxidative stress in DM including the involvement of mitochondria, cytochrome P450, NADPH oxidase, xanthine oxidase, lipoxygenase, cyclooxygenase, PKC, polyol pathway, advanced glycated end products (AGEs), and hexosamine pathway. Also in type 2 DM, increased abdominal fat mass is an inflammatory source that increases oxidative stress. Moreover, in DM activated rennin/ angiotensin system and decreased levels of antioxidants contribute to higher oxidative stress. Mechanisms other than increased oxidative stress that are involved in ETC dysfunction include increased blood lipids as well as decreased number and/or viability of endothelial progenitor cells (EPCs) used for blood vessel repair. Hyperglycemia also decreases endothelial nitric oxide synthase (eNOS) activity directly or via increasing asymmetric dimethylmethylarginine (ADMA), an endogenous inhibitor of eNOS, that increases in high oxidative stress secondary to hyperglycemia.  17 L-Citrulline eNOS O2 NO O2 OONO - NADPH, Ca++ L-Arg ↑SOD O2 1 2 BH4 3 * Exercise *   Figure 1-2: Schematic model for NO degradation by oxygen free radicals (O2•) in diabetes and the suggested preventive role of exercise. Increased O2• in db/db mice degrades NO to peroxynitrite (ONOO-) (larger shock arrow and yellow arrow). Peroxynitrite degrades BH4 and uncouples eNOS. eNOS then consumes L-Arg to generate extra O2• (black curved arrow) and further generates ONOO- from NO (smaller shock arrow and yellow arrow). By increasing SOD, exercise may help to remove oxygen free radicals (1) and prevent NO conversion to ONOO- (2). It can also prevent eNOS uncoupling secondary to BH4 depletion by ONOO- (3). This model explains how incubation of diabetic aortae with SOD in vitro may produce the same effects. Finally, it describes how adding L-Arg plus BH4 to the in vitro environment can prevent eNOS uncoupling and increase NO concentration. Exercise can upregulate SOD and therefore increase NO bioavailability.   18  O2* H2O2 H2O + O2 Catalase GSH & GSSG NADPH & NADP MDA & DASA & ASA Other Antioxidants SOD   Figure 1-3: The antioxidant defense mechanism pathways. SOD is the first line of defense against superoxide and therefore plays a key role in this process. Catalase and other antioxidants that are shown below the dotted line can help after superoxide is converted to hydrogen peroxide by SOD. (MDA: malondialdehyde, DAsA: dehydroascorbate, AsA: ascorbate, GSH: glutathione, GSSG: oxidized glutathione).  19 WT 40 db/db 40 80    Mice 5 W/O Exercised 20 Sedentary 20 Exercised 20 Sedentary 20 Euthanized @ 12 W/O 7 weeks  Figure 1-4: Algorithm indicating animal groups used in this study. Mice were exercised with low (3.6 m/ min) or moderate (5.2 m/ min) intensity protocols.  20 1.10 Bibliography  Abou-Seif,M.A. & Youssef,A.A. (2004) Evaluation of some biochemical changes in diabetic patients. Clin.Chim.Acta, 346, 161-170. Adachi,T., Nakamura,M., Yamada,H., Futenma,A., Kato,K., & Hirano,K. (1994) Quantitative and qualitative changes of extracellular-superoxide dismutase in patients with various diseases. Clin.Chim.Acta, 229, 123-131. Andrews,K.L., Pannirselvam,M., Anderson,T.J., Jenkins,A.J., Triggle,C.R., & Hill,M.A. (2005) The vascular endothelium in diabetes: a practical target for drug treatment? Expert.Opin.Ther.Targets., 9, 101-117. Arrigo,A.P. (1999) Gene expression and the thiol redox state. Free Radic.Biol.Med., 27, 936-944. Bagi,Z., Koller,A., & Kaley,G. (2003) Superoxide-NO interaction decreases flow- and agonist-induced dilations of coronary arterioles in Type 2 diabetes mellitus. Am.J.Physiol Heart Circ.Physiol, 285, H1404-H1410. Beckman,J.S. & Koppenol,W.H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am.J Physiol, 271, C1424-C1437. Blanc,A., Pandey,N.R., & Srivastava,A.K. (2003) Synchronous activation of ERK 1/2, p38mapk and PKB/Akt signaling by H2O2 in vascular smooth muscle cells: potential involvement in vascular disease (review). Int.J Mol.Med., 11, 229-234. Brownlee,M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813-820. Brownlee,M. (2005) The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 54, 1615-1625. Canadian Diabetes Association. The History of Diabetes. Canadian Diabetes Association . 2007a. Ref Type: Electronic Citation  Canadian Diabetes Association. The prevalence and costs of diabetes. Canadian Diabetes Association . 2007b.Ref Type: Electronic Citation  Carlsson,L.M., Jonsson,J., Edlund,T., & Marklund,S.L. (1995) Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia. Proc.Natl.Acad.Sci.U.S.A, 92, 6264-6268.  Chang,S.P., Chen,Y.H., Chang,W.C., Liu,I.M., & Cheng,J.T. (2004) Increase of anti-oxidation by exercise in the liver of obese Zucker rats. Clin.Exp.Pharmacol. Physiol, 31, 506-511. Chaytor,A.T., Edwards,D.H., Bakker,L.M., & Griffith,T.M. (2003) Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries. Proc.Natl.Acad.Sci.U.S.A, 100, 15212-15217.  21 Chen,H., Charlat,O., Tartaglia,L.A., Woolf,E.A., Weng,X., Ellis,S.J., Lakey,N.D., Culpepper,J., Moore,K.J., Breitbart,R.E., Duyk,G.M., Tepper,R.I., & Morgenstern,J.P. (1996) Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell, 84, 491-495. Chua,S.C., Jr., Chung,W.K., Wu-Peng,X.S., Zhang,Y., Liu,S.M., Tartaglia,L., & Leibel,R.L. (1996) Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science, 271, 994-996. Coleman,D.L. (1978) Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia, 14, 141-148. Coleman,D.L. (1982) Diabetes-obesity syndromes in mice. Diabetes, 31, 1-6. Cosentino,F., Hishikawa,K., Katusic,Z.S., & Luscher,T.F. (1997) High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation, 96, 25-28. Darley-Usmar,V., Wiseman,H., & Halliwell,B. (1995) Nitric oxide and oxygen radicals: a question of balance. FEBS Lett., 369, 131-135. Dimmeler,S., Hermann,C., Galle,J., & Zeiher,A.M. (1999) Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler.Thromb.Vasc.Biol., 19, 656-664. Du,X., Matsumura,T., Edelstein,D., Rossetti,L., Zsengeller,Z., Szabo,C., & Brownlee,M. (2003) Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J.Clin.Invest, 112, 1049-1057. Du,X.L., Edelstein,D., Rossetti,L., Fantus,I.G., Goldberg,H., Ziyadeh,F., Wu,J., & Brownlee,M. (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc.Natl.Acad.Sci.U.S.A, 97, 12222-12226. Elmi,S., Sallam,N.A., Rahman,M.M., Teng,X., Hunter,A.L., Moien-Afshari,F., Khazaei,M., Granville,D.J., & Laher,I. (2008) Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice. Vascul.Pharmacol., 48, 1-8. Engerman,R.L., Kern,T.S., & Larson,M.E. (1994) Nerve conduction and aldose reductase inhibition during 5 years of diabetes or galactosaemia in dogs. Diabetologia, 37, 141-144. Faraci,F.M. & Didion,S.P. (2004) Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler.Thromb.Vasc.Biol., 24, 1367-1373. Farooqi,I.S., Jebb,S.A., Langmack,G., Lawrence,E., Cheetham,C.H., Prentice,A.M., Hughes,I.A., McCamish,M.A., & O'Rahilly,S. (1999) Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N.Engl.J.Med., 341, 879-884. Flekac,M., Skrha,J., Hilgertova,J., Lacinova,Z., & Jarolimkova,M. (2008) Gene polymorphisms of superoxide dismutases and catalase in diabetes mellitus. BMC.Med.Genet., 9, 30.  22 Forstermann,U. & Munzel,T. (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113, 1708-1714. Fuchsjager-Mayrl,G., Pleiner,J., Wiesinger,G.F., Sieder,A.E., Quittan,M., Nuhr,M.J., Francesconi,C., Seit,H.P., Francesconi,M., Schmetterer,L., & Wolzt,M. (2002) Exercise training improves vascular endothelial function in patients with type 1 diabetes. Diabetes Care, 25, 1795-1801. Fukai,T., Folz,R.J., Landmesser,U., & Harrison,D.G. (2002) Extracellular superoxide dismutase and cardiovascular disease. Cardiovasc.Res., 55, 239-249. Gao,X., Belmadani,S., Picchi,A., Xu,X., Potter,B.J., Tewari-Singh,N., Capobianco,S., Chilian,W.M., & Zhang,C. (2007) Tumor necrosis factor-alpha induces endothelial dysfunction in Lepr(db) mice. Circulation, 115, 245-254. Gao,X., Zhang,H., Schmidt,A.M., & Zhang,C. (2008) AGE/RAGE Produces Endothelial Dysfunction in Coronary Arterioles in Type II Diabetic Mice. Am.J Physiol Heart Circ.Physiol. Green,D.J., Walsh,J.H., Maiorana,A., Best,M.J., Taylor,R.R., & O'Driscoll,J.G. (2003) Exercise- induced improvement in endothelial dysfunction is not mediated by changes in CV risk factors: pooled analysis of diverse patient populations. Am.J Physiol Heart Circ.Physiol, 285, H2679- H2687. Grundy,S.M., Benjamin,I.J., Burke,G.L., Chait,A., Eckel,R.H., Howard,B.V., Mitch,W., Smith,S.C., Jr., & Sowers,J.R. (1999) Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation, 100, 1134-1146. Gryglewski,R.J., Palmer,R.M., & Moncada,S. (1986) Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature, 320, 454-456. Halcox,J.P., Schenke,W.H., Zalos,G., Mincemoyer,R., Prasad,A., Waclawiw,M.A., Nour,K.R., & Quyyumi,A.A. (2002) Prognostic value of coronary vascular endothelial dysfunction. Circulation, 106, 653-658. Hambrecht,R., Wolf,A., Gielen,S., Linke,A., Hofer,J., Erbs,S., Schoene,N., & Schuler,G. (2000) Effect of exercise on coronary endothelial function in patients with coronary artery disease. N.Engl.J Med., 342, 454-460. Hammes,H.P., Martin,S., Federlin,K., Geisen,K., & Brownlee,M. (1991) Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc.Natl.Acad.Sci.U.S.A, 88, 11555-11558. Harris,M. (2004) Definition and Classification of Diabetes Mellitus and the criteria for diagnosis. Diabetes Mellitus: A Fundamental and Clinical Text (ed. by D. LeRoith, S. I. Taylor, & J. M. Olefsky), pp. 457-467. Lippincott Williams & Wilkins, Philadelphia. Hawkins,M., Barzilai,N., Liu,R., Hu,M., Chen,W., & Rossetti,L. (1997) Role of the glucosamine pathway in fat-induced insulin resistance. J Clin.Invest, 99, 2173-2182.  23 Heilig,C.W., Concepcion,L.A., Riser,B.L., Freytag,S.O., Zhu,M., & Cortes,P. (1995) Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype. J.Clin.Invest, 96, 1802-1814. Herberg,L. & Coleman,D.L. (1977) Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism, 26, 59-99. Hink,U., Li,H., Mollnau,H., Oelze,M., Matheis,E., Hartmann,M., Skatchkov,M., Thaiss,F., Stahl,R.A., Warnholtz,A., Meinertz,T., Griendling,K., Harrison,D.G., Forstermann,U., & Munzel,T. (2001) Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ.Res., 88, E14-E22. Hsueh,W.A. & Quinones,M.J. (2003) Role of endothelial dysfunction in insulin resistance. Am.J Cardiol., 92, 10J-17J.  Hummel,K.P., Coleman,D.L., & Lane,P.W. (1972) The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL-KsJ and C57BL-6J strains. Biochem.Genet., 7, 1-13. Inoue,N., Ramasamy,S., Fukai,T., Nerem,R.M., & Harrison,D.G. (1996a) Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells. Circ.Res., 79, 32- 37. Jensen,T., Bjerre-Knudsen,J., Feldt-Rasmussen,B., & Deckert,T. (1989) Features of endothelial dysfunction in early diabetic nephropathy. Lancet, 1, 461-463. Jolliffe,J.A., Rees,K., Taylor,R.S., Thompson,D., Oldridge,N., & Ebrahim,S. (2001) Exercise-based rehabilitation for coronary heart disease. Cochrane.Database.Syst.Rev., CD001800. Jung,O., Marklund,S.L., Geiger,H., Pedrazzini,T., Busse,R., & Brandes,R.P. (2003) Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice. Circ.Res., 93, 622-629. Kaiser,N., Sasson,S., Feener,E.P., Boukobza-Vardi,N., Higashi,S., Moller,D.E., Davidheiser,S., Przybylski,R.J., & King,G.L. (1993) Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes, 42, 80-89. Kamata,K. & Kobayashi,T. (1996) Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes. Br.J Pharmacol., 119, 583-589. Katusic,Z.S. (2001) Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am.J Physiol Heart Circ.Physiol, 281, H981-H986. Kingwell,B.A., Tran,B., Cameron,J.D., Jennings,G.L., & Dart,A.M. (1996) Enhanced vasodilation to acetylcholine in athletes is associated with lower plasma cholesterol. Am.J Physiol, 270, H2008-H2013. Kobayashi,K., Forte,T.M., Taniguchi,S., Ishida,B.Y., Oka,K., & Chan,L. (2000) The db/db mouse, a model for diabetic dyslipidemia: molecular characterization and effects of Western diet feeding. Metabolism, 49, 22-31.  24 Kolm-Litty,V., Sauer,U., Nerlich,A., Lehmann,R., & Schleicher,E.D. (1998) High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells. J Clin.Invest, 101, 160-169. Korshunov,S.S., Skulachev,V.P., & Starkov,A.A. (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett., 416, 15-18. Lam,T.Y., Seto,S.W., Lau,Y.M., Au,L.S., Kwan,Y.W., Ngai,S.M., & Tsui,K.W. (2006) Impairment of the vascular relaxation and differential expression of caveolin-1 of the aorta of diabetic +db/+db mice. Eur.J Pharmacol., 546, 134-141. Lee,A.Y. & Chung,S.S. (1999) Contributions of polyol pathway to oxidative stress in diabetic cataract. FASEB J, 13, 23-30. Lee,G.H., Proenca,R., Montez,J.M., Carroll,K.M., Darvishzadeh,J.G., Lee,J.I., & Friedman,J.M. (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature, 379, 632-635. Leibel,R.L., Chung,W.K., & Chua,S.C., Jr. (1997) The molecular genetics of rodent single gene obesities. J Biol.Chem., 272, 31937-31940. Licinio,J., Caglayan,S., Ozata,M., Yildiz,B.O., de Miranda,P.B., O'Kirwan,F., Whitby,R., Liang,L., Cohen,P., Bhasin,S., Krauss,R.M., Veldhuis,J.D., Wagner,A.J., DePaoli,A.M., McCann,S.M., & Wong,M.L. (2004) Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptin-deficient adults. Proc.Natl.Acad.Sci.U.S.A, 101, 4531-4536. Lui,A.H., McManus,B.M., & Laher,I. (2000) Endothelial and myogenic regulation of coronary artery tone in the mouse. Eur.J Pharmacol., 410, 25-31. MacCracken,J. & Hoel,D. (1997) From ants to analogues. Puzzles and promises in diabetes management. Postgrad.Med., 101, 138-5, 149. Maejima,K., Nakano,S., Himeno,M., Tsuda,S., Makiishi,H., Ito,T., Nakagawa,A., Kigoshi,T., Ishibashi,T., Nishio,M., & Uchida,K. (2001) Increased basal levels of plasma nitric oxide in Type 2 diabetic subjects. Relationship to microvascular complications. J.Diabetes Complications, 15, 135-143. Marklund,S.L. (1982) Human copper-containing superoxide dismutase of high molecular weight. Proc.Natl.Acad.Sci.U.S.A, 79, 7634-7638. Marklund,S.L. (1984) Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem.J., 222, 649-655. Marklund,S.L., Bjelle,A., & Elmqvist,L.G. (1986) Superoxide dismutase isoenzymes of the synovial fluid in rheumatoid arthritis and in reactive arthritides. Ann.Rheum.Dis., 45, 847-851. Marletta,M.A. (1993) Nitric oxide synthase structure and mechanism. J.Biol.Chem., 268, 12231-12234.  25 Marshall,S., Bacote,V., & Traxinger,R.R. (1991) Discovery of a metabolic pathway mediating glucose- induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. J Biol.Chem., 266, 4706-4712. Matoba,T. & Shimokawa,H. (2003) Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Pharmacol.Sci., 92, 1-6. McGavock,J.M., Eves,N.D., Mandic,S., Glenn,N.M., Quinney,H.A., & Haykowsky,M.J. (2004) The role of exercise in the treatment of cardiovascular disease associated with type 2 diabetes mellitus. Sports Med., 34, 27-48. McIntosh,C.H.S. & Pederson,R.A. (1999) Noninsulin-Dependent Animal Models of Diabetes Mellitus. Experimental Models of Diabetes (ed. by J. H. McNeill), pp. 337-398. CRC Press, Washington, DC. Miike,T., Kunishiro,K., Kanda,M., Azukizawa,S., Kurahashi,K., & Shirahase,H. (2008a) Impairment of endothelium-dependent ACh-induced relaxation in aorta of diabetic db/db mice-possible dysfunction of receptor and/or receptor-G protein coupling. Naunyn Schmiedebergs Arch.Pharmacol., 377, 401-410. Milstien,S. & Katusic,Z. (1999) Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem.Biophys.Res.Commun., 263, 681-684. Minami,A., Ishimura,N., Harada,N., Sakamoto,S., Niwa,Y., & Nakaya,Y. (2002) Exercise training improves acetylcholine-induced endothelium-dependent hyperpolarization in type 2 diabetic rats, Otsuka Long-Evans Tokushima fatty rats. Atherosclerosis, 162, 85-92. Montague,C.T., Farooqi,I.S., Whitehead,J.P., Soos,M.A., Rau,H., Wareham,N.J., Sewter,C.P., Digby,J.E., Mohammed,S.N., Hurst,J.A., Cheetham,C.H., Earley,A.R., Barnett,A.H., Prins,J.B., & O'Rahilly,S. (1997) Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature, 387, 903-908. Mulvany,M.J. & Halpern,W. (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ.Res., 41, 19-26. Myers,J., Prakash,M., Froelicher,V., Do,D., Partington,S., & Atwood,J.E. (2002) Exercise capacity and mortality among men referred for exercise testing. N.Engl.J Med., 346, 793-801. Nakamura,S., Makita,Z., Ishikawa,S., Yasumura,K., Fujii,W., Yanagisawa,K., Kawata,T., & Koike,T. (1997) Progression of nephropathy in spontaneous diabetic rats is prevented by OPB-9195, a novel inhibitor of advanced glycation. Diabetes, 46, 895-899. Nesto,R.W. (2004) Correlation between cardiovascular disease and diabetes mellitus: current concepts. Am.J Med., 116 Suppl 5A, 11S-22S. Nishikawa,T., Edelstein,D., Du,X.L., Yamagishi,S., Matsumura,T., Kaneda,Y., Yorek,M.A., Beebe,D., Oates,P.J., Hammes,H.P., Giardino,I., & Brownlee,M. (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 404, 787- 790.  26 Nobe,K., Suzuki,H., Sakai,Y., Nobe,H., Paul,R.J., & Momose,K. (2004) Glucose-dependent enhancement of spontaneous phasic contraction is suppressed in diabetic mouse portal vein: association with diacylglycerol-protein kinase C pathway. J.Pharmacol.Exp.Ther., 309, 1263- 1272. Okon,E.B., Szado,T., Laher,I., McManus,B., & van Breemen,C. (2003) Augmented contractile response of vascular smooth muscle in a diabetic mouse model. J.Vasc.Res., 40, 520-530. Paffenbarger,R.S., Jr., Hyde,R.T., Hsieh,C.C., & Wing,A.L. (1986) Physical activity, other life-style patterns, cardiovascular disease and longevity. Acta Med.Scand.Suppl, 711, 85-91. Pannirselvam,M., Anderson,T.J., & Triggle,C.R. (2003a) Endothelial cell dysfunction in type I and II diabetes: The cellular basis for dysfunction. Drug Development Research, 58, 28-41. Pannirselvam,M., Simon,V., Verma,S., Anderson,T., & Triggle,C.R. (2003b) Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br.J Pharmacol., 140, 701-706. Pannirselvam,M., Verma,S., Anderson,T.J., & Triggle,C.R. (2002) Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br.J Pharmacol., 136, 255-263. Pieper,G.M. (1998) Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension, 31, 1047-1060. Pieper,G.M. & Dondlinger,L.A. (1997) Plasma and vascular tissue arginine are decreased in diabetes: acute arginine supplementation restores endothelium-dependent relaxation by augmenting cGMP production. J Pharmacol.Exp.Ther., 283, 684-691. Pieper,G.M., Siebeneich,W., & Dondlinger,L.A. (1996) Short-term oral administration of L-arginine reverses defective endothelium-dependent relaxation and cGMP generation in diabetes. Eur.J Pharmacol., 317, 317-320. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (1997). Diabetes Care, 20, 1183-1197. Report of the expert committee on the diagnosis and classification of diabetes mellitus   (2003). Diabetes Care, 26 Suppl 1, S5-20. Sesso,H.D., Paffenbarger,R.S., Jr., & Lee,I.M. (2000) Physical activity and coronary heart disease in men: The Harvard Alumni Health Study. Circulation, 102, 975-980. Soro-Paavonen,A. & Forbes,J.M. (2006) Novel therapeutics for diabetic micro- and macrovascular complications. Curr.Med.Chem., 13, 1777-1788. Soulis-Liparota,T., Cooper,M., Papazoglou,D., Clarke,B., & Jerums,G. (1991) Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes, 40, 1328-1334.  27 Srinivasan,S., Hatley,M.E., Bolick,D.T., Palmer,L.A., Edelstein,D., Brownlee,M., & Hedrick,C.C. (2004) Hyperglycemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia, 47, 1727-1734. Su,J., Lucchesi,P.A., Gonzalez-Villalobos,R.A., Palen,D.I., Rezk,B.M., Suzuki,Y., Boulares,H.A., & Matrougui,K. (2008) Role of Advanced Glycation End Products With Oxidative Stress in Resistance Artery Dysfunction in Type 2 Diabetic Mice. Arterioscler.Thromb.Vasc.Biol. Tan,R.J., Lee,J.S., Manni,M.L., Fattman,C.L., Tobolewski,J.M., Zheng,M., Kolls,J.K., Martin,T.R., & Oury,T.D. (2006) Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury. Am.J.Respir.Cell Mol.Biol., 34, 226-232. Tartaglia,L.A., Dembski,M., Weng,X., Deng,N., Culpepper,J., Devos,R., Richards,G.J., Campfield,L.A., Clark,F.T., Deeds,J., & . (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell, 83, 1263-1271. Tuck,M.L. (2003) Nitric oxide in diabetes mellitus. J.Hypertens., 21, 1081-1083. Uchimura,K., Nagasaka,A., Hayashi,R., Makino,M., Nagata,M., Kakizawa,H., Kobayashi,T., Fujiwara,K., Kato,T., Iwase,K., Shinohara,R., Kato,K., & Itoh,M. (1999) Changes in superoxide dismutase activities and concentrations and myeloperoxidase activities in leukocytes from patients with diabetes mellitus. J.Diabetes Complications, 13, 264-270. UK Prospective Diabetes Study (UKPDS) Group (1998). Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet, 352, 837-853. Wang,S., Shiva,S., Poczatek,M.H., Darley-Usmar,V., & Murphy-Ullrich,J.E. (2002) Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells. J.Biol.Chem., 277, 9880-9888. World Health Organization. Diabetes fact sheet N312. World Health Organization . 2006. Ref Type: Electronic Citation  World Health Organization (1980). Second report of the WHO expert committee on diabetes mellitus. 646. 1980. Geneva, Switzerland. Zhang,C., Park,Y., Picchi,A., & Potter,B.J. (2008) Maturation-induces endothelial dysfunction via vascular inflammation in diabetic mice. Basic Res.Cardiol. Zou,M.H., Shi,C., & Cohen,R.A. (2002) Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin.Invest, 109, 817-826.  28 2 EXERCISE RESTORES AORTIC ENDOTHELIAL FUNCTION INDEPENDENT OF WEIGHT LOSS OR HYPERGLYCAEMIC STATUS IN db/db MICE 1           1  A version of this chapter has been published. Moien-Afshari F, Ghosh S, Elmi S, Rahman MM, Sallam N, Khazaei M, Kieffer TJ, Brownsey RW, Laher I. Diabetologia. 2008; 51(7):1327-37.  29 2.1 Introduction Cardiovascular disease is the leading cause of mortality in patients with diabetes (Laakso, 1999). Vascular abnormalities in these patients may be partly due to endothelial dysfunction, leading to atherosclerosis, hypertension, and hypercoagulability (Vinik et al, 2001). Although the exact molecular mechanisms are unclear, changes in lifestyle are routinely advocated for the management of type 2 diabetes (Hambrecht et al, 2000).  In diabetes, exercise induced improvements in vascular function are believed to be secondary to lowering of metabolic risk factors such as plasma lipids, or blood glucose (Kingwell et al, 1996b) or oxidative stress biomarkers (Chang et al, 2004). Regarding oxidative stress, reactive oxygen species (ROS) such as superoxide react with nitric oxide (NO) causing a loss of NO bioavailability (Kojda & Hambrecht, 2005). Such reactions can lead to the formation of peroxynitrite, a potent oxidant that causes irreversible oxidative modifications to the endothelium and ultimately cell death (Pacher & Szabo, 2006). Superoxide dismutases (SODs) are endogenous antioxidants, which compete with NO for superoxides and neutralise them.  Since NO is a relatively stable highly diffusible molecule, relative distribution of cellular SOD isoforms is important in determining the extent and location of peroxynitrite-induced oxidative damage in blood vessels (Cooke & Davidge, 2003; Kavdia, 2006). In addition to SODs, catalase is another endogenous antioxidant able to protect blood vessels from hydrogen peroxide-mediated oxidative stress in diabetes (Erdei et al, 2007). However, the relative importance of different intracellular SOD isoforms or catalase during diabetes remains unclear. Whereas mitochondrial free radicals and mitochondrial manganese SOD (Mn-SOD) may be important regulators of oxidative stress in the diabetic heart and endothelial cells (Ghosh et al, 2005; Nishikawa et al, 2000; Shen et al, 2006), Cu,Zn-SOD, the cytosolic isoform accounts for approximately 50% to 80% of total SOD activity in blood vessels (Didion et al, 2002; Fukai et al, 2000).  Based on previous findings in humans and animals, we hypothesized that exercise decreases oxidative stress independent  30 of body weight, or metabolic parameters and via increasing the expression of antioxidant enzymes in the aorta of db/db mice, a routinely used murine model of obesity and type 2 diabetes.  2.2 Methods 2.2.1 Animals and exercise regimen, boday weight, food and water consumption Protocols were designed in accordance with the University of British Columbia Animal Care Committee Guidelines. We randomly grouped 5-week-old male db/db (BKS.cg-m +/+ Leprdb/J) and wild-type (WT) mice (Jackson Laboratory, Bar Harbor, ME, USA) in sedentary (no-exercise), low- intensity exercise and moderate exercise groups (n= 8-10 per group). Body weights were recorded weekly. Mice assigned to exercise protocols were trained to run on a motorized exercise wheel system (Lafayette Instrument Co, Indiana, USA). To allow for animal acclimatization, exercise intensity was gradually increased over the first two weeks till a target of 1 h of daily exercise at a speed of 3.6 m/min in the low-intensity, or 5.2 m/min in the moderate intensity group was achieved.  For the duration of the 7-week training period, mice were exercised daily at a set time each day for 5 days a week. Sedentary db/db or WT mice were placed in non-rotating wheels for the same duration.  The experimental protocol was terminated when the mice reached 12 weeks of age. In 12-month-old mice, VO2max for the low-intensity and moderate-intensity exercise protocols used in this study are estimated to be 54% and 64% respectively (Schefer & Talan, 1996). In humans VO2max of 40-59% represents moderate-intensity exercise and VO2max of 60-84% represents vigorous exercise (Gormley et al, 2008). Boday weight was measured initially and at weekly thereafter. Food and water consumtion was measured in all groups as described in Appendix A.  2.2.2 Estimation of body fat using nuclear magnetic resonance (NMR) At 12 weeks of age, whole body fat measurements were made using a 7 tesla animal MRI scanner  31 (Bruker, Germany). The MRI scan was performed at the University of British Columbia MRI Research Center. The NMR signal from the entire body was acquired with a quadrature volume radio-frequency coil tuned to 300MHz. A standard Carr Purcell Meiboom Gill (CPMG) sequence (TE = 2.377 ms, TR = 10 s) was used to acquire 256 echoes from which the time 2 (T2) decay curve was extracted. The decay curves were fitted to a double exponential function using a software procedure developed in house with Igor (Wave-Metrics, Portland, OR, USA). The component corresponding to T2 ≈ 40 ms was identified as water in lean tissue, and the T2 ≈ 200 ms component was identified as body fat (Strader et al, 2004). The diffusion coefficient (dc) shift of the double exponential function was identified as a “free” water component corresponding to body fluids, e.g. urine and cerebrospinal fluid, with typical amounts of less than 5% of the total signal. The ratio of lean tissue: body fat expressed as weight: weight was calculated from the NMR data (Greig et al, 1999).  2.2.3 Collection of blood and tissue samples Animals were anesthetised with pentobarbital (50 mg/kg, i.p.) combined with heparin (50 U/kg) and blood samples were taken from the inferior vena cava by a 5ml syringe. A portion of each blood sample was separated in Eppendorf tubes for use in the 8-isoprostane enzyme immunoassay (see below). The remaining blood was immediately centrifuged (10 min at 4°C, 1,000g) in 1.5 ml Eppendorf tubes to separate plasma, which was stored at -76°C for later analysis. The animals were euthanized by cutting the abdominal aorta after collection of blood samples. Thoracic aortae were removed, placed in ice-cold physiologic salt solution (PSS) and then dissected and cleaned of connective tissue. Part of the aorta was immediately assayed for nitrite levels, whereas other pieces were snap-frozen in liquid nitrogen and stored at -76°C for western blotting analyses and protein carbonyl analyses under basal conditions.    32 2.2.4 Measurement of plasma parameters Plasma glucose levels were measured by Trinder Assay using a commercial kit (Diagnostic Chemicals, Oxford, CT, USA). Insulin levels were determined in plasma using an assay kit (Mercodia Ultrasensitive Mouse Insulin Assay; Alpco, Salem, NH, USA). Photometric measurements were performed by Dimension® Clinical Chemistry System (GMI, Ramsey, MN, USA) to obtain plasma triacyglycerol, total cholesterol, and HDL-cholesterol levels.  LDL-cholesterol was then calculated by the following formula: LDL-cholesterol = total cholesterol – (HDL-cholesterol + triacylglycerol/ 2.2).  2.2.5 Oral glucose tolerance test (OGTT) Mice underwent an OGTT when they reached 10 weeks of age (i.e. after 5 weeks of exercise).  After 6 h of fasting, mice were glucose loaded (1.5 g/kg) with a 40% glucose solution by oral gavage. Blood samples were taken at times 0, 10, 20, 60, and 120 min. Plasma was separated by centrifugation (1,000g) and stored at -76°C for later analyses of glucose and insulin. To reduce short-term treatment effects, animals were not exercised for 24 h prior to the OGTT.  2.2.6  Isometric force measurement Ring segments of aorta were threaded with stainless steel wire (0.02 mm diameter) and attached to the tissue holders of a four-channel wire myograph (JP Trading, Aarhus, Denmark). Tissues were allowed to equilibrate for 60 min at 37°C, during which time the PSS was replaced at 20-min intervals. During the equilibration, the resting tension was gradually increased to 5 mN and remained at this resting tension for 20 to 30 min. Each tissue was maximally activated with a solution of KCl (80 mmol/l) that was prepared by equimolar substitution of NaCl in PSS. Following washout with fresh PSS and return of tension to basal preload, phenylephrine (1 µmol/l) was added to establish a stable contraction. Thereafter, cumulative additions of acetylcholine (ACh) (1 nmol/l to 10 µmol/l) were made. After  33 washout, the ACh concentration-response curve was repeated in the presence of SOD (150 U/ml) or L- arginine (L-Arg) (103 µmol/l) plus tetrahydrobiopterin (BH4) (10 µmol/l). The same protocol was repeated for sodium nitroprusside (SNP) (1 nmol/l to 10 µmol/l) after washout. Vasodilatory responses were recorded on a computer using MyoDaq Acquisition software (v. 2.01, Danish MyoTechnology, Aarhus, Denmark) and expressed as per cent dilation of phenylephrine-induced constriction (Leung et al, 2007).  2.2.7 8-isoprostane enzyme immunoassay Just before the animals were killed, blood samples were collected in ice-cooled heparinized Eppendorf tubes, to which 10 mg butylated hydroxy toluene (BHT) was added. The tubes were immediately centrifuged (1,000g) and the plasma fraction separated and stored at -76°C until further use. Plasma levels of free 8-isoprostane were determined using a commercially available enzyme immunoassay kit (8-isoprostane EIA kit, Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s protocol.  2.2.8 Spectrophotometric quantification of tissue nitrite Pieces of aorta were incubated for 30 min in 0.5 ml Krebs-Henseleit solution at 37°C. Following the equilibration period, the solution was changed to 0.5 ml Krebs-Henseleit (37°C) containing ACh (10 µmol/l). After 5 min, 100 ml of the perfusate was mixed with 100 ml of Griess reagent from an assay kit (Calbiochem, San Diego, CA, USA). To convert all nitrate in the sample to nitrite, samples were treated with nitrate reductase and NADPH. Standards were prepared with sodium nitrite at concentrations ranging from 1 to 35 mmol/l.  Spectrophotometric measurements were made using a microplate reader set at 550 nm. The values were expressed per mg dry weights of the tissues.    34 2.2.9 Western Blot Thoracic aortae were initially cut into small pieces and homogenized in ice-cold homogenization buffer. The protein contents of the homogenates were then quantified using a Bradford protein assay. The homogenates were diluted and boiled with sample loading dye. Samples of this corresponding to 50 µg protein, were used in SDS-polyacrylamide gel electrophoresis. After transfer, nitrocellulose membranes were blocked overnight in 5% skimmed milk in Tris-buffered saline containing 0.1% (vol./vol.) Tween-20 (TBS-T).  Membranes were incubated for two hours at room temperature with antibodies raised in (1) rabbit (endothelial nitric oxide synthase [eNOS], phospho-eNOS [#SC654; #SC12972R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or catalase [#219010; Calbiochem, San Diego, CA, USA], sheep (Cu,Zn-SOD or Mn-SOD; #574597, #574596; Calbiochem or (2) mouse (nitrotyrosine; #189542; Cayman Chemicals).  Following triple washes in TBS-T, membranes were incubated for 2 h at room temperature with secondary goat anti-rabbit, and donkey anti-sheep or goat anti-mouse horseradish peroxidase-conjugated antibodies, and visualized using electrochemiluminescent detection kit. Values were expressed as arbitrary units per mg protein.  2.2.10 Protein Carbonyl levels Protein carbonyls were assayed as an index of oxidative modification of proteins.  Briefly, 50 µl of the plasma fractions were added to an equal volume of 10% (vol./vol.) trichloroacetic acid, centrifuged for 5 min at 6,000g and 4°C, and the supernatant fraction discarded.  The precipitated proteins were resuspended in 0.2% 2,4-dinitrophenyl hydrazine, and incubated for 1 h at 37°C.  Subsequently, proteins were precipitated again with trichloroacetic acid, centrifuged at 4,000g, washed with ethanol/ethyl acetate (1:1), dissolved in 6 mmol/l guanidine hydrochloride, and the absorbance measured spectrophotometrically at 370 nm (Dalle-Donne et al, 2006; Ghosh et al, 2005).   35 2.2.11 Statistical analysis and calculations Results are expressed as mean ± SEM.  Data were analyzed using NCSS-2000 (Kaysville, UT, USA) computer software. Repeated measures ANOVA with multiple comparisons using Bonferroni’s test or one-way ANOVA was performed where appropriate. GraphPad Prism (version 3.02-2000, San Diego, CA, USA) was used for linear regression, curve fitting and dose-response analysis. The results of statistical tests were considered significant if p values were less than 0.05.  2.3 Results 2.3.1 Influence of exercise on body weight and body fat The effects of exercise on body weight are shown in Figure 2-1a,b. Body weight in db/db mice were initially higher than in WT mice at 5 weeks of age and continued to increase to nearly twice that of WT mice at 12 weeks (Fig 2-1a). Low-intensity exercise did not influence body weight in db/db mice compared with non-exercised groups (Fig 2-1a).  In contrast, even after one week of moderate-intensity exercise, the weights of db/db mice were significantly (~10%) lower than those of sedentary db/db mice (Fig 2-1b). Neither exercise protocol affected body weight of WT mice (Fig 2-1a,b). When the mice were 12 weeks old, the lean to fat ratio was significantly higher in WT than db/db mice; neither of exercise protocols significantly affected the lean to fat ratio in the db/db mice (WT, 1.75±0.05 vs. db/db, 0.58±0.04, db/db low-intensity exercise, 0.6±0.03, db/db moderate-intensity exercise, 0.6±0.02, p<0.05). Food consumption was significantly higher in db/db mice compared to WT mice, however, within the db/db or WT groups significant differences in food comsumption between the exercised and sedentary mice were not identified (Appendix A). Water consumption was also significantly higher in db/db compared to WT mice. Exercise decreased the amount of water consumption in db/db mice from week 7 to 10, which was inversly related to the intensity of exercise (see Appendix A).   36 2.3.2 Influence of exercise on plasma lipid profile Plasma cholesterol, triacylglycerol, LDL-cholesterol and HDL-cholesterol were measured as indicators of whole-body lipid profile (Table 2-1). Triacylglycerol, cholesterol and LDL-cholesterol were elevated in sedentary db/db mice compared with sedentary WT mice. Neither low nor moderate- intensity exercise changed plasma lipids in the WT mice. However, exercise with both protocols decreased triacylglycerol in db/db mice. Moderate-intensity exercise also lowered total cholesterol and LDL-cholesterol, whereas low-intensity exercise increased HDL-cholesterol levels in db/db mice plasma (Table 2-1).  2.3.3 Influence of exercise on glycemic status Both plasma insulin and glucose values were elevated in db/db compared with WT mice.  Neither form of exercise altered these parameters in either db/db or WT mice (Table 2-1). As exercise has often been linked to improved whole body insulin resistance, an OGTT was performed after 5 weeks of exercise in db/db and WT mice (Fig 2-1c). Neither exercise regimen (low or moderate intensity) altered plasma glucose levels in db/db or WT mice within 120 min after an oral glucose load (Fig 2-1c).  2.3.4 Endothelium-dependent and -independent vasodilation following exercise To evaluate endothelial function, endothelium dependent and -independent vasodilation was evaluated using ACh and SNP respectively. The tracings illustrating tension (mN) of aortic rings from different mouse groups are depicted in Fig 2-2, while Fig 2-3 depicts ACh and SNP concentration-response curves in aortic rings preconstricted with phenylephrine with their Emax and EC50 values given in Table 2-2. The endothelium dependent vasodilation produced by ACh was impaired in aortic rings from db/db mice compared with WT counterparts (Figs 2-2a,b and 2-3a,c; Table 2-2). Exercising in db/db mice with either low or moderate intensity increased endothelium-dependent vasodilation (Figs 2-2c,d and 2-3a,c; Table 2-2). The EC50 for ACh-induced vasodilation was similar in all groups (Table 2-2).  37 Finally, endothelium-independent vasodilation induced by SNP was similar in db/db and WT mice and exercise did not alter this response in any of the experimental groups (Fig 2-3b,d). Low and moderate intensity exercise did not have a significant effect on ACh and SNP vasodilatory responses in aortic rings of WT mice (see appendix B, Fig 7-1 A and B).  2.3.5 Aortic nitric oxide bioavailability following exercise We used two strategies to investigate endothelial-dependent vasodilation in db/db mice.  First, we incubated aortic rings with SOD (150 U/ml), to improve NO availability by removing superoxide. Second, we incubated arteries with the nitric oxide synthase precursor, L-Arg (10-3 mol/l), combined with an eNOS cofactor, BH4 (10-5 mol/l), to augment NO production (Forstermann & Munzel, 2006). Vasodilation in response to ACh in sedentary db/db mice improved significantly with both SOD (Fig 2-4a) and L-Arg/BH4 supplementation (Fig 2-4b), implying the presence of increased superoxide and/or impaired NO production/availability in db/db mice. In contrast, neither SOD incubation nor L- Arg/BH4 altered ACh responses in exercised db/db mice (Fig 2-4c to f), suggesting that in these mice superoxide generation was decreased or endogenous SOD activity improved following exercise. Vasodilation in response to ACh in sedentary or exercised WT groups was not changed by SOD or L- Arg/BH4 (data not shown). Emax for ACh responses were significantly increased in db/db mice after incubation with SOD or L-Arg plus BH4. However, such treatment had no effect in exercised db/db mice (Table 2-3); the EC50 values did not change in any group before and after incubations with these cofactors (Table 2-3).  2.3.6 Changes in eNOS production and tissue nitrite levels following exercise We evaluated protein expression of eNOS and SOD isoforms under basal conditions (without treatment) in db/db aortae. Total eNOS protein was unchanged in sedentary db/db mice (compared with WT mice), but was upregulated to an equal extent by the exercise protocols (Fig 2-5a). Evaluation of  38 phosphorylated eNOS at Ser1177 (Dudzinski & Michel, 2007) revealed that diabetes decreases eNOS phosphorylation at Ser1177 in the aortae of sedentary db/db compared with those of WT mice (Fig 2- 5b); however, eNOS phosphorylation at Ser1177 was increased with both exercise intensities, with low-intensity exercise demonstrating a greater benefit.  When expressed as a ratio of the ‘active’ eNOS (phospho-eNOS) to total eNOS protein, sedentary db/db mice demonstrated decreased NOS activity, which was reversed by both exercise protocols (Fig 2-5c). Sedentary db/db mice also had lower plasma nitrite levels than WT mice. Exercise-induced tissue eNOS activation was associated with corresponding increases in aorta NO release, as measured by tissue nitrite levels, which increased by almost five- to six-fold with both forms of exercise (Fig 2-5d).  2.3.7 Differential regulation of intracellular antioxidants following exercise Catalase levels were unchanged in all WT and db/db mice groups (Fig 2-6a).  As shown in Figure 2-6b, low-intensity exercise caused an upregulation of Cu,Zn-SOD, the cytosolic SOD isoform, in aortae from diabetic mice.  On the other hand, MnSOD, the mitochondrial isoform, was decreased in db/db mice and was selectively upregulated by a moderate-intensity exercise regimen (Fig 2-6c).  2.3.8 Influence of exercise on whole body and tissue-specific oxidative stress Plasma free 8-isoprostane is formed by whole body lipid peroxidation reactions and tissue protein carbonyls are formed by protein oxidation.  Plasma free 8-isoprostane (Fig 2-7a) and aorta protein carbonyls (Fig 2-7b) were higher in db/db than in WT mice. Both low- and moderate-intensity exercise lowered the levels of plasma 8-isoprostane and aorta protein carbonyls in db/db mice to levels similar to those in WT mice. Nitrotyrosine (biomarker for peroxynitrite-induced protein nitration) immunostaining increased following diabetes and decreased maximally with moderate-intensity exercise (Fig 2-7c) (Pacher & Szabo, 2006).   39 2.4 Discussion It is estimated that approximately 70% of people with diabetes will die as a result of a vascular event (Wing et al, 2001). Endothelial dysfunction occurs early in diabetes and improving endothelial dysfunction is an important goal of therapeutic strategies to combat the disease (Vinik et al, 2001). Increased physical activity is routinely recommended in the management of human type 2 diabetes (Wing et al, 2001) and is believed to improve glycemic control and plasma lipids, while simultaneously decreasing insulin resistance and body weight (Boule et al, 2001).  Although loss of body weight is often an important consideration in life style changes, beneficial effects of exercise, especially on cardiovascular complications, can also occur independently of body weight loss as shown in this study and by others (Gregg et al, 2004).  Thus, the molecular mechanisms by which exercise is beneficial in type 2 diabetes remains largely unknown. There are several proposed mechanisms for the cardiovascular benefits of exercise in diabetes. Exercise improves cardiovascular function through decreasing risk factors such as plasma lipids, blood glucose, or body mass index (Boule et al, 2001; Gregg et al, 2004; Kingwell et al, 1996). However, although long-term vigorous exercise decreases some of these metabolic parameters, exercise is also known to improve vascular function without significantly altering those risk factors (Green et al, 2003). In our study, db/db mice exercised at low-intensity had improved vascular function without lowering of cholesterol, LDL-cholesterol, glucose or insulin levels.  However, low-intensity exercise did reduce circulating triacylglycerol levels, while at the same time increasing HDL-cholesterol levels, which may be related to improved vascular endothelial function in exercised mice (Kusterer et al, 1999) as it has been discussed before (Hausenloy & Yellon, 2008; Vinals et al, 1999).  Exercise-induced increases in vascular shear stress may also be an important stimulus for NO release in vivo (Gielen et al, 2001). Since we were able to record improved vascular function in isolated blood vessel segments where there was no shear stress, our data suggest a more enduring effect of chronic exercise.  Reductions in  40 hyperglycemia and insulin resistance, independently of body weight loss, have also been suggested to improve endothelial function and vasodilation (Nassis et al, 2005). In our study, following 5 weeks of exercise at different intensities, plasma insulin or glucose levels in a 2-h OGTT in db/db mice were not different from those in in sedentary animals.  OGTT was expected to improve by exercise as several adaptations secondary to exercise may alleviate insulin resistance in DM. For example, exercise helps decrease abdominal fat and loss of muscle mass that are highly associated with the development of insulin resistance. Exercise also increases skeletal muscle insulin action via potentiating insulin- regulatable glucose transporters, GLUT4, as well as enzymes responsible for the phosphorylation, storage and oxidation of glucose. Moreover, exercise changes muscle morphology by converting fast twitch glycolytic IIb fibers to fast twitch oxidative IIa fibers with a greater capillary density and more insulin-sensitivity thereby alleviating glucose tolerance (Ivy, 1997). The failure to improve insulin sensitivity has been shown in some previous studies in this model (Tang & Reed, 2001) and could be related to the exercise protocol used, since repeated short periods of daily exercise are more effective in reducing OGTT results than a longer period of exercise of the same duration every day (Eriksen et al, 2007). Blood vessels from diabetic animals and humans exhibit attenuated endothelium-dependent relaxation in response to ACh (Oyama et al, 1986).  Acetylcholine interacts with endothelial muscarinic M3 receptors and causes NO release and vasodilatation (Boulanger et al, 1994). It is possible that vascular dysfunction in diabetes is a result of impaired muscarinic receptor activity (Carrier & Aronstam, 1987). We show here that decreased endothelium-dependent vasodilation in db/db mice is unrelated to alterations in the receptor sensitivity to ACh. The decline, in the present study, of ACh Emax in the absence of changes in EC50 indicates that ACh receptor function remained relatively unaltered in diabetic mice. It is also unlikely that the activity of guanylate cyclase is altered in diabetes, as indicated by similar responses to SNP, an exogenous donor of NO (Williams et al, 1996) and by the fact that in human diabetes the vasodilator response to SNP remained intact even though there was a marked  41 decline in vasodilation in response to Ach (Maiorana et al, 2001). These the vasodilator response to SNP remained intact even though there was a marked decline in vasodilation in response to ACh (Maiorana et al, 2001). We (in this study) and others report that diabetes does not alter the total protein production of eNOS (Okon et al, 2003). However, some studies suggest a downregulation of eNOS activity during diabetes (Srinivasan et al, 2004).  Phosphorylation of eNOS is important for post-translational regulation of eNOS activity (Dimmeler et al, 1999; Dudzinski & Michel, 2007; Fulton et al, 1999). The activation of eNOS catalytic function by Ser1177 phosphorylation via Akt/PKB protein kinase inhibits the dissociation of calmodulin from eNOS and so increases the rate of eNOS electron transfer to produce NO (Dimmeler et al, 1999; Dudzinski & Michel, 2007; Fulton et al, 1999).  It has been previously shown that eNOS function is down regulated in DM by decreased phophorylation of Ser1177 (Du et al, 2001).In the present study, investigation of eNOS phosphorylation also revealed that following diabetes Ser1177 phosphorylation was decreased in the aortae of db/db mice, and a novel finding that an upregulation of phosphorylation levels occurred following exercise, suggesting greater eNOS activity without changes in body weight or glucose related parameters, which correlated well with aorta nitrite levels. Apart from eNOS expression and phosphorylation, decreased availability of the eNOS substrate L-Arg or cofactors such as BH4, also occurs in diabetes (Cai et al, 2005) and has been reported to be significantly lower in diabetic patients (Giugliano et al, 1997; Heitzer et al, 2000).  In our study, although direct measurements of L-Arg and BH4 were not performed, incubation with these agents potentiated the relaxation induced by ACh in aortae of sedentary db/db mice.  One of the major reasons for a lack of L-Arg and BH4, is increased peroxynitrite, a free radical and a strong oxidant, in the diabetic aortae. The formation of peroxynitrite occurs through a reaction between excess superoxide and NO, and takes place three times faster than the reaction between superoxide and its corresponding antioxidant, SOD (Pacher & Szabo, 2006). BH4 can be degraded directly by peroxynitrite (Forstermann  42 & Munzel, 2006). Additionally, BH4 deficiency and peroxynitrite can uncouple eNOS (Dudzinski & Michel, 2007).  Uncoupled eNOS consumes L-Arg to generate more superoxide instead of NO, forming a positive feedback loop and progressive loss of available NO.  In support of this hypothesis, we found increased levels of nitrotyrosine, a biomarker for peroxynitrite in the aortae of sedentary db/db mice. Three isoforms of SOD are currently known, but their relative role in protecting against hyperglycemia-induced vascular dysfunction is unclear.  In our study, following diabetes and various intensities of exercise, we found differential regulation of these SOD isoforms.  SOD-2 has manganese as cofactor, and is located mainly in the mitochondria, which it protects from oxidative damage (Faraci & Didion, 2004).  On the other hand, cytosolic Cu,Zn-SOD may counteract NADPH oxidase and xanthine oxidase activity, which are the prime generators of cytosolic superoxides (Faraci & Didion, 2004).  In this study, we observed a specific downregulation of aortic Mn-SOD following diabetes. Interestingly, although Cu,Zn-SOD did not change following diabetes as such, low-intensity exercise increased Cu,Zn-SOD protein production in db/db aortae.  Although the exact reasons for such an upregulation are unknown, it has been reported that during mild to moderate training exercise laminar blood flow increases shear stress and upregulates Cu,Zn-SOD (Inoue et al, 1996; Rush et al, 2003). Although whole-body 8-isoprostane, a marker of lipid peroxidation, decreased with low-intensity exercise, aorta nitrotyrosine and protein carbonyls were not affected by Cu,Zn-SOD upregulation. It may be speculated that being a cytosolic enzyme, Cu,Zn-SOD was unable to neutralize mitochondrial oxidative stress, which is the prime regulator of tissue peroxynitrite levels and protein oxidation in diabetes (Ghosh et al, 2006; Rush et al, 2003). However, with higher intensities of exercise, we report a specific upregulation of Mn-SOD, the mitochondrial SOD isoform. During higher intensities of exercise, whole-body and muscle oxygen consumptions increases up to 20- and 100-fold respectively (Wing et al, 2001; Meydani & Evans,  1993; Rush et al, 2003), leading to increased release of superoxide from the mitochondrial electron transport chain.  As Cu,Zn-SOD is downregulated with  43 increased exposure to oxygen (Hass & Massaro, 1987; Rush et al, 2003), upregulation of Mn-SOD under these conditions is common across various tissues (Chang et al, 2004; Hollander et al, 1999; Rush et al, 2003; Yamashita et al, 1999), and may play a role in compensating a lack of Cu,Zn-SOD upregulation, thus providing greater protection by preventing formation of nitrotyrosine and  protein oxidation, as shown in our study. In conclusion, the primary defect in the diabetic aortae seems to be the prevalence of free radicals like superoxide and peroxynitrite, which lower NO bioavailability by degrading this compound and decreasing eNOS substrate (L-Arg) and cofactor (BH4) availability. We demonstrate that a loss of body weight, body fat or improvement in glucose parameters are not obligatory in exercise-induced reversal of the above vascular defects. Additionally, exercise may upregulate aorta eNOS activity independently of its total protein production in diabetic blood vessels. Finally, in this study, we demonstrate for the first time that differential regulation of SOD isoforms occurs in the diabetic aortae, depending on exercise intensity. This along with improved NO bioavailability may be pivotal in the reversal of diabetic endothelial dysfunction by lifestyle modification approaches such as exercise. We were not able to show significant differences in food consumption between exercised and sedentary mice which could partly be related to the low sensitivity of our measurement. The significant decrease in water consumption in exercised db/db mice could be related to decreased microalbuminuria and prevention of diabetic nephropathy by exerrcise (Ghosh et al, 2009).  44     Parameters WT WT low- intensity exe WT mod- intensity exe db/db db/db low- intensity exe db/db mod- intensity exe A. Related to lipid profile Triglycerides (mmol/l) 0.5±0.07 0.71±0.12 0.61±0.14 1.32±0.14° 0.62±0.07* 0.50±0.08* Cholesterol mmol/l) 2.7±.0.20 2.65±0.08 3.04±0.04 3.97±0.20° 4.1±0.10 2.9±0.16#, * LDL (mmol/l) 0.91±0.07 0.88±0.05 0.99±0.08 1.48±0.16° 1.55±0.06 0.83±0.23#, * HDL (mmol/l) 1.44±0.13 1.48±0.05 1.65± 0.15 1.66±0.27 2.26±0.13* 1.74±0.12 B. Related to glycemic status Glucose (mmol/l) 6.44±0.29 6.46±0.18 5.72±0.26 47.56±3.83° 43.96±1.16° 48.24±4.00° Insulin (µg/l)  1.49±0.14 1.64±0.33 1.15±0.17 3.63±0.53° 3.60±0.46° 3.72±0.71°  Table 2-1: Plasma parameters of diabetic (db/db) and wild type (WT) mice. Values are means ± SE for 6-8 mice in each group.  Plasma parameters were measured at the time of sacrifice (12 weeks old). A. Parameters related to lipid profile. B. Parameters related to glycemic status. *Significantly different from sedentary db/db mice, #Significantly different from low-intensity exercised db/db mice. °Significantly different than WT. p< 0.05.  WT, wild type; LDL, low density lipoprotein; HDL, high density lipoprotein; mmol, millimolar; mod, moderate; exe, exercise.      45    -Log EC50 Emax Figure 2-3 a,b ACh SNP ACh SNP WT 6.97±0.24 7.48±0.15  44.39±3.62 79.09±3.10 db/db 6.53±0.30 7.39±0.08 5.59±0.65* 78.52±1.97 db/db low-intensity exe  6.57±0.25 7.28±0.08 28.78±3.14** 85.58±2.35 Figure 2-3 c,d ACh SNP ACh SNP WT  6.57±0.05  7.45±0.06  59.03±1.33 89.65±1.67 db/db 6.68±0.11 7.28±0.05  18.18±0.81* 87.12±1.57 db/db mod-intensity exe 6.25±0.02  7.12±0.04  48.53±0.58** 93.63±1.38  Table 2-2: Emax and EC50 values for ACh and SNP concentration-response curves. Results are the means±SE.  While the Emax values for SNP were not statistically different between the groups, the Emax for ACh was significantly lower in db/db mice compared to WT mice (n =9-10 per group, *p<0.01, one-way ANOVA). Compared to control db/db mice, Emax was preserved in db/db mice exercised with low and moderate intensity (n =9-10 per group, **p<0.05, one-way ANOVA). EC50 values for ACh and SNP response curves were not statistically different amongst groups. ACh, acetylcholine; SNP, sodium nitroprusside; EC50, median effective concentration; Emax, maximal effect; mod, moderate; exe, exercise. .     46  - Log EC50 (mol/l) Group Control  SOD L-Arg + BH4 Co WT 6.57±0.05    6.40±0.05 6.16±0.07 59 db/db 6.68±0.11 6.10±0.06 6.31±0.05 18 db/db low-intensity exe 6.89±0.02 6.75±0.04 6.67±0.05 64 db/db mod-intensity exe 6.25±0.02 6.22±0.05 6.30±0.03 48                 Table 2-3:  Emax and EC50 values for ACh concentration-response curves incubation. Values are means ± SE for 8-10 mice in each group.  Incuba statistically change Emax quantities for ACh response in db/db mice ex However, Emax for ACh responses were significantly increased in db/db plus BH4 (*p<0.01, one-way ANOVA). EC50 did not change in any gro type; SOD, superoxide dismutase; L-Arg, L-arginine; BH4, tetrahydrEmax (% vasodilaton)   ntrol SOD L-Arg + BH4     .03±1.33 66.83±1.65 57.37±2.11 .18±0.81 59.12±1.97* 46.58±1.13* .04±0.55 65.52±1.04 63.15±1.22 .53±0.58 52.24±1.25 51.31±0.78  before and after SOD and L-Arg plus BH4 tion with SOD or L-Arg plus BH4 did not ercised with low and moderate intensity. mice after incubation with SOD or L-Arg up before and after incubations. WT, wild obiopterin, mod, moderate; exe, exercis 47 B od y W ei gh t ( g) 0 11 22 33 44 55 WT WT low-intensity exe db/db db/db low-intensity exe a. b. Age (weeks) c. Figure 1Time after glucose ingestion (min) 0 20 40 60 80 100 120 Pl as m a G lu co se  (m g/ dl ) 0 200 400 600 800 1000 1200 WT WT low-intensity exe WT mod-intensity exe db/db db/db low-intensity exe db/db mod-intensity exe B od y W ei gh t ( g) 0 11 22 33 44 55 WT WT mod-intensity exe db/db db/db mod-intensity exe * * * ** * * # # # # Time in weeks 5 6 7 8 9 10 11 12 5 6 7 8 9 10 11 12    48 Figure 2-1: Age- and exercise-related changes in body weights of mice and OGTT values (a) Low- intensity exercise did not significantly change the body weight in db/db mice over 7 weeks  (b) db/db mice exercised with moderate intensity gained less weight compared to their control littermates.  This difference was apparent from six weeks of age onward (n =8-10 per group, *p <0.05, repeated measures ANOVA). Weight gain in WT mice was not affected by either low- or moderate-intensity exercise. (c) OGTT showing glucose changes after oral ingestion of 1.5 g/kg glucose at the 10th week of age in fasted mice. There was a significant difference between db/db and WT groups at all time points (n =9-10 per group, #p<0.01, repeated measures ANOVA). mod, moderate; exe, exercise. Results are means±SEM.  49   50 Figure 2-2: Traces illustrating tension (mN) of aortic rings from the following mouse groups: (a) WT, (b) db/db, (c) exercised db/db with low intensity and (d) exercised db/db with moderate intensity. Arteries were preconstricted with PE (1 µmol/l) and challenged with cumulative concentrations of ACh (10-3-10 µmol/l) and SNP (10-3-10 µmol/l).  In WT mice (a) both ACh and SNP caused vasodilation of aortic rings by ~80%. There was a marked attenuation in ACh-induced vasodilation in db/db mice (b) (~25% dilation) while SNP-mediated vasodilation was unchanged. Both low- and moderate-intensity exercise improved ACh-induced aortic dilation in db/db mice (c and d). PE, phenylephrine; ACh, acetylcholine; SNP, sodium notroprusside; mN, millinewton; mod, moderate; exe, exercise.   51  a b -10 -9 -8 -7 -6 -5 -4 25 50 75 100 Log [S NP ] (mol/l) -10 -9 -8 -7 -6 -5 -4 0 25 50 75 W T db/db low -inte ns ity e xe db/db Log [AC h]  (m ol/l) P er ce n t va so d il at io n c d -10 -9 -8 -7 -6 -5 -4 25 50 75 100 Log [SN P] (m ol/l) -10 -9 -8 -7 -6 -5 -4 0 25 50 75 W T db/db db/db  m od-inte ns ity e x e Log [AC h]  (mol/l) P er ce n t va so d il at io n * * ** ** e Figure 3  Figure 2-3: ACh and SNP concentration-response curves from aortic rings preconstricted with PE (1 µmol/l). Endothelium-dependent vasodilation in response to ACh was significantly impaired in aortae of db/db mice compared to WT littermates. Both low- and moderate-intensity exercise improved endothelium-dependent vasodilation in db/db mice (a and c). Endothelium-independent vasodilation of aortic rings induced by SNP was not statistically different in groups (b and d) (n =9- 10 per group, *p <0.05, **p<0.01, repeated measures ANOVA). ACh, acetylcholine; SNP, sodium nitroprusside; mod, moderate; exe, exercise.  52  53  a b c d e f -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db + L-Arg + BH4 db/db Log [ACh] (mol/l) Pe rc en t v as od ila tio n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db + SOD db/db Log [ACh] (mol/l) Pe rc en t v as od ila tio n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db low-intensity exe db/db low-intensity exe + SOD Log [ACh] (mol/l) Pe rc en t v as od ila tio n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db low-intensity exe db/db low-intensity exe + L-Arg + BH4 Log [ACh] (mol/l) Pe rc en t v as od ila tio n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db mod-intensity exe + SOD db/db mod-intensity exe Log [ACh] (mol/l) Pe rc en t v as od ila tio n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 db/db mod-intensity exe + L-Arg + BH4 db/db mod-intensity exe Log [ACh] (mol/l) Pe rc en t v as od ila tio n * * Figure 4  Fig Figure 2-4: Effect of SOD and L-Arg plus BH4 incubation on ACh-induced vasodilation. SOD and L-Arg plus BH4 incubation of aortic rings improved endothelium-dependent vasodilation in db/db mice (a and b) but had no effect in db/db mice exercised with low intensity (c and d), and moderate intensity (e and f) (n =8-10 per group, *p <0.05, repeated measures ANOVA).  WT, wild type; SOD, superoxide dismutase; L-Arg, L-arginine; BH4, tetrahydrobiopterin, mod, moderate; exe, exercise.  54  p ho sp ho -e NO S/ eN O S ra tio 0.0 0.2 0.4 0.6 0.8 To ta l e NO S (A .U ./m g pr ot ei n) 0 10 20 30 40 50 WT db/db db/db low-intensity exe db/db mod-intensity exe  p ho sp ho -e NO S (S er -1 17 7)  (A .U ./m g pr ot ei n) 0 5 10 15 20 25 Figure 5 # 0 2 4 6 8 Ao rt a ni tr ite  ( µ m ol /l/ m g dr y w ei gh t) a. c. d. # b. * * $ * ** * * * # t sity exe  Figure 2-5: Endothelial nitric oxide synthase (eNOS) protein expression and aortic nitrite levels. (a) Total eNOS and (b) phosphorylated (Ser 1177) eNOS protein expression were measured by Western blot followed by densitometry. The upper panel depicts representative bands from each group.  (c) Ratio between phosphorylated eNOS and total eNOS protein signifying ‘active’ eNOS levels. (d) Spectrophotometric quantification of nitrite (NO2-) in the aortae. Results are the means ± SE of 6 rats in each group.  *Significantly different from sedentary db/db mice; # Significantly different from sedentary WT mice, $ Significantly different than db/db mice exercised with low- intensity p< 0.05. , mod, moderate; exe, exercise. A.U., arbitrary units. Phospho-eNOS, phosphorylated eNOS. 55 C u/ Zn  S O D  (A .U ./m g pr ot ei n) 0.0 0.5 1.0 1.5 2.0 0 1 2 3 4 5 M n SO D  (A .U ./m g pr ot ei n) C at al as e (A .U ./m g pr ot ei n) 0 1 2 3 4 WT db/db db/db low-intensity exe db/db mod-intensity exe a. b. c. * ** Figure 6  Figure 2-6: Western Blot analysis of antioxidant protein expression (a) Catalase (b) Cu,Zn-SOD (c) Mn-SOD protein expression were measured by Western blot followed by densitometry. The upper panel depicts representative bands from each group.  Results are the means ± SE of 6 rats in each group.  *Significantly different from sedentary db/db mice p< 0.05.  mod, moderate; exe, exercise. A.U., arbitrary units. Cu, copper; Zn, zinc; Mn, manganese; SOD, superoxide dismutase   56 010 20 30 40 50 WT db/db db/db low-intensity exe db/db mod-intensity exe N itr ot yr os in e (A .U ./m g pr ot ei n) 0 5 10 15 20 25 30 * # Pr ot ei n ca rb on yl  c on te nt  (O .D ./m g pr ot ei n) 0 70 140 210 Figure 7 8- is op ro st an e (p g/ m l) * * a. b. c. / b / b low-intensity exe db/db mod-intensity exe  Figure 2-7: Oxidative stress related parameters (a) spectrophotometric determination of plasma free 8-isoprostane levels (b) western blot of aorta nitrotyrosine protein expression. The upper panel depicts representative bands from each group. (c) spectrophotometric determination of aorta protein carbonyls.  Results are the means ± SE of 6 rats in each group.  *Significantly different from all other groups, #Significantly different from low-intensity exercised db/db mice p< 0.05.  mod, moderate; exe, exercise; A.U., arbitrary units; pg, picogram; O.D., optical density.  57 2.5 Bibliography  Boulanger,C.M., Morrison,K.J., & Vanhoutte,P.M. (1994) Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat. Br.J Pharmacol., 112, 519-524. Boule,N.G., Haddad,E., Kenny,G.P., Wells,G.A., & Sigal,R.J. (2001) Effects of exercise on glycemic control and body mass in type 2 diabetes mellitus: a meta-analysis of controlled clinical trials. JAMA, 286, 1218-1227. Cai,S., Khoo,J., Mussa,S., Alp,N.J., & Channon,K.M. (2005) Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation. Diabetologia, 48, 1933-1940. Carrier,G.O. & Aronstam,R.S. (1987) Altered muscarinic receptor properties and function in the heart in diabetes. J Pharmacol.Exp.Ther., 242, 531-535. Chang,S.P., Chen,Y.H., Chang,W.C., Liu,I.M., & Cheng,J.T. (2004) Increase of anti-oxidation by exercise in the liver of obese Zucker rats. Clin.Exp.Pharmacol.Physiol, 31, 506-511. Cooke,C.L. & Davidge,S.T. (2003) Endothelial-dependent vasodilation is reduced in mesenteric arteries from superoxide dismutase knockout mice. Cardiovasc.Res., 60, 635-642. Dalle-Donne,I., Aldini,G., Carini,M., Colombo,R., Rossi,R., & Milzani,A. (2006) Protein carbonylation, cellular dysfunction, and disease progression. J Cell Mol.Med., 10, 389-406. Didion,S.P., Ryan,M.J., Didion,L.A., Fegan,P.E., Sigmund,C.D., & Faraci,F.M. (2002) Increased superoxide and vascular dysfunction in CuZnSOD-deficient mice. Circ.Res., 91, 938-944. Dimmeler,S., Fleming,I., Fisslthaler,B., Hermann,C., Busse,R., & Zeiher,A.M. (1999) Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature, 399, 601-605. Du,X.L., Edelstein,D., Dimmeler,S., Ju,Q., Sui,C., & Brownlee,M. (2001) Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J.Clin.Invest, 108, 1341-1348. Dudzinski,D.M. & Michel,T. (2007) Life history of eNOS: partners and pathways. Cardiovasc.Res., 75, 247-260. Erdei,N., Bagi,Z., Edes,I., Kaley,G., & Koller,A. (2007) H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice. Am.J Physiol Heart Circ.Physiol, 292, H649-H656. Eriksen,L., Dahl-Petersen,I., Haugaard,S.B., & Dela,F. (2007) Comparison of the effect of multiple short-duration with single long-duration exercise sessions on glucose homeostasis in type 2 diabetes mellitus. Diabetologia, 50, 2245-2253. Faraci,F.M. & Didion,S.P. (2004) Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler.Thromb.Vasc.Biol., 24, 1367-1373. Forstermann,U. & Munzel,T. (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113, 1708-1714.  58 Fukai,T., Siegfried,M.R., Ushio-Fukai,M., Cheng,Y., Kojda,G., & Harrison,D.G. (2000) Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J Clin.Invest, 105, 1631-1639. Fulton,D., Gratton,J.P., McCabe,T.J., Fontana,J., Fujio,Y., Walsh,K., Franke,T.F., Papapetropoulos,A., & Sessa,W.C. (1999) Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature, 399, 597-601. Ghosh,S., Kewalramani,G., Yuen,G., Pulinilkunnil,T., An,D., Innis,S.M., Allard,M.F., Wambolt,R.B., Qi,D., Abrahani,A., & Rodrigues,B. (2006) Induction of mitochondrial nitrative damage and cardiac dysfunction by chronic provision of dietary omega-6 polyunsaturated fatty acids. Free Radic.Biol.Med., 41, 1413-1424. Ghosh,S., Khazaei,M., Moien-Afshari,F., Ang,L.S., Granville,D.J., Verchere,C.B., Dunn,S.R., McCue,P., Mizisin,A., Sharma,K., & Laher,I. (2009) Moderate exercise attenuates caspase-3 activity, oxidative stress, and inhibits progression of diabetic renal disease in db/db mice. Am.J.Physiol Renal Physiol, 296, F700-F708. Ghosh,S., Pulinilkunnil,T., Yuen,G., Kewalramani,G., An,D., Qi,D., Abrahani,A., & Rodrigues,B. (2005) Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion. Am.J Physiol Heart Circ.Physiol, 289, H768-H776. Gielen,S., Schuler,G., & Hambrecht,R. (2001) Exercise training in coronary artery disease and coronary vasomotion. Circulation, 103, E1-E6. Giugliano,D., Marfella,R., Coppola,L., Verrazzo,G., Acampora,R., Giunta,R., Nappo,F., Lucarelli,C., & D'Onofrio,F. (1997) Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation, 95, 1783-1790. Gormley,S.E., Swain,D.P., High,R., Spina,R.J., Dowling,E.A., Kotipalli,U.S., & Gandrakota,R. (2008) Effect of intensity of aerobic training on VO2max. Med.Sci.Sports Exerc., 40, 1336- 1343. Green,D.J., Walsh,J.H., Maiorana,A., Best,M.J., Taylor,R.R., & O'Driscoll,J.G. (2003) Exercise- induced improvement in endothelial dysfunction is not mediated by changes in CV risk factors: pooled analysis of diverse patient populations. Am.J.Physiol Heart Circ.Physiol, 285, H2679-H2687. Gregg,E.W., Gerzoff,R.B., Thompson,T.J., & Williamson,D.F. (2004) Trying to lose weight, losing weight, and 9-year mortality in overweight U.S. adults with diabetes. Diabetes Care, 27, 657-662. Greig,N.H., Holloway,H.W., De Ore,K.A., Jani,D., Wang,Y., Zhou,J., Garant,M.J., & Egan,J.M. (1999) Once daily injection of exendin-4 to diabetic mice achieves long-term beneficial effects on blood glucose concentrations. Diabetologia, 42, 45-50. Hambrecht,R., Wolf,A., Gielen,S., Linke,A., Hofer,J., Erbs,S., Schoene,N., & Schuler,G. (2000) Effect of exercise on coronary endothelial function in patients with coronary artery disease. N.Engl.J Med., 342, 454-460. Hass,M.A. & Massaro,D. (1987) Differences in CuZn superoxide dismutase induction in lungs of neonatal and adult rats. Am.J Physiol, 253, C66-C70.  59 Hausenloy,D.J. & Yellon,D.M. (2008) Targeting residual cardiovascular risk: raising high-density lipoprotein cholesterol levels. Heart, 94, 706-714. Heitzer,T., Krohn,K., Albers,S., & Meinertz,T. (2000) Tetrahydrobiopterin improves endothelium- dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus. Diabetologia, 43, 1435-1438. Hollander,J., Fiebig,R., Gore,M., Bejma,J., Ookawara,T., Ohno,H., & Ji,L.L. (1999) Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training. Am.J Physiol, 277, R856-R862. Inoue,N., Ramasamy,S., Fukai,T., Nerem,R.M., & Harrison,D.G. (1996) Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells. Circ.Res., 79, 32-37. Ivy,J.L. (1997) Role of exercise training in the prevention and treatment of insulin resistance and non-insulin-dependent diabetes mellitus. Sports Med., 24, 321-336. Kavdia,M. (2006) A computational model for free radicals transport in the microcirculation. Antioxid.Redox.Signal., 8, 1103-1111. Kingwell,B.A., Tran,B., Cameron,J.D., Jennings,G.L., & Dart,A.M. (1996a) Enhanced vasodilation to acetylcholine in athletes is associated with lower plasma cholesterol. Am.J Physiol, 270, H2008-H2013. Kojda,G. & Hambrecht,R. (2005) Molecular mechanisms of vascular adaptations to exercise. Physical activity as an effective antioxidant therapy? Cardiovasc.Res., 67, 187-197. Kusterer,K., Pohl,T., Fortmeyer,H.P., Marz,W., Scharnagl,H., Oldenburg,A., Angermuller,S., Fleming,I., Usadel,K.H., & Busse,R. (1999) Chronic selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in rats. Cardiovasc.Res., 42, 783-793. Laakso,M. (1999) Hyperglycemia and cardiovascular disease in type 2 diabetes. Diabetes, 48, 937- 942. Leung,F.P., Yung,L.M., Leung,H.S., Au,C.L., Yao,X., Vanhoutte,P.M., Laher,I., & Huang,Y. (2007) Therapeutic concentrations of raloxifene augment nitric oxide-dependent coronary artery dilatation in vitro. Br.J Pharmacol., 152, 223-229. Maiorana,A., O'Driscoll,G., Cheetham,C., Dembo,L., Stanton,K., Goodman,C., Taylor,R., & Green,D. (2001) The effect of combined aerobic and resistance exercise training on vascular function in type 2 diabetes. J Am.Coll.Cardiol., 38, 860-866. Meydani M & Evans WJ (1993) Free radicals, exercise, and aging. Free Radicals in Aging (ed. by Yu BP), pp. 183-204. CRC Press, Boca Raton, Fl. Nassis,G.P., Papantakou,K., Skenderi,K., Triandafillopoulou,M., Kavouras,S.A., Yannakoulia,M., Chrousos,G.P., & Sidossis,L.S. (2005) Aerobic exercise training improves insulin sensitivity without changes in body weight, body fat, adiponectin, and inflammatory markers in overweight and obese girls. Metabolism, 54, 1472-1479. Nishikawa,T., Edelstein,D., Du,X.L., Yamagishi,S., Matsumura,T., Kaneda,Y., Yorek,M.A., Beebe,D., Oates,P.J., Hammes,H.P., Giardino,I., & Brownlee,M. (2000) Normalizing  60 mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 404, 787-790. Okon,E.B., Szado,T., Laher,I., McManus,B., & van Breemen,C. (2003) Augmented contractile response of vascular smooth muscle in a diabetic mouse model. J.Vasc.Res., 40, 520-530. Oyama,Y., Kawasaki,H., Hattori,Y., & Kanno,M. (1986) Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur.J Pharmacol., 132, 75-78. Pacher,P. & Szabo,C. (2006) Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes. Curr.Opin.Pharmacol., 6, 136-141. Rush,J.W., Turk,J.R., & Laughlin,M.H. (2003) Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium. Am.J Physiol Heart Circ.Physiol, 284, H1378-H1387. Schefer,V. & Talan, M.I. (1996) Oxygen consumption in adult and AGED C57BL/6J mice during acute treadmill exercise of different intensity. Exp.Gerontol., 31, 387-392. Shen,X., Zheng,S., Metreveli,N.S., & Epstein,P.N. (2006) Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes, 55, 798-805. Srinivasan,S., Hatley,M.E., Bolick,D.T., Palmer,L.A., Edelstein,D., Brownlee,M., & Hedrick,C.C. (2004) Hyperglycaemia-induced superoxide production decreases eNOS expression via AP- 1 activation in aortic endothelial cells. Diabetologia, 47, 1727-1734. Strader,A.D., Reizes,O., Woods,S.C., Benoit,S.C., & Seeley,R.J. (2004) Mice lacking the syndecan- 3 gene are resistant to diet-induced obesity. J Clin.Invest, 114, 1354-1360. Tang,T. & Reed,M.J. (2001) Exercise adds to metformin and acarbose efficacy in db/db mice. Metabolism, 50, 1049-1053. Vinals,M., Martinez-Gonzalez,J., & Badimon,L. (1999) Regulatory effects of HDL on smooth muscle cell prostacyclin release. Arterioscler.Thromb.Vasc.Biol., 19, 2405-2411. Vinik,A.I., Erbas,T., Park,T.S., Nolan,R., & Pittenger,G.L. (2001) Platelet dysfunction in type 2 diabetes. Diabetes Care, 24, 1476-1485. Williams,S.B., Cusco,J.A., Roddy,M.A., Johnstone,M.T., & Creager,M.A. (1996) Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am.Coll.Cardiol., 27, 567-574. Wing,R.R., Goldstein,M.G., Acton,K.J., Birch,L.L., Jakicic,J.M., Sallis,J.F., Jr., Smith-West,D., Jeffery,R.W., & Surwit,R.S. (2001) Behavioral science research in diabetes: lifestyle changes related to obesity, eating behavior, and physical activity. Diabetes Care, 24, 117- 123. Yamashita,N., Hoshida,S., Otsu,K., Asahi,M., Kuzuya,T., & Hori,M. (1999) Exercise provides direct biphasic cardioprotection via manganese superoxide dismutase activation. J Exp.Med., 189, 1699-1706.    61 3 EXERCISE RESTORES CORONARY VASCULAR FUNCTION INDEPENDENT OF MYOGENIC TONE OR HYPERGLYCEMIC STATUS IN db/db MICE2                       2 A version of this chapter has been published. Moien-Afshari F, Ghosh S, Elmi S, Khazaei M, Rahman MM, Sallam N, Laher I. Am J Physiol Heart Circ Physiol. 2008; 295(4):H1470-80.  62 3.1 Introduction Cardiovascular disease is the leading cause of mortality in patients with diabetes (Laakso, 1999). There is a markedly reduced endothelial dependent vasodilatation and an increase in myogenic tone of resistance arteries in animal models of type 2 diabetes mellitus (DM) (Bagi et al, 2005; Ito et al, 2006; Ungvari et al, 1999).  These changes are likely to reduce tissue blood perfusion.  Blood flow is regulated by the influence of several constrictors (e.g. increased intravascular pressure, endothelial constrictors etc) and dilators (e.g. reduced intravascular pressure, endothelial dilators etc) (Moien-Afshari et al, 2004).  In many cases, endothelial dysfunction precedes the onset of cardiovascular disease in type 2 DM.  It is likely that loss of endothelial regulation results in a mismatch of myocardial supply and demand, thus provoking cardiac ischemia and myocardial infarction (Mahgoub & Abd-Elfattah, 1998; Nitenberg et al, 1998). The cardiovascular benefits of regular exercise in patients with type 2 DM are well accepted. Exercise alters myocardial redox status, calcium handling, improves energy metabolism and induces the formation of heat shock proteins and other cardioprotective molecules (Ascensao et al, 2007; Kemi et al, 2008).  Exercise also reduces insulin resistance, an important cause for the elevated plasma glucose and insulin levels in humans and animals with diabetes (Nassis et al, 2005).  However, the mechanisms by which exercise promotes improved coronary microcirculatory function in type 2 DM hearts are incompletely understood, especially in relation to altered myogenic tone and endothelial function in coronary resistance arteries.  We hypothesized that exercise reduces pressure induced myogenic constriction of coronary arteries while at the same time also improving endothelial function in db/db mice, a frequently used animal model of type 2 DM. We found that the vascular benefits conferred by moderate levels of exercise in our study were independent of changes in myogenic tone or hyperglycemic status, and primarily involved increased NO bioavailability in the coronary microcirculation.    63 3.2 Materials and methods 3.2.1 Animals All experimental procedures were approved by the Animal Care Committee of the University of British Columbia.  Five weeks old male db/db (BKS.Cg-m +/+ Leprdb/J) and age matched control (BKS.cg-m +/+ Leprdb/+/J) mice, simply referred to as wild type (WT) in this work, were purchased from Jackson Laboratory (USA).  Mice were housed in standard animal facility conditions with 12h light/dark cycles, 26ºC temperature and were allowed free access to standard chow and water.  Wild type and db/db mice were divided into exercised and sedentary subgroups (8-10 in each group).  3.2.2 Exercise protocol Mice (5 weeks old) assigned to the exercise group were trained to run on a motorized exercise wheel system (Lafayette Instrument Co, IN, USA) for 8 weeks.  The extent of exercise was incrementally increased to allow for animal acclimatization during the first two weeks of training. The initial exercise speed was a 2.5 meters/min for one hour (150m) per day.  This was gradually increased to a target of one-hour exercise at a speed of 5.2 meters/min, which represents a daily forced exercise of 312 meters.  Mice were exercised daily, five days per week for 8 weeks at a set time each day. Mice were housed in the animal facility between exercise sessions.  Sedentary animals were placed in the non-rotating wheels for the same duration as the exercised group.  3.2.3 Blood and tissue samples At 13 weeks of age mice were anaesthetised by intraperitoneal injection of pentobarbital sodium (Somnotol 30 mg/kg) and heparin sodium (50 U/kg).  Blood samples were collected from the inferior vena cava using a 5ml syringe and a 21-gauge needle and centrifuged (14000 r/min) for 10 minutes for plasma generation.  Plasma was stored in separate Eppendorf tubes at -76°C for later biochemical assay.  Following blood collection, animals were euthanized by cutting the abdominal aorta. Hearts were excised and placed in ice-cold physiologic salt solution (PSS, see solutions and  64 chemicals).  After weighing the hearts, coronary septal arteries were dissected for pressure myograph studies, the rest of the hearts were saved at –76°C for SOD proteins, catalase, nitrotyrosine, and eNOS quantification.  A piece of thigh adductor muscle was flash frozen using liquid nitrogen and kept at -76°C for citrate synthase assay.  3.2.4 Oral glucose tolerance test (OGTT) Mice underwent an OGTT when they reached 10 weeks of age (i.e. after 5 weeks of exercise). After six hours of fasting, mice were glucose loaded (1.5 g/kg) with a 40% glucose solution by oral gavage. Blood samples were taken at times 0, 10, 20, 60, and 120 min. Plasma was separated by centrifugation and stored at -76°C for later analyses of glucose. To reduce short-term treatment effects, animals were not exercised for twenty-four hours prior to the OGTT.  3.2.5 Citrate synthase (CS) enzyme assay Frozen thigh adductor muscles were homogenized on ice in 100mM TRIS buffer (pH 8) containing 0.1% triton-X, 0.5mM EDTA, pH 8) using a glass homogenizer (da Silva et al, 2007).  The homogenates were centrifuged at 4°C for 5 min at 13,200 rpm to remove tissue debris.  The supernatant was assayed for CS activity at 30°C at the linear portion of the activity curve (1-2mins). Reaction mixtures consisted of 50 mM TRIS pH 8, 0.1 mM oxaloacetate, 0.1 mM acetyl CoA, and 0.1 mM 5,5-dithiobis (2-nitrobenzoate) (DTNB).  Briefly, the reaction was initiated by adding 25 µl of muscle extract and linking the release of free CoA to DTNB, a colorimetric agent.  The CS activity was monitored at 412 nm using a spectrophotometer (PerkinElmer, Lambda 35 UV/VIS). Calculations of activity used a millimolar extinction coefficient of 13.6 and were corrected for background acetyl CoA deacylase activity by determining the rate of change in absorbance at 412nm in the absence of oxaloacetate.  The CS activity was expressed as micromoles per minute per milligram protein of the extract (measured by Bradford assay).   65 3.2.6 Western Blot analysis Pieces of whole hearts were ground in liquid nitrogen and homogenized in a polytron homogenizer for 30 secs × 3 in ice-cold homogenization buffer (20 mM Tris HCl, 250mM EGTA, 200mM EDTA, 100mM PMSF, 100mM NaF, 2-mercaptoethanol, leupeptin, aprotinin, NP-40, 10%SDS and 5%DCA).  The protein contents of the homogenates were quantified using a Bradford Protein Assay.  The homogenates were diluted, boiled with sample loading dye, and samples corresponding to 50 µg protein were used in SDS-polyacrylamide gel electrophoresis.  After transfer, nitrocellulose membranes were blocked in 5% skim milk overnight in Tris-buffered saline containing 0.1% Tween-20 (TBS-T).  Membranes were incubated with antibodies raised in rabbit [endothelial nitric oxide synthase (eNOS; or NOS3), catalase, extracellular SOD (SOD3)], sheep [Cu,Zn superoxide dismutase (Cu,Zn-SOD; or SOD1), Mn superoxide dismutase (Mn-SOD; or SOD2)], and mouse (nitrotyrosine) for 2 hours at room temperature.  Following 3× wash in TBS-T, the membranes were incubated for 2 hours at room temperature with secondary goat anti-rabbit, goat anti-mouse, donkey anti-sheep or donkey anti-goat HRP-conjugated antibodies, and visualized using ECL detection kit (Ghosh et al, 2007). Controls for equal protein loading were performed using an anti-goat polyclonal antibody raised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Genscript Corporation, Piscataway, NJ, USA).  3.2.7 Immunofluorescence Part of the left ventricle was fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 µm sections prepared.  Double immunostaining was performed for both von Willebrand factor (vWF) and MnSOD.  For the first vWF labelling, sections were deparaffinized in xylene, rehydrated using various grades of ethanol.  Non-specific binding of immunoglobulins was blocked by incubating sections in 10% BSA for 20 minutes.  vWF immunolabeling was performed using a mouse monoclonal anti-vWF antibody (1:50, 2 hours at 37°C) (Novocastra Labs, UK) with Alexa Fluor 594® conjugated goat anti-mouse IgG (Molecular Probes; 90 min at 37°C).  For the second  66 labelling, the same sections were incubated with primary sheep anti-Mn-SOD antibody (Calbiochem) (1:200, overnight at room temperature), and secondary donkey anti-sheep antibody conjugated with FITC (Sigma).  Sections were finally counterstained with DAPI (to visualize nuclei), and examined using confocal microscopy.  Slides were visualized using FITC, DAPI and Texas Red filters under a confocal microscope.  3.2.8 Resistance artery preparation Mouse coronary septal arteries were located through a right ventricular wall opening, dissected, and cleaned of adherent cardiac muscle tissue (Leung et al, 2007).  For all functional studies, a 0.8-1.2 mm segment of the artery (inner diameter 60-150 µm) at the level of the superior papillary muscle was excised and mounted at both ends onto glass cannulae in a pressure myograph chamber (Living Systems Instrumentation, Burlington, Vermont, USA).  Both ends of the artery were tied using single strands teased from a 4-0 surgical silk thread and the chamber was placed on an inverted microscope stage to measure the arterial diameter.  One of the cannulae was occluded to prevent flow through the vessel and the other was connected through a pressure transducer to a peristaltic feedback pump to maintain constant pressure (20 mmHg) and also to monitor transmural diameter. The vessels were continuously superfused with bubbled (95%O2, 5%CO2) PSS (pH: 7.35-7.4) at 37°C.  Pressure-constriction curves were determined in all artery segments.  Intravascular pressure was incrementally increased to 80mmHg.  Vessels that did not develop a leak were equilibrated for one hour at this pressure to develop myogenic tone (vessels that did not develop spontaneous constriction were excluded).  Following the development of pressure–induced constriction, transmural pressure was reduced to 10 mmHg and then increased in a stepwise manner at 5-minute intervals to 120mmHg.  The internal diameter was recorded at each step and then compared with diameters occurring in a calcium free PSS at the end of the experiment to determine the degree of myogenic tone at each pressure.  The artery was incubated for half an hour with bosentan (10µM) and the protocol was repeated to determine the effect of endogenous endothelin on the myogenic  67 response (Rahman et al, 2007).  Then, at 20mmHg pressure, arteries were preconstricted up to 50% of their initial diameter using a thromboxane agonist (U46619, 10-7M) (Leung et al, 2007). Cumulative concentrations (1nM-10µM) of ACh and SNP were added to the bath separately.  ACh concentration-response curve was repeated in arteries incubated with either superoxide dismutase (SOD, 120U/ml) or L-arginine (L-Arg, 10-3M) and tetrahydrobiopterin (BH4, 10µM) (incubated for 45 minutes).  After wash out, U46610 concentration-response curves were generated by adding cumulative concentrations (5nM -5µM). Finally, arterial diameter was measured in response to stepwise increase in intraluminal pressure in calcium free PSS containing EGTA to calculate arterial distensibility as described below.  3.2.9 Glucose and Insulin Plasma glucose levels were measured by Trinder Assay using a commercial kit purchased from Diagnostic Chemicals Limited (Oxford, Connecticut, USA).  Insulin levels were determined in plasma using Mercodia Ultrasensitive Mouse Insulin Assay Kit from Alpco (Salem, New Hampshire, USA).  Photometric measurements were performed by the Dimension® Clinical Chemistry System (GMI, Ramsey, Minnesota, USA).  3.2.10 Chemicals and solutions All buffer reagents were purchased from BDH.  U46619 was purchased from Cayman Chemicals (Michigan, USA). ACh, SNP, Cu,Zn-SOD, L-Arg, and BH4 were purchased from Sigma (St. Louis, MO).  Bosentan was a generous gift from Actelion Phamaceuticals LTD., Switzerland.  The composition of the PSS (mM) was: NaCl (119), KCl (4.7), KH2PO4 (1.18), MgSO4 (1.17), NaHCO3 (24.9), EDTA (0.023), CaCl2 (1.6), dextrose (11.1).  The composition of calcium free solution was similar to PSS but did not include calcium and contained EGTA (10-3M).    68 3.2.11 Statistical analysis and calculations Results of all calculations are expressed as mean ± SEM.  Data were analyzed using NCSS-2000 computer software.  Repeated measures ANOVA or one-way ANOVA with multiple comparisons using Bonferroni’s test was performed where appropriate. GraphPad Prism (version 3.02-2000) was used for curve fit and dose-response analysis.  The results of statistical tests were considered significant if p-values were less than 0.05.  Myogenic tone was calculated as percentage of arterial constriction at each pressure step using the formula: Percent Constriction = 100 × (DCa free, P – DP)/ DCa free, P where DCa free, P is the arterial diameter at pressure P in calcium free PSS and DP is the diameter in PSS at pressure P.  Percentage of relaxation/ dilation of the arteries in response to ACh and SNP was calculated as: 100 × (D20U [Drug] – D20U) /(D20Ca free – D20U) where D20U [Drug], is the diameter of U46619 preconstricted vessel at pressure 20mmHg in PSS in the presence of a particular concentration of drug (ACh or SNP), and D20U is the diameter in PSS at pressure 20 mmHg in the presence of U46619, and D20Ca free is the passive diameter of vessel in calcium free PSS at pressure 20 mmHg.  Passive vascular distensibility was calculated as distensibility at pressure P = 100× passive DP/DCa free, 10. Passive DP = vascular diameter in Ca free PSS at pressure P mmHg.  3.3 Results 3.3.1 Exercise, body weight, heart weight and plasma parameters Moderate intensity exercise decreased the body weights of db/db mice (~10%) compared to their sedentary littermates, whereas exercise did not alter the weights of WT mice.  Heart weight appeared lower in db/db mice compared to WT mice; however, the difference was not significant (158 ± 6mgr vs.177 ± 6mgr). Exercise did not increase heart weight significantly in either group (170 ± 7mgr and 184 ± 6mgr respectively) (Table 3-1a). Plasma glucose and plasma insulin levels of db/db mice were significantly higher compared with age matched lean WT mice.  Eight weeks of moderate intensity exercise did not significantly alter plasma glucose and insulin levels in db/db  69 mice (Table 3-1a).  As an indicator of the physiological effectiveness of our exercise protocol, citrate synthase activity was significantly increased in both exercised WT and db/db mice (Table 3- 1a). An OGTT was performed after 5 weeks of exercise in both db/db and WT mice (Table 3-1b). Exercise did not alter plasma glucose levels in db/db or WT mice within 120 min after an oral glucose load (Table 3-1b). There was a significant difference between db/db and WT groups at all time points (n =9-10 per group, #p<0.01, repeated measures ANOVA).  3.3.2 Exercise decreases whole body and tissue oxidative stress Whole body oxidative stress was estimated by measuring plasma 8-isoprostane, a lipid peroxidation by-product (Montuschi et al, 2007).  Plasma levels of 8-isoprostane were elevated in db/db mice and were significantly reduced by exercise (Table 3-1a).  Estimation of antioxidant protein expression in the whole heart by western blotting revealed that Mn-SOD was significantly decreased and nitrotyrosine was significantly increased in the db/db mice compared to their age- matched lean WT mice.  Exercise significantly increased levels of Mn-SOD (Fig 3-1A) and EC- SOD levels (Fig 3-1F) and lowered nitrotyrosine levels in db/db hearts (Fig 3-1D). However, exercise did not alter the protein expression levels of Cu,Zn-SOD and catalase in either db/db and WT mouse hearts (Fig 3-1B and 3-1C).  The increase in nitrotyrosine levels occurred in the absence of changes in cardiac eNOS levels, which were unaffected by either diabetes or exercise (Fig 3-1E). To ensure that the increases in SOD were localized to the coronary arteries, we utilized an immunofluorescence approach. Representative images of colocalization experiments of Mn-SOD (green) in von Willebrand factor (vWF) positive cells (red) representing the endothelium are shown in Fig 3-2. Fig 3-2A to 3-2C represents lean WT control hearts, whereas Fig 3-2D to 3-2F, 3-2G to 3-2I and 3-2J to 3-2L represents exercised WT, db/db and exercised db/db hearts respectively. A, D, G, J represents sections of hearts and coronary arteries stained for vWF (shown with white arrow in 3-2A). B, E, H, and K represent Mn-SOD (shown with white arrows). C, F, I and L represents composite figures with DAPI counterstaining in blue. Fig 3-1E, F, K, and L demonstrate increased  70 Mn-SOD staining both in the myocardium and endothelial cells in exercised WT and db/db hearts respectively (shown with block arrows in 3-2K and 3-2L).  3.3.3 Exercise and coronary arteriolar tone Pressure-constriction curves (transmural pressure 10 to 120 mmHg) were not statistically different in sedentary and exercised db/db and WT mice (Fig 3-3) (Table 3-2). Bosentan did not have a significant effect on myogenic tone in coronary arteries of sedentary and exercised WT or db/db mice (Fig 3-3C to 3-3F).  We did not find any significant difference in arterial distensibility or passive diameter at pressure 80mmHg between db/db and WT mice (Fig 3-3B) (Table 3-2). Agonist-induced constriction curves by commulative concentratons of U46619 (5×10-9- 5×10-6M) in the presence and absence of L-NAME were also similarI in WT and db/db coronary arteries and exercise did not cause a significant change in the response (Table 3-2) and (Appendix C, Fig 8-2).  3.3.4 Exercise and endothelium dependent arteriolar relaxation Endothelium-dependent coronary vasodilation induced by cumulative concentrations of ACh was significantly attenuated in coronary arteries of db/db mice compared to WT mice (Fig 3-4 and 3-5). The EC50 values for ACh response were similar in coronary arteries of db/db and WT mice in either sedentary (-log EC50: 7.12±0.08 vs. 7.26±0.08 respectively) or exercised (-log EC50: 7.18±0.11 vs. 7.28±0.09 respectively) groups.  The maximal relaxation in response to ACh (percent Emax) was significantly lower in db/db mice (Emax: 51.3±1.7) compared to lean WT mice (Emax: 91.3±2.7). Exercise significantly improved percent Emax of ACh response in db/db mice (85.3±3.5) (Fig 3-4 and 3-5).  The endothelium-independent coronary vasodilatory responses to an exogenous NO donor (sodium nitroprusside, SNP) was not different in WT and db/db mice (Fig 3-4 and 3-5). There were no statistical differences in the Emax and EC50 of the responses to SNP within the sedentary (Emax, WT: 86.9±3.4 vs. db/db: 82.7±4.9; -logEC50, WT: 7.2±0.1 vs. db/db: 7.0±0.2) and  71 exercised groups (percent Emax, exercised WT: 85.8±2.9 vs. exercised db/db: 85.1±3.6; -logEC50, exercised WT: 7.0±0.1 vs. exercised db/db: 6.9±0.1).  3.3.5 Exercise and endothelial NO bioavailability We repeated ACh concentration-response curves in the presence of L-Arg (eNOS substrate, 103µM) and BH4 (eNOS cofactor, 10µM) and Cu,Zn-SOD (120U/ml).  Incubation of db/db coronary septal arteries with L-Arg+BH4 or SOD significantly improved endothelium-dependent relaxation (Fig 3- 6A) without affecting ACh induced vasodilation in arteries from exercised db/db mice (Fig 3-6B) or sedentary and exercised WT mice (data not shown).  Incubation with L-Arg+BH4 or SOD did not change EC50 of ACh in db/db arteries (-log EC50 in db/db: 7.12+0.08 vs. db/db+L-Arg+BH4: 6.93±0.08 vs. db/db+SOD: 7.09±0.11) (Fig 3-6C), but significantly improved percent Emax of the ACh response  (db/db: 51.3±1.7 vs. db/db + L-Arg+BH4: 96.1±3.2 vs. db/db + SOD: 91.3±3.9) (Fig 3-6D).  3.4 Discussion Although the cardiac benefits of life style improvements such as exercise are well known in the management of diabetes, the majority of studies focussed on cardiac muscle alterations either via improvements in metabolic and mitochondrial activity or upregulation of genes and proteins that lead to cardioprotection (Ascensao et al, 2007; Church et al, 2004; Hu et al, 2001; Wing et al, 2001).  The role of improved coronary artery microcirculatory function in exercise induced cardiac health benefits in mouse models of diabetes are relatively unexplored due largely to the technical difficulties in studying the coronary resistance arteries from animals such as the db/db mice.  Our results indicate a marked endothelial dysfunction in coronary septal arteries of db/db mice, a model of type 2 DM, which can be reversed by exercise.  At the time of sacrifice (13 weeks of age), plasma glucose and insulin were significantly higher in db/db mice compared to WT mice, probably as a result of increased insulin resistance. Endothelial dysfunction is a hallmark of diabetes (Cohen  72 et al, 2007; Graier et al, 1999; Pieper et al, 1995). Reductions in hyperglycemia and insulin resistance improves endothelial function and vasodilation (Nassis et al, 2005). In our study, although there was a significant decrease in body weight, 8 weeks of chronic moderate intensity exercise was unable to decrease plasma insulin or glucose levels in db/db mice, demonstrating that moderate levels of exercise did not change hyperglycaemic status. We assessed effect of exercise on blood sugar by two methods at two different time points, OGTT at 10 weeks of age as well as glucose levels at the time of euthanasia, which showed exercise did not have a significant effect on hyperglycemia in db/db mice. The lack of change in OGTT could be related to the exercise protocol used, since repeated short periods of daily exercise is more effective in reducing OGTT than a longer period of exercise of the same duration everyday (Eriksen et al, 2007). Regarding other metabolic effects of exercise in the mice, we reported previously that the exercise intensity used in this study lowers triglycerides and LDL cholesterol without changing HDL (Moien-Afshari et al, 2008). This effect may be related to improved vascular endothelial function in exercised mice. Hyperglycemia in diabetes results in increased production of reactive oxygen and nitrogen species in the cell, leading to oxidative stress (Nishikawa et al, 2000; Pacher & Szabo, 2006). Free radicals play a major role in endothelial dysfunction that occurs in hyperglycaemic conditions (Cohen et al, 2007; Graier et al, 1999; Pieper & Siebeneich, 1998). Oxidative stress arises from an imbalance between the production of free radicals and their neutralization by endogenous antioxidants. Because our exercise regime did not lead to any reduction in high plasma glucose levels in db/db mice, we reasoned that the stimulus for production of free oxygen radicals was not likely to have been diminished by exercise.  Therefore we anticipated that an increase in antioxidant defences occurs with exercise. Free radicals such as superoxide quenches nitric oxide (NO) and decreases the bioavailability of this endothelial vasodilator (Kojda & Hambrecht, 2005). Additionally, superoxides also lead to the formation of peroxynitrite, a potent inducer of irreversible oxidative damage (Pacher & Szabo, 2006).  Superoxide dismutases (SODs) are endogenous antioxidants, able to compete with NO for reacting with superoxides and are thus able to neutralize them.  Exercise  73 decreases whole body oxidative stress, as indicated by lowered plasma 8-isoprostane levels in exercised db/db mice. As the total protein levels in coronary arteries were too low for direct immunoblotting experiments, we used whole hearts from db/db mice to demonstrate that exercise increased the expression of Mn-SOD and decreased nitrotyrosine levels (used as a biomarker for increased peroxynitrite activity) in the heart (Pacher & Szabo, 2006).  We further confirmed our finding of increased Mn-SOD in coronary arteries following exercise in both WT and db/db hearts using immunofluorescence techniques. Our finding of potentiation of Mn-SOD expression by exercise in diabetes is important since increased mitochondrial SOD eliminates oxygen free radicals generated by mitochondria, which is the primary source of these compounds in diabetes (Brownlee, 2001). This will reduce the extent of free radical induced cellular damage by mitigating the four major sources of vascular/ endothelial dysfunction in diabetes including polyol pathway, advanced glycation end-products, protein kinase C, and activated hexosamine pathway (Brownlee, 2001). The mechanisms whereby exercise induces the expression of Mn-SOD is unknown, however, there are some probable explanations. For example, bouts of oxidative stress induced by exercise increases the levels of Mn-SOD (Yamashita et al, 1999). Mn-SOD can also be induced by cytokines such as TNFα and IL-1β (Yamashita et al, 1999) that are increased secondary to elevation of NFκB (Verma et al, 1995) in altered redox states (Hayashi et al, 1993) such as in exercise. It appears that only moderate-intensity exercise (and not low-intensity exercise) increases Mn-SOD (Moien-Afshari et al, 2008). This could be related to higher oxidative stress threshold required for induction of Mn- SOD (Yamashita et al, 1999). Other than the heart muscle, it is likely that oxidative stress in coronary arteries from db/db mice is also related to a reduced SOD expression / activity since addition of exogenous SOD was able to completely restore ACh induced vasodilation in arteries from sedentary db/db mice, which is in agreement with previous reports on reversal of endothelial dysfunction in diabetic vessels by exogenous SOD (Bagi et al, 2003; Pannirselvam et al, 2002; Pieper & Siebeneich, 1998). Exercise probably increases SOD expression/activity in diabetic  74 coronary arterioles (Rush et al, 2000), which is evident from reduced effectiveness of exogenous SOD on endothelium dependent vasodilatation in coronary arteries from exercised db/db mice. Although unlikely in the light of unchanged catalase levels, it is also possible that hydrogen peroxide accumulation, formed from the increased dismutation of superoxides by various SODs following exercise, may also be partly responsible for vasorelaxation in the study groups. Hydrogen peroxide (H2O2), a product of O2• dismutation generated from uncoupled eNOS, plays an important role in endothelium-dependent relaxation under conditions of BH4 deficiency (Cosentino et al, 2001). Therefore, conversion of H2O2 to H2O and O2 by catalase significantly decreases endothelium-dependent relaxation in BH4 deficient states (Cosentino et al, 2001). Under such conditions, SOD can improve endothelium-dependent relaxation by converting eNOS generated O2• to H2O2. In db/db mice, there is a deficiency of BH4, making it likely that that catalase levels would decrease and addition of SOD would increase endothelium-dependent relaxation in response to ACh. However there are studies in mesenteric arteries from db/db mice where both catalase and SOD failed to improve ACh induced vasodilation (Pannirselvam et al, 2002), in contrast to findings made in coronary arteries and aorta (Bagi et al, 2003; Moien-Afshari et al, 2008) suggesting possible regional variations. It is important to note that in vivo S-nitrosohemoglobin plays an important vasodilatory role via releasing NO in hypoxemic conditions that usually occurs in areas of vasoconstriction. In the diabetic environment, however, glycosylation of hemoglobin inhibits release of NO from this protein which may decrease vasodilation mediated by S-nitrosohemoglubin and so contributing to vascular dysfunction (Singel & Stamler, 2005). We are unable to assess the effect of exercise on this mechanism in db/db mice since our experiments were performed in vitro in the absence of circulating blood. Exaggerated coronary myogenic tone or increased arterial stiffness in coronary arteries from diabetic hearts may lead to increased cardiac ischemia. (Shehadeh & Regan, 1995). Laguad et al. and Frisebee et al. (Frisbee et al, 2002; Lagaud et al, 2001), in separate studies on db/db mouse and Zucker rat models of type 2 DM, reported that myogenic tone is increased in mesenteric and  75 skeletal muscle arterioles respectively. Crijns et al. demonstrated increased arterial stiffness in streptozocin-induced type 1 diabetic rats (Crijns et al, 1999). Our results suggest that in the coronary vascular bed, myogenic response and passive distensibility are the same in db/db mice compared to WT mice.  Exercise did not have a significant effect on myogenic tone or arterial passive distensibility in WT or db/db mice.  Moreover, our study shows that the constrictor responses of smooth muscle cells to pressure and a thromboxane agonist were similar in diabetic and WT mice. Therefore, an increased myogenic response or decreased vascular compliance cannot completely account for reduced cardiac function in db/db mice (How et al, 2006). We observed a marked reduction in ACh-induced, endothelium-dependent NO-mediated vasoidilation in coronary septal arteries isolated from db/db mice. It is possible that since the arterioles were preconstricted with U46619, the lower dilator response to ACh was related to a greater preconstriction by U46619, as reported in some arteries from db/db mice (Khazaei et al, 2007; Verma et al, 2002). However, the concentration response curves to U46619 were similar in sedentary and exercised db/db and WT mice in our study.  Alterations in ACh receptor activity in DM may also lead to decreases in ACh-mediated relaxation in db/db mice (Carrier & Aronstam, 1987; Carrier & Aronstam, 1990). However, the decline in ACh Emax in the absence of a change in EC50 in our results indicates that ACh receptor function remained relatively unaltered in diabetic arteries. The reduced NO-mediated relaxation in arteries of db/db mice was not due to decreased expression of NO generating enzyme protein (eNOS), in agreement with other studies showing an unaltered or even upregulated eNOS protein expression in DM (Cosentino et al, 1997; Okon et al, 2003). Although eNOS is present, it is possible that the activity and/ or regulation of this enzyme may be negatively altered in DM (Forstermann & Munzel, 2006).  Moreover, it is unlikely that smooth muscle cell sensitivity to NO or activation of vascular smooth muscle cell guanylate cyclase is altered in diabetic mice since the vasodilation produced by SNP was unaltered by exercise.  76 Alterations in the co-factor and substrate regulation in eNOS activity have been reported in diabetes (Cai et al, 2005; Giugliano et al, 1996).  Moderate levels of exercise in db/db mice corrected the relative deficiency of L-Arg and BH4 in db/db coronary arteries. In sedentary db/db mice, incubation of coronary arteries with L-Arg plus BH4 reversed the impaired ACh response. Such a deficiency of cofactors (Cai et al, 2005; Pannirselvam et al, 2002; Pannirselvam et al, 2003) and substrates (Pieper et al, 1996; Pieper & Dondlinger, 1997) of NOS were reported previously and considered to be due to enhanced consumption of the substrate (Giugliano et al, 1996) or directdegradation of BH4 by excessive free radicals such as peroxynitrite (Milstien & Katusic, 1999), a common feature of DM (Pacher & Szabo, 2006). It is likely that exercise ameliorates oxidative stress both in the whole body and the heart (e.g. reduction in nitrotyrosine, a biomarker for peroxynitrite activity) and coronary arteries (e.g. endothelium-dependent relaxation was not further improved by incubation with L-Arg and BH4 or SOD) in exercised db/db mice. The incidence of ischemic heart disease is greater in diabetic patients (Shehadeh & Regan, 1995; Turner et al, 1998). Alterations in myogenic regulation of arteriolar diameter are likely to detrimentally affect regional myocardial blood flow. Since the pressure-constriction curves were similar in coronary arteries from control and diabetic mice, we speculate that a greater myogenic tone in coronary arteries of diabetic mice is unlikely to be the primary cause of cardiac ischemia in this model of type 2 DM (Anzawa et al, 2006). The markedly reduced ACh induced, NO mediated coronary vasodilation in db/db mice was related to a greater oxidative stress and reduced NO bioavailability in diabetes, likely leading to an imbalance between cardiac oxygen supply and demand during activity.  Moreover, during ischemia/reperfusion during myocardial infarction in diabetes, poorer outcome is often predicted due to more extensive myocardial damage as a consequence of low antioxidant levels (Ghosh et al, 2005; Tao et al, 2007).  According to our study, moderate levels of exercise not only increases myocardial antioxidant levels but also increases NO bioavailability, thus leading to improved endothelium dependent vasodilation, and better perfusion of the diabetic heart.  77   WT WT exe db/db db/db exe  Heart weight, mg 177 ± 6  184 ± 6  158 ± 6 170 ± 7 Body weight, g 27.01 ± 0.39 26.5 ± 0.54 49.68 ± 0.36 46.20 ± 1.18* Plasma glucose, mmol/l 6.45 ± 0.31 5.74 ± 0.24 47.53 ± 3.90° 48.29 ± 4.11° Plasma Insulin, µg/l 1.51 ± 0.15 1.16 ± 0.16 3.64 ± 0.55° 3.73 ± 0.73° Citrate synthase, µmol/min/mg protein  52.46 ± 3.14 68.43 ± 2.93° 51.41 ± 2.39 67.12 ± 1.77*  Plasma 8-isoprostane, pg/ml 8.88 ± 5.11 7.9 ± 4.11 43.49 ± 3.21° 16.58 ± 3.52*  Table 3-1a: General Characteristics of Animals Values are means ± SE for 8-10 mice in each group.  Parameters were measured at the time of euthanasia (13 weeks old). °p< 0.05 vs. WT, *p< 0.01 vs. db/db; n = 8-10 in each group.      Glucose (mmol/L) Time (Minutes) WT WT exe db/db db/db exe 0 6.78 ± 0.63  6.44 ± 0.60  37.97 ± 1.91* 35.03 ± 2.77* 10 14.12 ± 1.03 15.65 ± 0.41 42.70 ± 5.28* 49.84 ± 2.73* 20 13.04 ± 1.37 14.16 ± 0.92 48.16 ± 2.43* 49.56 ± 3.96* 60 9.15 ± 1.04 8.76 ± 0.46 40.61 ± 2.58* 41.75 ± 3.05* 120 6.95 ± 0.40 6.72 ± 0.57 32.84 ± 1.00* 28.20 ± 1.59*  Table 3-1b: Oral Glucose Tolerance Test performed in mice that were exercised for five weeks. Mice were fasted for six hours and glucose loaded with 1.5 g/kg. (*p<0.01 db/db and db/db exe compared to WT and WT exe, repeated measures ANOVA, n =9-10 per group)  78  Table 3-2: Vasomotor responses of isolated coronary arteries. Values are means ± SE for 8-10 mice in each group.  Data are from 13-week-old animals.   WT WT exe db/db db/db exe  Passive diameter at 80 mmHg (percent constriction) 168.4 ± 2.7 174.3 ± 4.9 167.0 ± 4.8 168.2 ± 4.9 Myogenic tone at 80 mmHg (percent constriction) 34.4 ± 4.1 34.0 ± 3.9 41.1 ± 4.5 37.5 ± 5.2 U46619 [- logEC50 (M)] 6.56 ± 0.15 6.80 ± 0.14 6.52 ± 0.18 6.76 ± 0.13 U46619  Emax (percent constriction)  75.91 ± 6.18 72.60 ± 4.78 68.20 ± 3.34 75.38 ± 4.65    79 AWT WT  Ex e db /db  db /db  Ex e 0 1 2 3 4 5 O D ×  m m 2 * * A B WT WT  Ex e db /db db /db  Ex e 0 1 2 3 4 O D ×  m m 2 B C WT WT  Ex e db /db db /db  Ex e 0.0 0.5 1.0 1.5 O D ×  m m 2 C D WT WT  Ex e db /db db /db  Ex e 0 1 2 3 4 O D ×  m m 2 D * * E WT WT  Ex e db /db db /db  Ex e 0.0 2.5 5.0 7.5 O D ×  m m 2 E WT WT  Ex e db /db db /db  Ex e 0.0 0.5 1.0 1.5 O D ×  m m 2 * * F db /db  Ex e  x e  x e db / db /db  Ex e db /db  Ex e db /db db /db db / db /db db /db b/d b    80 Figure 3-1: Protein expression of antioxidants and eNOS in the whole heart. A- Mn-SOD is significantly decreased in whole hearts extract of db/db mice compared to lean WT mice. Exercise significantly increased the level of Mn-SOD in the db/db mice hearts. B- Cu,Zn-SOD expression is not different in the hearts from the exercise and sedentary groups. C- Catalase expression shows no significant difference amongst the groups. D- Nitrotyrosine levels are significantly higher in db/db hearts. Exercise decreased the levels significantly in db/db hearts. E- eNOS expression shows no significant difference amongst the groups. F.- Extracellular SOD expression increases with exercise in db/db hearts compared to sedentary littermates and lean WT mice. (*p<0.05, n = 6 in all groups, one-way ANOVA).                 81    Figure 3-2: Intracardiac localization of Mn-SOD by immunofluorescence.  Formalin-fixed heart sections were analyzed by double immunostaining for von Willebrand factor (red, vWF) to label endothelial cells and Mn-SOD (green, SOD2).  Images A-C represent lean WT control hearts, whereas D-F, G-I and J-L represents exercised WT, db/db and exercised db/db hearts respectively. A, D, G, J represents sections of hearts and coronary arteries stained for vWF (shown with white arrow in A). B, E, H, and K represent Mn-SOD (shown with white arrows). E and K demonstrate increased Mn-SOD staining both in the myocardium and endothelial cells (shown in block arrows in K and L). C, F, I and L represent composite figures with DAPI counterstaining (blue), representing nucleus. All images were taken at 630X magnification.  82 A B C F D E 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 WT db/db Transmural Pressure (mmHg) Pe rc en t C on st ri ct io n 0 20 40 60 80 100 120 140 100 120 140 160 180 200 WT WT Exe db/db db/db Exe Transmural Pressure (mmHg) Pa ss iv e Di st en si bi lit y (%  D ia m  a t 1 0m m H g) 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 WT with Bosentan WT Transmural Pressurem (mmHg) Pe rc en t C on st ri ct io n 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 db/db Exe db/db Exe with Bosentan Transmural Pressure (mmHg) Pe rc en t C on st ri ct io n 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 db/db db/db with bosentan Transmural Pressure (mmHg) Pe rc en t C on st ri ct io n 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 WT Exe WT Exe  w ith Bosentan Transmural Pressure (mmHg) Pe rc en t C on st ri ct io n   Figure 3-3: Myogenic tone and passive distensibility in coronary septal arteries: A- myogenic tone in coronary arteries of db/db mice and WT mice are not significantly different. B- passive distensibility of coronary arteries of sedentary and exercised db/db mice and WT mice do not show statistical difference. C and E- myogenic tone with or without bosentan in coronary arteries of sedentary and exercised WT mice respectively. D and F- myogenic tone with or without bosentan in coronary arteries of sedentary and exercised db/db mice respectively. Incubation with bosentan indicated that endogenous endothelin-1 does not have a significant effect on vascular myogenic tone. (n = 8-10 in each group)  83 ACh/ SNP 1nM-10µM U46619 0.1 µM 70 140 Internal Diameter ( µm) C- db/db Exercised ACh/ SNP 1nM-10µM U46619 0.1 µM 53 97 Internal Diameter ( µm) ACh/ SNP 1nM-10µM U46619 0.1 µM 79 128 Internal Diameter ( µm) B- db/db A- WT // b / b Exercised A- T  Figure 3-4: Endothelium-dependent and -independent response traces in coronary septal arteries. Traces illustrating diameter of septal coronary arteries from WT (A), db/db (B), and db/db exercised (C) mice that were preconstricted with U46619 (10-7M) and challenged with cumulative concentrations of ACh (endothelium-dependent vasodilator) and SNP (endothelium-independent vasodilator). In WT mice (A) both ACh and SNP cause relaxation (increase in diameter) of coronary arteries by about 80-90%. However, there is a marked attenuation in ACh-induced relaxation in db/db mice (B) (~ 50% relaxation) while SNP-mediated relaxation is unchanged. Exercise improved ACh-induced aortic relaxation to 80% in db/db mice (C).  84 -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 Log [ACh] M Pe rc en t R el ax at io n -10 -9 -8 -7 -6 -5 -4 20 40 60 80 100 WT db/db db/db Exe Log [SNP] M Pe rc en t R el ax at io n A B D ACh ACh SNPSNP 0.0 1.5 3.0 4.5 6.0 7.5 db/db Exe db/db WT EC 50  -l og [D ru g] (M ) C 0 25 50 75 100 E ( m ax ) ( % ) * *  Figure 3-5: Concentration response curves for endothelium-dependent (ACh mediated, A) and endothelium independent (SNP mediated, B) vasorelaxation in coronary arteries. There is a marked decline in ACh induced vasodilation in db/db mice compared to WT mice (* p<0.05, n = 8-10 in each group, repeated measures ANOVA). Exercise preserved endothelium dependent ACh mediated vasodilation in db/db mice. C- EC50 and D- Emax of the ACh and SNP response in the coronary arteries. EC50 is not statistically different amongst groups for both ACh and SNP response. Emax is significantly decreased in db/db mice compared to WT group (* p<0.05, n = 8-10 in each group, repeated measures ANOVA). Exercise significantly increased Emax in db/db mice. For SNP response there was no significant difference in Emax response amongst groups.  85 *A B DC * No  dr ug SO D L- Ar g +  B H 4 0.0 1.5 3.0 4.5 6.0 7.5 db/db Exe db/db EC 50  -l og [A C h] (M ) No  dr ug SO D L- Ar g +  B H 4 0 25 50 75 100 E ( m ax ) ( % ) -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 db/db Exe db/db Exe + L-Arg + BH4 db/db Exe + SOD Log [ACh] M Pe rc en t R el ax at io n -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 db/db db/db + L-Arg + BH4 db/db + SOD Log [ACh] M Pe rc en t R el ax at io n   Figure 3-6: Effect of L-Arg + BH4 and SOD incubation on ACh mediated vasorelaxation in coronary septal arteries of sedentary and exercised db/db mice. Decreased ACh induced vasodilation response was significantly improved after incubation with L-Arg + BH4 or SOD in db/db mice (A and B) and not in exercised db/db mice. The Emax and EC50 values for ACh concentration-response curves before and after L-Arg + BH4 and SOD incubation are shown in C- and D.  (* p<0.01, n = 8-10 in each group, repeated measures ANOVA).  86 3.5 Bibliography Anzawa,R., Bernard,M., Tamareille,S., Baetz,D., Confort-Gouny,S., Gascard,J.P., Cozzone,P., & Feuvray,D. (2006) Intracellular sodium increase and susceptibility to ischaemia in hearts from type 2 diabetic db/db mice. Diabetologia, 49, 598-606. Ascensao,A., Ferreira,R., & Magalhaes,J. (2007) Exercise-induced cardioprotection--biochemical, morphological and functional evidence in whole tissue and isolated mitochondria. Int.J Cardiol., 117, 16-30. Bagi,Z., Erdei,N., Toth,A., Li,W., Hintze,T.H., Koller,A., & Kaley,G. (2005) Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins. Arterioscler.Thromb.Vasc.Biol., 25, 1610-1616. Bagi,Z., Koller,A., & Kaley,G. (2003) Superoxide-NO interaction decreases flow- and agonist- induced dilations of coronary arterioles in Type 2 diabetes mellitus. Am.J Physiol Heart Circ.Physiol, 285, H1404-H1410. Brownlee,M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813-820. Cai,S., Khoo,J., Mussa,S., Alp,N.J., & Channon,K.M. (2005) Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation. Diabetologia, 48, 1933-1940. Carrier,G.O. & Aronstam,R.S. (1987) Altered muscarinic receptor properties and function in the heart in diabetes. J Pharmacol.Exp.Ther., 242, 531-535. Carrier,G.O. & Aronstam,R.S. (1990) Increased muscarinic responsiveness and decreased muscarinic receptor content in ileal smooth muscle in diabetes. J Pharmacol.Exp.Ther., 254, 445-449. Church,T.S., Cheng,Y.J., Earnest,C.P., Barlow,C.E., Gibbons,L.W., Priest,E.L., & Blair,S.N. (2004) Exercise capacity and body composition as predictors of mortality among men with diabetes. Diabetes Care, 27, 83-88. Cohen,G., Riahi,Y., Alpert,E., Gruzman,A., & Sasson,S. (2007) The roles of hyperglycaemia and oxidative stress in the rise and collapse of the natural protective mechanism against vascular endothelial cell dysfunction in diabetes. Arch.Physiol Biochem., 113, 259-267. Cosentino,F., Barker,J.E., Brand,M.P., Heales,S.J., Werner,E.R., Tippins,J.R., West,N., Channon,K.M., Volpe,M., & Luscher,T.F. (2001) Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler.Thromb.Vasc.Biol., 21, 496-502. Cosentino,F., Hishikawa,K., Katusic,Z.S., & Luscher,T.F. (1997) High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation, 96, 25-28. Crijns,F.R., Wolffenbuttel,B.H., De Mey,J.G., & Struijker Boudier,H.A. (1999) Mechanical properties of mesenteric arteries in diabetic rats: consequences of outward remodeling. Am.J Physiol, 276, H1672-H1677.  87 da Silva,P.A., Lambertucci,R.H., Gorjao,R., Dos Reis,S.L., & Curi,R. (2007) Effect of a single session of electrical stimulation on activity and expression of citrate synthase and antioxidant enzymes in rat soleus muscle. Eur.J Appl.Physiol, 102, 119-126. Eriksen,L., Dahl-Petersen,I., Haugaard,S.B., & Dela,F. (2007) Comparison of the effect of multiple short-duration with single long-duration exercise sessions on glucose homeostasis in type 2 diabetes mellitus. Diabetologia, 50, 2245-2253. Forstermann,U. & Munzel,T. (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113, 1708-1714. Frisbee,J.C., Maier,K.G., & Stepp,D.W. (2002) Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats. Am.J Physiol Heart Circ.Physiol, 283, H2160-H2168. Ghosh,S., Novak,E.M., & Innis,S.M. (2007) Cardiac proinflammatory pathways are altered with different dietary n-6 linoleic to n-3 alpha-linolenic acid ratios in normal, fat-fed pigs. Am.J Physiol Heart Circ.Physiol, 293, H2919-H2927. Ghosh,S., Pulinilkunnil,T., Yuen,G., Kewalramani,G., An,D., Qi,D., Abrahani,A., & Rodrigues,B. (2005) Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion. Am.J Physiol Heart Circ.Physiol, 289, H768-H776. Giugliano,D., Ceriello,A., & Paolisso,G. (1996) Oxidative stress and diabetic vascular complications. Diabetes Care, 19, 257-267. Graier,W.F., Posch,K., Fleischhacker,E., Wascher,T.C., & Kostner,G.M. (1999) Increased superoxide anion formation in endothelial cells during hyperglycemia: an adaptive response or initial step of vascular dysfunction? Diabetes Res.Clin.Pract., 45, 153-160. Hayashi,T., Ueno,Y., & Okamoto,T. (1993) Oxidoreductive regulation of nuclear factor kappa B. Involvement of a cellular reducing catalyst thioredoxin. J Biol.Chem., 268, 11380-11388. How,O.J., Aasum,E., Severson,D.L., Chan,W.Y., Essop,M.F., & Larsen,T.S. (2006) Increased myocardial oxygen consumption reduces cardiac efficiency in diabetic mice. Diabetes, 55, 466-473. Hu,F.B., Stampfer,M.J., Solomon,C., Liu,S., Colditz,G.A., Speizer,F.E., Willett,W.C., & Manson,J.E. (2001) Physical activity and risk for cardiovascular events in diabetic women. Ann.Intern.Med., 134, 96-105. Ito,I., Jarajapu,Y.P., Guberski,D.L., Grant,M.B., & Knot,H.J. (2006) Myogenic tone and reactivity of rat ophthalmic artery in acute exposure to high glucose and in a type II diabetic model. Invest Ophthalmol.Vis.Sci., 47, 683-692. Kemi,O.J., Ellingsen,O., Smith,G.L., & Wisloff,U. (2008) Exercise-induced changes in calcium handling in left ventricular cardiomyocytes. Front Biosci., 13, 356-368. Khazaei,M., Moien-Afshari,F., Kieffer,T.J., & Laher,I. (2007) Effect of exercise on augmented aortic vasoconstriction in the db/db mouse model of type-II diabetes. Physiol Res. Kojda,G. & Hambrecht,R. (2005) Molecular mechanisms of vascular adaptations to exercise. Physical activity as an effective antioxidant therapy? Cardiovasc.Res., 67, 187-197.  88 Laakso,M. (1999) Hyperglycemia and cardiovascular disease in type 2 diabetes. Diabetes, 48, 937- 942. Lagaud,G.J., Masih-Khan,E., Kai,S., van Breemen,C., & Dube,G.P. (2001) Influence of type II diabetes on arterial tone and endothelial function in murine mesenteric resistance arteries. J Vasc.Res., 38, 578-589. Leung,F.P., Yung,L.M., Leung,H.S., Au,C.L., Yao,X., Vanhoutte,P.M., Laher,I., & Huang,Y. (2007) Therapeutic concentrations of raloxifene augment nitric oxide-dependent coronary artery dilatation in vitro. Br.J Pharmacol., 152, 223-229. Mahgoub,M.A. & Abd-Elfattah,A.S. (1998) Diabetes mellitus and cardiac function. Mol.Cell Biochem., 180, 59-64. Milstien,S. & Katusic,Z. (1999) Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem.Biophys.Res.Commun., 263, 681-684. Moien-Afshari,F., Ghosh,S., Khazaei,M., Kieffer,T.J., Brownsey,R.W., & Laher,I. (2008) Exercise restores endothelial function independently of weight loss or hyperglycaemic status in db/db mice. Diabetologia. Moien-Afshari,F., Skarsgard,P.L., McManus,B.M., & Laher,I. (2004) Cardiac transplantation and resistance artery myogenic tone. Can.J Physiol Pharmacol., 82, 840-848. Montuschi,P., Barnes,P., & Roberts,L.J. (2007) Insights into oxidative stress: the isoprostanes. Curr.Med.Chem., 14, 703-717. Nassis,G.P., Papantakou,K., Skenderi,K., Triandafillopoulou,M., Kavouras,S.A., Yannakoulia,M., Chrousos,G.P., & Sidossis,L.S. (2005) Aerobic exercise training improves insulin sensitivity without changes in body weight, body fat, adiponectin, and inflammatory markers in overweight and obese girls. Metabolism, 54, 1472-1479. Nishikawa,T., Edelstein,D., Du,X.L., Yamagishi,S., Matsumura,T., Kaneda,Y., Yorek,M.A., Beebe,D., Oates,P.J., Hammes,H.P., Giardino,I., & Brownlee,M. (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 404, 787-790. Nitenberg,A., Paycha,F., Ledoux,S., Sachs,R., Attali,J.R., & Valensi,P. (1998) Coronary artery responses to physiological stimuli are improved by deferoxamine but not by L-arginine in non-insulin-dependent diabetic patients with angiographically normal coronary arteries and no other risk factors. Circulation, 97, 736-743. Okon,E.B., Szado,T., Laher,I., McManus,B., & van Breemen,C. (2003) Augmented contractile response of vascular smooth muscle in a diabetic mouse model. J Vasc.Res., 40, 520-530. Pacher,P. & Szabo,C. (2006) Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes. Curr.Opin.Pharmacol., 6, 136-141. Pannirselvam,M., Simon,V., Verma,S., Anderson,T., & Triggle,C.R. (2003) Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br.J Pharmacol., 140, 701-706.  89 Pannirselvam,M., Verma,S., Anderson,T.J., & Triggle,C.R. (2002) Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br.J Pharmacol., 136, 255-263. Pieper,G.M. & Dondlinger,L.A. (1997) Plasma and vascular tissue arginine are decreased in diabetes: acute arginine supplementation restores endothelium-dependent relaxation by augmenting cGMP production. J Pharmacol.Exp.Ther., 283, 684-691. Pieper,G.M., Jordan,M., Adams,M.B., & Roza,A.M. (1996) Restoration of vascular endothelial function in diabetes. Diabetes Res.Clin.Pract., 31 Suppl, S157-S162. Pieper,G.M., Meier,D.A., & Hager,S.R. (1995) Endothelial dysfunction in a model of hyperglycemia and hyperinsulinemia. Am.J Physiol, 269, H845-H850. Pieper,G.M. & Siebeneich,W. (1998) Oral administration of the antioxidant, N-acetylcysteine, abrogates diabetes-induced endothelial dysfunction. J Cardiovasc.Pharmacol., 32, 101-105. Rahman,M.M., Elmi,S., Chang,T.K., Bai,N., Sallam,N.A., Lemos,V.S., Moien-Afshari,F., & Laher,I. (2007) Increased vascular contractility in isolated vessels from cigarette smoking rats is mediated by basal endothelin release. Vascul.Pharmacol., 46, 35-42. Rush,J.W., Laughlin,M.H., Woodman,C.R., & Price,E.M. (2000) SOD-1 expression in pig coronary arterioles is increased by exercise training. Am.J Physiol Heart Circ.Physiol, 279, H2068- H2076. Shehadeh,A. & Regan,T.J. (1995) Cardiac consequences of diabetes mellitus. Clin.Cardiol., 18, 301-305. Singel,D.J. & Stamler,J.S. (2005) Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin. Annu.Rev.Physiol, 67, 99-145. Tao,L., Gao,E., Jiao,X., Yuan,Y., Li,S., Christopher,T.A., Lopez,B.L., Koch,W., Chan,L., Goldstein,B.J., & Ma,X.L. (2007) Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress. Circulation, 115, 1408-1416. Turner,R.C., Millns,H., Neil,H.A., Stratton,I.M., Manley,S.E., Matthews,D.R., & Holman,R.R. (1998) Risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: United Kingdom Prospective Diabetes Study (UKPDS: 23). BMJ, 316, 823-828. Ungvari,Z., Pacher,P., Kecskemeti,V., Papp,G., Szollar,L., & Koller,A. (1999) Increased myogenic tone in skeletal muscle arterioles of diabetic rats. Possible role of increased activity of smooth muscle Ca2+ channels and protein kinase C. Cardiovasc.Res., 43, 1018-1028. Verma,I.M., Stevenson,J.K., Schwarz,E.M., Van Antwerp,D., & Miyamoto,S. (1995) Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev., 9, 2723- 2735. Verma,S., Arikawa,E., Lee,S., Dumont,A.S., Yao,L., & McNeill,J.H. (2002) Exaggerated coronary reactivity to endothelin-1 in diabetes: reversal with bosentan. Can.J Physiol Pharmacol., 80, 980-986. Wing,R.R., Goldstein,M.G., Acton,K.J., Birch,L.L., Jakicic,J.M., Sallis,J.F., Jr., Smith-West,D., Jeffery,R.W., & Surwit,R.S. (2001) Behavioral science research in diabetes: lifestyle  90 changes related to obesity, eating behavior, and physical activity. Diabetes Care, 24, 117- 123. Yamashita,N., Hoshida,S., Otsu,K., Asahi,M., Kuzuya,T., & Hori,M. (1999) Exercise provides direct biphasic cardioprotection via manganese superoxide dismutase activation. J Exp.Med., 189, 1699-1706.   91 4 EFFECT OF EXERCISE ON AUGMENTED AORTIC VASOCONSTRICTION IN THE db/db MOUSE MODEL OF TYPE 2 DIABETES3   3. A version of this chapter has been published. Kazaei M, Moien-Afshari F, Kieffer TJ, Laher I. Physiol Res 2008; 57(6):847-56.   92 4.1 Introduction Diabetes is associated with an increased incidence of cardiovascular disease (Garcia et al., 1974). It is likely that vascular smooth muscle cell responsiveness is altered in diabetes where there is an attenuated response to nitric oxide (Tesfamariam & Cohen, 1992), while at the same time the constrictor response to phenylephrine (PE) is enhanced (Agrawal and McNeill, 1987, Kamata et al. 1988). These abnormal vasomotor regulatory effects may in part underlie the cardiovascular morbidity and mortality that is a common complication of non-insulin dependent diabetic mellitus. Several studies revealed that contractile response to α-adrenergic stimuli in diabetic rats are enhanced (Abebe et al. 1990; MacLeod, 1985, Taylor et al. 1994), but the effect of exercise on increased contractile response of vascular smooth muscle cell is unknown. Exercise is generally thought to provide cardioprotection (Sesso et al. 2000; Paffenbarger, Jr. et al. 1993, Myers et al. 2002; Jolliffe et al.2001) and has several well-documented cardiovascular and systemic effects. The effects of exercise include improvements in endothelial function, left ventricular diastolic function, arterial stiffness, systematic inflammation, and reduced total and abdominal fat; it is thought that these effects of exercise may be largely responsible for improving insulin sensitivity and endothelial function in diabetes (Stewart, 2004a, Stewart, 2004b). The beneficial effects of life style modifications such as exercise in diabetic subjects is as important as controlling plasma glucose (Knowler et al. 2002). In this study, we used db/db mice, an animal model of non-insulin dependent diabetic model (Guo et al. 2005; Kamata et al. 1988, Pannirselvam et al. 2002, Pannirselvam et al. 2003) to test the hypothesis that exercise decreases the constrictor response of vascular smooth muscle cells to adrenergic stimulation.  4.2 Methods 4.2.1 Animal groups Twenty male db/db (BKS.cg-m +/+ Leprdb/J) and age-matched wild-type (WT) mice (aged 5 weeks) were purchased from Jackson Laboratories (USA). All experimental protocols were  93 approved by the Animal Care Committee of the University of British Columbia. Animals were housed in groups of 5 per cage with a 12h light/dark cycle at 26ºC and allowed access to food and drinking water ad libitum. Each group was randomized to exercised (n=10) and sedentary subgroups (n=10).  4.2.2 Exercise training program Mice in the exercise group were trained to run on a motorized exercise wheel system (Lafayette Instrument Co, IN, USA). The initial two-week period involved a training period during which the exercise intensity was gradually increased. The initial exercise speed was 2.5 meters/min for one hour (150m) and incrementally changed to 5.2 meters/min (312 m) (Table 4-1), which is well below the exercise tolerance level in mice (Verma-Ahuja et al. 2000). Mice were exercised five days per week for the duration of the experiment (7 weeks, 2 weeks training included) at a set time each day (De Angelis et al., 2004; Tang & Reed, 2001). The integrated digital interface on the motorized exercise training wheel system controlled the wheel speed and duration of exercise. Sedentary animals were placed in non-rotating wheels daily for the same duration as the exercise group.  4.2.3 Isometric force measurement Mice were euthanized when aged 12 weeks: mice were anaesthetized by injection of pentobarbital sodium (Somnotol 30 mg/kg, i.p.) and heparin sodium (50 U/kg; i.p). The thoracic aortae were excised and placed in ice-cold physiologic salt solution (PSS, see solutions and chemicals) where they were carefully cleaned of fat and surrounding connective tissue. Segments of aortae were threaded with stainless steel wire (0.02mm diameter) and attached to tissue holders of a 4-channel wire myograph (JP Trading, Aarhus, Denmark) containing PSS solution aerated with 95% O2-5% CO2. Tissues were allowed to equilibrate for 60 min at 37°C, during which time the PSS was replaced at 20-30 minute intervals. During the equilibration, the resting tension was gradually increased to 5mN (milli newton) and kept at this level for 20-30 minutes. Each tissue was  94 maximally activated with a solution of KCl (80mM) that was prepared by equimolar substitution of NaCl in PSS. Following washout with fresh PSS and return of tension to basal preload, phenylephrine (PE, 1 µmol/l) was added to establish a stable contraction. Thereafter, cumulative additions of ACh (1 nmol/l to 10 µmol/l) were made. The same protocol was repeated for SNP (1 nmol/l to 10 µmol/l). Following washout, constrictor responses to phenylephrine (PE, 1nM to10µM) were obtained using cumulative additions. After washout, the PE concentration-response curves were repeated in the presence of each of the following: nitric oxide synthase (NOS) blocker, N-nitro-L-arginine methyl ester (L-NAME, 200µM), endothelin dual receptor (A and B) antagonist (bosentan, 10 µM), protein kinase C (PKC) inhibitor (calphostin C, 5µM), cyclooxygenase inhibitor (indomethacin, 10µM) or the Rho-kinase inhibitor (Y-27632, 0.1µM). In other tissues, concentration-response curves were also made to indolactam, a PKC activator, (10-8 to 10-5M). All data were recorded on a computer using MyoDaq Acquisition software (Danish Myo Technology, Aarhus, Denmark).  4.2.4 Measurement of plasma parameters Animals were fasted for 12 h before euthanasia. Blood sample was collected from the inferior vena cava and immediately dispensed into tubes (Microtainer, Becton Dickinson, USA) and centrifuged at 8000g for 10 min for plasma generation. Plasma samples were then collected in separate Eppendorf tubes and stored at –70°C for further analysis. Plasma lipid concentrations were measured using a Dimension® Clinical Chemistry System (GMI, Ramsey, Minnesota, USA). Plasma glucose and insulin levels were measured using commercially available assay kits.  4.2.5 Citrate synthase assay To document the presence of an endurance-trained state, citrate synthase activity assays were performed on skeletal muscle. After sacrificing the animals, thigh adductor muscles were gently removed and frozen, and citrate synthase activity was measured as previously described (Korzick et  95 al. 2004, Spier et al. 1999).  4.2.6 Drugs and Chemicals Acetylcholine, sodium nitroprusside, phenylephrine, L-NAME, calphostin C, indomethacin and Y- 27632 were purchased from Sigma Chemical Co (St. Louis, MO). The composition of the PSS (mM) was: NaCl (119), KCl (4.7), KH2PO4 (1.18), MgSO4 (1.17), NaHCO3 (24.9), EDTA (0.023), CaCl2 (1.6), dextrose (11.1). Isotonic substitutions (replacement of Na+ with equimolar concentrations of K+) were used when using PSS solutions with increased K+ concentrations.  4.2.7 Statistical analysis and calculations Results are expressed as mean ± SEM. Data analysis and curve fitting were made with NCSS-2000 software and GraphPad Prism (version 3.02-2000), respectively. ANOVA and repeated measures ANOVA with multiple comparisons using Bonferroni’s test or student t-test was performed where appropriate. A value of p< 0.05 was considered as being statistically significant.  4.3 Results 4.3.1 Body weight Fig 4-1 illustrates age related changes in the weight of mice in the experimental groups. At the beginning of the study (mice aged 5 weeks), the body weights of db/db mice were greater than WT, and this increased to an approximately two-fold difference at the end of the study. Exercised db/db mice had lower body weights compared to their sedentary counterparts (p<0.05).  4.3.2 Plasma Parameters At the end of the study, plasma glucose and insulin concentrations in db/db mice were significantly greater than in WT mice. Exercise did not alter either plasma glucose or insulin levels (p>0.05) (Table 4-2). Plasma levels of cholesterol, LDL, and TG were higher in db/db sedentary compared to  96 WT (p<0.05). Exercise significantly decreased cholesterol, LDL and TG in diabetic mice (p<0.05) without significantly changing the HDL concentration (p>0.05).  4.3.3 Efficacy of exercise training program The levels of citrate synthase activity were significantly higher in the thigh adductor muscles of exercised db/db mice (69.44 ± 4.05 µmol/min/mg Pr) compared to the sedentary db/db mice group (51.42 ± 2.41 µmol/min/mg Pr) (p<0.01, n=5-7 per group). In addition, there was a significant difference between WT and exercised WT mice (52.44 ± 3.05 vs. 70.33 ± 4.10 µmol/min/mg Pr; p<0.05, n=6-7).  4.3.4 Endothelium-dependent and -independent vasodilation The maximum response of endothelium-dependent vasodilation produced by ACh was impaired in aortic rings from db/db mice compared to their control counterparts (Fig 4-2). Exercise significantly improved endothelium-dependent vasodilation in db/db (p<0.01). Endothelium-independent vasodilation induced by SNP was similar in db/db and WT mice and exercise did not alter this response in any of the experimental groups (data not shown).  4.3.5 Aortic contractile responses Contractility of mice aortae to 80mM KCl was not significantly different in sedentary and exercised db/db and WT mice (Fig 4-4B). The maximal force generated in response to the alpha-adreneregic receptor agonist PE (1nM to10µM) was markedly greater in db/db aortae. Exercise did not attenuate this augmented contractile response in db/db mice (Fig.4-3 & 4-4A) and also did not change PE constriction response in WT mice (Appendix B, Fig 7-1C). Fig 4-4C illustrates the Emax and EC50 of PE-response in all groups. Both sensitivity (EC50) and maximum constriction (Emax) to PE was greater in diabetic mice compared to WT; these parameters were unaffected by exercise.   97 4.3.6 Effect of cyclooxygenase inhibitor (indomethacin) The endothelium of the mouse aorta produces sufficient PGH2/TXA2 to initiate large contractions (Okon et al. 2002). To examine the role of PGH2/TXA2 in the augmented contractile response in diabetic mice aortae, we used indomethacin, a cyclooxygenase inhibtor. Incubation of aortae with indomethacin (10µM) did not attenuate either the enhanced PE-induced constriction in db/db and exercised db/db mice or the altered the sensitivity to PE (data not shown).  4.3.7 Effect of endothelin-1 receptor antagonist (bosentan), and Rho-kinase inhibitor (Y- 27632) To examine the possible role of endothelin-1 and Rho-kinase in the augmented PE-induced contractions in db/db aortae, PE-concentration response curves were reproduced in the presence of either bosentan or Y-27632. Pre-treatment with bosentan (10-5M) or Y-27632 (10-7M), did not change the maximal PE-induced constriction or EC50 in db/db mice. Similar results were obtained in the exercised db/db group (data not shown).  4.3.8 Effect of NOS blocker (L-NAME) The role of basal NO in the PE-induced contractions in WT and db/db aortae was studied by comparing PE-concentration response curves in the absence and presence of L-NAME, a NOS inhibitor. Pre-treatment with L-NAME (200µM), did not change the maximal PE-induced constriction or EC50 in db/db mice. Similar results were obtained in the exercised db/db mice (data not shown).  4.3.9 Effect of PKC inhibitor (Calphostin-C) The PE concentration-response curves in the aorta from WT mice were not affected by pretreatment of vessels with calphostin-C, a PKC inhibitor (5×10-6M). In contrast, calphostin-C reduced the  98 increased PE-induced constriction in both db/db sedentary and exercised db/db mice to levels similar to that observed in WT (p<0.05) (Fig 4-5).  4.3.10 PKC activator (indolactam) concentration-response curve Cumulative concentrations of indolactam (10-8 to 10-5), a PKC activator, induced significantly greater constrictor responses in aortic rings of db/db compared to WT mice. Exercise did not affect this exaggerated response to PKC activation (Fig 4-6).  4.4 Discussion The aim of this study was to examine the effect of exercise on PE-constrictor responses in db/db mice. We demonstrate that the constrictor responses of smooth muscle cells to the alpha-adrenergic stimulant, PE, and the PKC activator, indolactam, are markedly enhanced in aortae of diabetic mice and that exercise did not attenuate this exaggerated constrictor response. Levels of citrate synthase activity in the thigh adductor muscle (Korzick et al. 2004, Spier et al. 1999) were significantly increased in exercised db/db mice, confirming the systemic effects of the exercise protocol used in this study. The db/db mice had raised plasma glucose and insulin levels, likely as a result of insulin resistance in this model of type 2 diabetes. Exercise did not change plasma glucose and insulin levels, at least at the time of euthanasia of these diabetic mice. However, exercise reduced the raised plasma levels of LDL cholesterol and triglycerides in db/db mice, which may at least be partially related to the improved endothelial function in diabetic exercised mice (Bartus et al. 2005, Bae et al. 2001). However, exercise failed to improve serum HDL levels in diabetic mice. Vascular responses to depolarization with KCl were similar in db/db and WT mice, both in the sedentary and exercised groups. Thus, it is unlikely that a generalized increase in responsiveness of arteries or changes in calcium-activated contractile mechanisms are involved in the augmented PE contractile response in diabetic mice. Alterations of vascular smooth muscle function have been implicated in the development of vascular complications and circulatory dysfunction in diabetes  99 such as the increased aortic contractile response to PE (Zhu et al. 2001, Okon et al. 2003, Kawasaki, 1997, Guo et al. 2005; Abebe et al. 1990). While augmented responses to PE have been reported by several groups, there are also some reports showing no changes or even decreased contractions of aorta (Mulhern and Docherty, 1989, Keegan et al. 1995). The endothelium produces vasoconstrictors such as eicosanoids and endothelin-1 (Tesfamariam et al. 1989, Vanhoutte, 1994). Serum isolated from db/db mice induces COX-2 expression and increases TXA2 production in primary cultured vascular smooth muscle cells (Guo et al. 2005, Xavier et al. 2003), suggesting that they may at least partially contribute to the vascular smooth muscle contractile hyperactivity in db/db mice (Guo et al. 2005). Endothelin-1 is also a powerful paracrine regulator of vascular smooth muscle tone (Yanagisawa et al. 1988). We speculated that the augmented constrictor response in db/db mice could be due to increased activity of endothelin-1 (Arikawa et al. 2001) and/or TXA2/PGH2 (Abebe et al. 1990; Tesfamariam et al. 1989). We excluded these possibilities by demonstrating that bosentan and indomethacin did not attenuate the enhanced PE-constrictor responses in db/db mice. Another possibility is that a decrease in basal NO production in diabetic mice may be responsible for the augmented PE response. We examined this possibility by repeating PE concentration-response curves after incubation with L-NAME. In the presence of L-NAME, PE- induced constriction remained significantly higher in db/db and exercised db/db mice compared to WT mice. This suggests that lower NO production may not underlie the enhanced contractile response to alpha-adrenergic stimulation in diabetic mice. Contractile responses can be modulated by agonists independently of changes in intracellular Ca2+, a process known as Ca2+ sensitization (Bradley and Morgan, 1987; Himpens et al. 1990, Morgan and Morgan, 1984). Rho-kinase inhibits myosin light chain phosphatase activity and has a key role in Ca2+ sensitization (Somlyo and Somlyo, 2000, Satoh et al. 1994). Therefore, it is possible that changes in Ca2+ sensitivity, for example mediated by Rho-kinase or PKC pathways (Sandu et al. 2000, Buus et al. 1998) could be responsible for the enhanced PE-constriction in diabetes. To investigate these possibilities, we inhibited Rho-kinase and PKC using Y-27632 and calphostin-C,  100 respectively. Y-27632 did not change, while calphostin-C suppressed, the augmented contractile response to PE. Inhibition of PKC restored the contractions in db/db mice to levels observed in WT. In addition, cumulative concentrations of indolactam-V, a PKC activator, induced higher constrictor responses in db/db mice compared to WT. Therefore, increased Ca2+ sensitization due to increased protein kinase C activation likely mediates the enhanced alpha-adrenergic-mediated contractile response. Others have suggested that exposure of vascular smooth muscle to elevated concentrations of glucose increases protein kinase activity through activation by DAG (Abebe and MacLeod, 1991) and that this may be an important causal factor in diabetic vascular dysfunction (Haller et al. 1995, Inoguchi et al. 1992, Koya and King, 1998). Our data suggest that the exercise improves endothelial function in db/db mice without affecting blood glucose or insulin level. We also demonstrated that augmented PE-induced constriction in db/db mice is not relieved by lifestyle medications such as exercise. It is likely that increased PKC activity may underlie the enhanced constrictor response in db/db mice. Exercise does not appear to modulate PKC activation of the db/db mouse aorta.                     101   Day Exercise speed (m/min) 1 2.5 2 2.6 3 2.8 4 3.0 5 3.2 8 3.4 9 3.6 10 3.9 11 4.2 12 4.6 13 5.2 14 5.2 15 5.2 . . . . . Continues same for 5 weeks                                   Table 4-1: Exercise training protocol for mice.   102    Plasma parameters WT Exercised WT db/db  Exercised db/db  Triglycerides 0.50±0.07 0.61±0.14 1.32±0.14* 0.50±0.08** Cholesterol 2.7±0.20 3.04±0.04 3.97±0.20* 2.9±0.16** LDL 0.91±0.07 0.99±0.08 1.48±0.16* 0.830±0.23** HDL 1.44±0.13 1.65± 0.15 1.66±0.27 1.74±0.12 Glucose 6.44±0.29 5.72±0.26 47.56±3.83* 48.24±4.00 Insulin 1.41±0.53 1.51±0.36 3.63±0.52* 3.72±0.70*  Table 4-2: Serum parameters at the end of study in all experimental groups (n=6-8 per group; * p<0.05 when compared to WT sedentary, ** p<0.05 when compared to db/db sedentary).  103 4 5 6 7 8 9 10 11 12 13 10 20 30 40 50 60 WT WT Exercised db/db db/db Exercised Age (weeks) W ei gh t ( gr ) *   Figure 4-1: Age- and exercise-related changes in body weight of WT and db/db mice (*p<0.05 significant difference from exercised db/db; repeated measures ANOVA, n = 8-10 per group).  104 010 20 30 40 50 60 WT WT  ex erc ise d db /db db /db  ex erc ise d M ax im um  r es po ns e to  A Ch (%  o f r el ax at io n) WT WT ex db/db db/db e db /db Ex erc ise d  E / ° **  Figure 4-2: The maximum response to ACh in aortic rings of WT, db/db and exercised db/db mice. Endothelium-dependent relaxation was significantly impaired in db/db aortae compared to WT and this response was significantly improved in exercised db/db animals (ANOVA; *p<0.01, WT & exercised WT vs. db/db; ºp<0.01, db/db vs. exercised db/db; n = 6-7 per group).  105                              B. db/db A. WT 5mN C. Exercised db/db  Washout 12.5 mN  5mN PE 12.5mN 5mN 7.5mN  Washout Washout   PE PE Figure 4-3: Traces illu (a), db/db (b), exercise markedly greater in db/   strating tension (mN) of aortic rings from the following mouse groups: WT d db/db (c). The maximal force generated in response to PE (10-9-10-5M) was db aortae. Exercise did not attenuate the augmented contractile response. 106 -10 -9 -8 -7 -6 -5 -4 2.5 5.0 7.5 10.0 12.5 15.0 W T db/db db/db exercised * * * * * * Log[PE] Fo rc e (m N ) 0.0 2.5 5.0 7.5    db/db Exe rcise d    WT    db/db EC 50  -l og  [P E]  (M ) 0.0 2.5 5.0 7.5 10.0 12.5    db/db Exercised    WT    db/db * E m ax  (m N ) * 10 11 12 13 14 15 WT db/db db/db Exercised Fo rc e (m N ) / b ised /db b/db b/db Exercised db/db Exercised db/db A B C * *   Figure 4-4: A- PE-induced constriction in aortae of WT, db/db, and exercised db/db mice. The constrictor responses are potentiated in db/db compared to WT mice and exercise did not attenuate this augmented response (repeated measures ANOVA; *p<0.01, n = 8-10 per group). B- Vascular contractile responses to depolarization with KCl were similar in db/db and WT mice. C- Emax and EC50 values for PE concentration-response curves. Emax and EC50 values were significantly higher in db/db mice compared to WT (ANOVA; p<0.05, n = 8-10 per group). Exercise did not alter the Emax and EC50 values in db/db mice (ANOVA; *p<0.01, n = 8-10 per group).  107  A C -10 -9 -8 -7 -6 -5 -4 0.0 2.5 5.0 7.5 10.0 12.5 15.0 WT WT with calphostin Log[PE] (M) Fo rc e (m N ) -10 -9 -8 -7 -6 -5 -4 2.5 5.0 7.5 10.0 12.5 15.0 db/db Exercised with calphostin db/db Exercised Log[PE] (M) Fo rc e (m N ) B D -10 -9 -8 -7 -6 -5 -4 2.5 5.0 7.5 10.0 12.5 15.0 db/db with calphostin db/db Log[PE] (M) Fo rc e (m N ) -10 -9 -8 -7 -6 -5 -4 0.0 2.5 5.0 7.5 10.0 12.5 15.0 WT db/db db/db Exercised Log[PE] (M) Fo rc e( m N )* * db/db /db with lphostin d /  xercise /db db/db rcised with cal stin /db Exercised ith calphostin /db Exercised db/db it calphosti db/db    Figure 4-5: PE concentration-response curves in the presence and absence of calphostin-C in WT (A), db/db (B), and exercised db/db mice (C). Treatment with calphostin C restored PE- induced constriction in diabetic mice to the levels that were similar to those in WT mice (D) (repeated measures ANOVA; *p<0.01; n = 5-6 per group).   108  -9 -8 -7 -6 -5 -4 0 5 10 15 20 WT db/db Exercised db/db Log[Indolactam] (M) Fo rc e (m N ) * db/db db/db WT    Figure 4-6: Indolactam concentration-response curve. The constrictor response to indolactam was markedly higher in diabetic (control and exercised) compared to WT mice (repeated measures ANOVA; *p<0.05, n = 4 per group)                       109 4.5 Bibliography  Abebe W., Harris K.H. & Macleod K.M. (1990) Enhanced contractile responses of arteries from diabetic rats to alpha 1-adrenoceptor stimulation in the absence and presence of extracellular calcium.  J.Cardiovasc.Pharmacol. 16: 239-248.  Abebe W. & Macleod K.M. (1991) Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism.  Can.J.Physiol Pharmacol. 69: 355-361.  Agrawal D.K. & Mcneill J.H. (1987) Vascular responses to agonists in rat mesenteric artery from diabetic rats.  Can.J.Physiol Pharmacol. 65: 1484-1490.  Arikawa E., Verma S., Dumont A.S. & Mcneill J.H. (2001) Chronic bosentan treatment improves renal artery vascular function in diabetes. J.Hypertens. 19: 803-812.  Bae J.H., Bassenge E., Kim K.B., Kim Y.N., Kim K.S., Lee H.J., Moon K.C., Lee M.S., Park K.Y. & Schwemmer M. (2001) Postprandial hypertriglyceridemia impairs endothelial function by enhanced oxidant stress. Atherosclerosis 155: 517-523.  Bartus M., Lomnicka M., Lorkowska B., Franczyk M., Kostogrys R.B., Pisulewski P.M. & Chlopicki S. (2005) Hypertriglyceridemia but not hypercholesterolemia induces endothelial dysfunction in the rat. Pharmacol.Rep. 57: 127-137.  Bradley A.B. & Morgan K.G. (1987) Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin. J.Physiol 385: 437-448.  Buus C.L., Aalkjaer C., Nilsson H., Juul B., Moller J.V. & Mulvany M.J.( 1998) Mechanisms of Ca2+ sensitization of force production by noradrenaline in rat mesenteric small arteries. J.Physiol 510: 577-590.  De Angelis K., Wichi R.B., Jesus W.R., Moreira E.D., Morris M., Krieger E.M. & Irigoyen M.C. (2004) Exercise training changes autonomic cardiovascular balance in mice. J Appl.Physiol 96: 2174-2178.  Garcia M.J., Mcnamara P.M., Gordon T. & Kannel W.B. (1974) Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes 23: 105- 111.  Guo Z., Su W., Allen S., Pang H., Daugherty A., Smart E. & Gong M.C. (2005) COX-2 up- regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovasc.Res. 67: 723-735.  Haller H., Baur E., Quass P., Behrend M., Lindschau C., Distler A. & Luft F.C. (1995) High glucose concentrations and protein kinase C isoforms in vascular smooth muscle cells. Kidney Int. 47: 1057-1067.  Himpens B., Kitazawa T. & Somlyo A.P. (1990) Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Arch. 417: 21-28.  Inoguchi T., Battan R., Handler E., Sportsman J.R., Heath W. & King G.L. (1992) Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart  110 of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc.Natl.Acad.Sci.U.S.A 89: 11059-11063.  Jolliffe J.A., Rees K., Taylor R.S., Thompson D., Oldridge N. & Ebrahim S. (2001) Exercise-based rehabilitation for coronary heart disease. Cochrane.Database.Syst.Rev. CD001800.  Kamata K., Miyata N. & Kasuya Y. (1988) Mechanisms of increased responses of the aorta to alpha-adrenoceptor agonists in streptozotocin-induced diabetic rats. J.Pharmacobiodyn. 11: 707-713.  Kawasaki H. (1997) Pharmacological studies on alterations in contractile reactivity in aortas isolated from experimental diabetic rats]. Hokkaido Igaku Zasshi 72: 649-665.  Keegan A., Walbank H., Cotter M.A. & Cameron N.E. (1995) Chronic vitamin E treatment prevents defective endothelium-dependent relaxation in diabetic rat aorta. Diabetologia 38: 1475-1478.  Knowler W.C., Barrett-Connor E., Fowler S.E., Hamman R.F., Lachin J.M., Walker E.A. & Nathan D.M. (2002) Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N.Engl.J.Med. 346: 393-403.  Korzick D.H., Laughlin M.H. & Bowles D.K. (2004) Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries. J.Appl.Physiol 96: 1425-1432.  Koya D. & King G.L: Protein kinase C activation and the development of diabetic complications. Diabetes 47: 859-866,1998.  Macleod K.M. (1985) The effect of insulin treatment on changes in vascular reactivity in chronic, experimental diabetes. Diabetes 34: 1160-1167.  Morgan J.P. & Morgan K.G. (1984) Calcium and cardiovascular function. Intracellular calcium levels during contraction and relaxation of mammalian cardiac and vascular smooth muscle as detected with aequorin. Am.J.Med. 77: 33-46.  Mulhern M. & Docherty J.R. (1989) Effects of experimental diabetes on the responsiveness of rat aorta. Br.J.Pharmacol. 97: 1007-1012.  Myers J., Prakash M., Froelicher V., DO D., Partington S. & Atwood J.E. (2002) Exercise capacity and mortality among men referred for exercise testing. N.Engl.J.Med. 346: 793-801.  Okon E.B., Golbabaie A. & Van breemen C. (2002) In the presence of L-NAME SERCA blockade induces endothelium-dependent contraction of mouse aorta through activation of smooth muscle prostaglandin H2/thromboxane A2 receptors. Br.J.Pharmacol. 137: 545-553.  Okon E.B., Szado T., Laher I., Mcmanus B. & Van breemen C. (2003) Augmented contractile response of vascular smooth muscle in a diabetic mouse model. J.Vasc.Res. 40: 520-530.  Paffenbarger R.S., Jr., Hyde R.T., Wing A.L., Lee I.M., Jung D.L. & Kampert J.B. (2003) The association of changes in physical-activity level and other lifestyle characteristics with mortality among men. N.Engl.J.Med. 328: 538-545.   111 Pannirselvam M., Simon V., Verma S., Anderson T. & Triggle C.R. (2003) Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br.J.Pharmacol. 140: 701-706.  Pannirselvam M., Verma S., Anderson T.J. & Triggle C.R. (2002) Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br.J.Pharmacol. 136: 255-263.  Sandu O.A., Ragolia L. & Begum N. (2000) Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation. Diabetes 49: 2178-2189.  Satoh S., Kreutz R., Wilm C., Ganten D. & Pfitzer G. (1994) Augmented agonist-induced Ca(2+)- sensitization of coronary artery contraction in genetically hypertensive rats. Evidence for altered signal transduction in the coronary smooth muscle cells. J.Clin.Invest 94: 1397-1403.  Sesso H.D., Paffenbarger R.S., Jr. & Lee I.M. (2000) Physical activity and coronary heart disease in men: The Harvard Alumni Health Study. Circulation 102: 975-980.  Somlyo A.P. & Somlyo A.V. (2000) Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J.Physiol 522:177-185.  Spier S.A., Laughlin M.H. & Delp M.D. (1999) Effects of acute and chronic exercise on vasoconstrictor responsiveness of rat abdominal aorta. J.Appl.Physiol 87:1752-1757.  Stewart K.J. (2004a) Exercise training: can it improve cardiovascular health in patients with type 2 diabetes? Br.J.Sports Med. 38: 250-252.  Stewart K.J. (2004b) Role of exercise training on cardiovascular disease in persons who have type 2 diabetes and hypertension. Cardiol.Clin. 22: 569-586.  Tang T. & Reed M.J. (2001) Exercise adds to metformin and acarbose efficacy in db/db mice. Metabolism 50:1049-1053.  Taylor P.D., Oon B.B., Thomas C.R. & Poston L. (1994) Prevention by insulin treatment of endothelial dysfunction but not enhanced noradrenaline-induced contractility in mesenteric resistance arteries from streptozotocin-induced diabetic rats. Br.J.Pharmacol. 111:35-41.  Tesfamariam B. & Cohen R.A. (1992) Free radicals mediate endothelial cell dysfunction caused by elevated glucose.  Am.J.Physiol 263: H321-H326.  Tesfamariam B., Jakubowski J.A. & Cohen R.A. (1989) Contraction of diabetic rabbit aorta caused by endothelium-derived PGH2-TxA2. Am.J.Physiol 257: H1327-H1333.  Vanhoutte P.M. (1994) Endothelin-1. A matter of life and breath. Nature 368: 693-694.  Verma-Ahuja S., Husain K., Verhulst S., Espinosa J.A. & Somani S.M. (2000) Delayed effects of pyridostigmine and exercise training on acetylcholinesterase and muscle tension in mouse lower extremity. Arch.Toxicol. 74: 539-546.   112 Xavier F.E., Davel A.P., Rossoni L.V. & Vassallo D.V. (2003) Time-dependent hyperreactivity to phenylephrine in aorta from untreated diabetic rats: role of prostanoids and calcium mobilization. Vascul.Pharmacol. 40:67-76.  Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K. & Masaki T. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415.  Zhu B.H., Guan Y.Y., Min J. & He H. (2001) Contractile responses of diabetic rat aorta to phenylephrine at different stages of diabetic duration. Acta Pharmacol.Sin. 22: 445-449.  113 5 CONCLUSIONS  5.1 Future Directions  1) This study showed that exercise could prevent endothelial dysfunction in db/db mice with exercise beginning at 5 weeks of age when endothelial dysfunction is possibly not well established. In future it is important to examine if exercise can reverse endothelial dysfunction in db/db mice with established endothelial dysfunction.  2) To investigate if exercise only increases removal of oxygen free radicals or it also decreases the production of the radicals. Decreased ACh concentration-response can be performed before and after incubation of coronary arteries with an SOD enzyme inhibitor, diethyldithiocarbamil (DDC), which chelates Cu/Zn.  If exercise only increases SOD expression without decreasing O2• generation, it is expected that upon incubation with the SOD inhibitor, the ACh-induced endothelium-dependent relaxation that was improved by exercise in db/db mice will decline to a larger extent compared to WT mice and reach to the level of sedentary db/db mice or lower.  3) Life style modification includes diet and exercise. Effect of exercise on endothelial function was examined in this study, it is important to examine the effect of diet/ food control on endothelial function in db/db mice.  4) TNFα has been shown to play an important role in diabetic endothelial dysfunction in db/db mouse (Gao et al, 2007). Therefore, it is important to show in future if exercise decreases/ changes the level of TNFα in db/db mice.  5) Diabetic microvascular disease causes nephropathy, neuropathy and retinopathy. The current study showed that exercise improved diabetic nephropathy at the functional and  114 structural level. However, effect of exercise on diabetic nephropathy, retinopathy and neuropathy was not assessed. This is an important project for future investigation.  6) Endothelial progenitor cells are a special type of stem cells that have been found in the bone marrow and peripheral blood. These cells are incorporated into injured vessels and become mature endothelial cells during re-endothelialization processes. There is evidence that the number of circulating endothelial progenitor cells are decreased in diabetes. It has also been shown that exercise increases the number of circulating endothelial progenitor cells (Miller- Kasprzak 2007). Therefore it is important to investigate if exercise in db/db mice increases endothelial progenitor cells migration from bone marrow to blood vessels as one of the mechanismsof improving endothelial cell function.  7) To investigate if exercise effects can be reproduced by inducing shear stress in sedentary condition with minimal changes in energy expenditure. Increases in shear stress and oxidative stress (both stimulating antioxidant defense) occur simultaneously with exercise. Therefore, by investigating the effect of exercise on endothelial cell function, it is not possible to separate the effects of in vivo increase in shear stress from other mechanisms (e.g. increased antioxidant defense) that are stimulated by increased metabolic rate. In future, it is important to investigate whether increasing shear stress with parasympathetic blockade that does not increase energy expenditure as much as exercise, can stimulate the protective pathways of endothelium including eNOS mediated NO production and antioxidant defense mechanisms and thereby improve endothelial dysfunction in diabetes.  5.2 Conclusions  Type 2 diabetes mellitus is the leading cause of morbidity and mortality in North America. Macrovascular complications of diabetes increase the risk of myocardial infarction and stroke and the microvascular complications cause diabetic neuropathy, retinopathy and nephropathy. The  115 earliest stage for diabetic vascular complications is endothelial dysfunction. Therefore, preventing endothelial dysfunction may slow down vasculopathy and as a result diabetic complications. Also contractile response to α-adrenergic stimulation is increased in diabetes, which may accelerate atherosclerosis by increasing blood pressure. Lifestyle modification, including diet and exercise, is the cornerstone of diabetic management and decreases diabetic vascular complications. The mechanism by which lifestyle modification and particularly exercise may decrease diabetes vasculopathy is not fully determined. It is also not clear if exercise must induce weight loss to preserve endothelial function. The mechanisms by which exercise may preserve endothelial function in diabetes were explored in conduit and resistance arteries in this study. In conduit arteries (aorta) the primary cause of endothelial dysfunction was associated with the prevalence of free radicals such as superoxide and peroxynitrite, which lowered NO bioavailability by degrading this compound and decreasing eNOS substrate (L-Arg) and cofactor (BH4) availability. Additionally, exercise upregulated aorta eNOS activity independently of its total protein production in diabetes. Finally, it was demonstrated for the first time that differential regulation of SOD isoforms occurs in the diabetic aorta, depending on exercise intensity. This along with improved NO bioavailability may be pivotal in the reversal of diabetic endothelial dysfunction by lifestyle modification approaches such as exercise. Most importantly, results showed that a loss of body weight, body fat or improvement in glucose parameters was not obligatory in exercise- induced reversal of the vascular defects. In resistance arteries (coronary), the markedly reduced endothelial function was also related to a greater oxidative stress and reduced NO bioavailability, likely leading to an imbalance between cardiac oxygen supply and demand during activity. According to this study, moderate levels of exercise increased NO bioavailability, thus leading to improved endothelium-dependent vasodilation, and expectedly better perfusion of the diabetic heart. Exercise also enhanced myocardial antioxidant levels, which can improve the poorer outcome that is often predicted during ischemia/reperfusion occurring with myocardial infarction in diabetes as a consequence of low  116 antioxidant levels. Other than endothelial function, alterations in myogenic regulation of arteriolar diameter are also likely to detrimentally affect regional myocardial blood flow in diabetes. However, results showed that myogenic tone was not affected in coronary arteries of diabetic mice. Therefore, a greater myogenic tone is unlikely to be the primary cause of cardiac ischemia in this model of type 2 DM. This study also demonstrated that the augmented phenylephrine-induced constriction in db/db mice conduit arteries is likely caused by increased PKC activity. Exercise attenuates the oxidative stress in diabetic aorta by potentiating the antioxidant defence, however, this does not appear to modulate PKC activation. PKC has been shown to contribute to enhanced oxidative stress in diabetes, however based on the results increased oxidative stress in diabetes does not seem to enhance PKC activity to create a vicious circle. Previous studies have shown that tight sugar control or weight loss may postpone diabetic vasculopathy, however, these measures are limited since tight glucose control may lead to lethal hypoglycaemic episodes and poor compliance is very common with plans for maintaining low body weight. The results of this study are extremely important since they indicate that aerobic exercise (equivalent to moderate and vigorous intensity in humans) prevents diabetic cardiovascular complications in the absence of a tight sugar control or low body weight. The beneficial effect of exercise was most likely mediated via enhancing the antioxidant defence. This finding raises the importance of searching for an agent with either a direct antioxidant effect or indirect potentiation of the intrinsic antioxidant defence, which can be administered safely to diabetic population for prophylaxis of diabetic vascular complications.   117 5.3 Bibliography Miller-Kasprzak,E. & Jagodzinski,P.P. (2007) Endothelial progenitor cells as a new agent contributing to vascular repair. Arch.Immunol.Ther.Exp.(Warsz.), 55, 247-259.  Gao,X., Belmadani,S., Picchi,A., Xu,X., Potter,B.J., Tewari-Singh,N., Capobianco,S., Chilian,W.M., & Zhang,C. (2007) Tumor necrosis factor-alpha induces endothelial dysfunction in Lepr(db) mice. Circulation, 115, 245-254.  118 6 APPENDIX A- MEASUREMENT OF WATER AND FOOD CONSUMPTION IN SEDENTARY AND EXERCISING db/db AND WT MICE  6.1 Methods For food intake measurement, mice were kept in special cages where they had free access to food administered via a basket attached to the cage from outside. The bottom of the cage had a meshwork-like structure that was not covered by wood chips bedding allowing the chow that was taken into the cage by mice but not ingested to drop in a tray underneath the cage. Food consumption was calculated using the following formula: Food consumed per mouse per day = [food added to the food basket through the week – (food left in the basket at the end of the week + food collected from under-cage tray)]/number of mice per cage × 7. Water intake was measured using the following formula: water intake per mouse per day = (water added to the water bottle through the week – water left in the bottle at the end of the week)/ number of mice per cage × 7.  6.2 Results Food intake was significantly higher in the diabetic mice compared to the WT mice at 6 weeks of age and continued to be higher up to 12 weeks of age. The amount of food consumption in WT and db/db mice measured with our method was comparable with the amount reported to Jackson Mice. Although at 6 weeks of age the amount of food consumption by db/db mice exercised with moderate-intensity seems to be significantly lower compared to other db/db groups (sedentary or low-intensity exercise) it does not seems to be a consistent pattern at other week points. Therefore, this may be related to equipment error.   119 6.3 Discussion  Higher food consumption by db/db mice is expected since these animals lack leptin receptor and therefore the satiety signaling at the level of hypothalamus is absent. It may be expected that exercised db/db mice and WT mice consume larger amount of food due to higher energy expenditure. However, our results did not support this notion. It is possible that our setting was not sensitive enough to pick up a small increase in the amount of food consumed by exercised mice. Water intake was significantly higher in db/db mice compared to WT mice even at 6 weeks of age. This difference became larger after 7 weeks of age. At 6 weeks of age, exercise in db/db mice did not have a significant effect on water consumption. However, from 7 to 10 weeks of age, exercise significantly decreased water consumption in db/db mice in a dose dependent manner. After 11 weeks of age, exercise did not show a beneficial effect on decreasing water consumption. Higher water intake in db/db mice was expected due to diabetes-related polyuria. The mechanism for decreased water consumption in exercised db/db mice could be due to decreased polyuria due to e: 1-better sugar control (not supported by the findings in this model), 2-prevention of diabetic nephropathy/ proteinuria.  120 6 7 8 9 10 11 12 0 5 10 15 20 25 db/db mod-intensity exe db/db low-intensity exe db/db WT WT low-intensity exe WT mod-intensity exe Age (weeks) W at er  (m l) A B 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 Age (weeks)  M ou se  C ho w  (g r) * * * * * ** * * * * * ** ° ° ° ° • • • •  Figure 6-1: Food and water consumption in mice. A- Food consumption in gr/day/mouse. *p<0.05 compared to db/db, db/db low-intensity exe and db/db mod-intensity exe. B- Water consumption in ml/day/mouse. *p<0.001 compared to db/db, db/db low-intensity exercise and db/db mod-intensity exercise.  •p<0.01 compared to db/db low-intensity exercise and db/db mod-intensity exercise. °p<0.05 compared to db/db mod-intensity exercise.  121 7 APPENDIX B- EFFECT OF LOW AND MODERATE INTENSITY EXERCISE ON AORTIC FUNCTION IN WT MICE -10 -9 -8 -7 -6 -5 -4 0 25 50 75 Log [ACh] (mol/l) Pe rc en t V as od ila tio n -10 -9 -8 -7 -6 -5 -4 25 50 75WT WT low-intensity Exe WT mod-intensity Exe Log [SNP] (mol/l) A B -10 -9 -8 -7 -6 -5 2.5 5.0 7.5 10.0 WT WT low-intensity Exe WT mod-intensity Exe C Log [PE] M Fo rc e (m N )  Figure 7-1: Ach, SNP and PE dose response curves in aortae of sedentary WT mice and WT mice exercised with low and moderate intensity. Low and moderate intensity exercise did not change ACh (A), or SNP (B) vasorelaxation response in WT aortic rings preconstricted with PE. Low and moderate intensity exercise also did not change PE-induced constriction in aortic rings of WT mice (C).  122 8 APPENDIX C- EFFECT OF LOW-INTENSITY EXERCISE ON CORONARY ENDOTHELIAL FUNCTION IN db/db MICE  A B -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 Log [ACh] M Pe rc en t R el ax at io n -10 -9 -8 -7 -6 -5 -4 20 40 60 80 100 Control db/db db/db Exe Log [SNP] M Pe rc en t R el ax at io n * C D ACh AChSNP SNP 0 25 50 75 100 E ( m ax ) ( % ) ** 0.0 1.5 3.0 4.5 6.0 7.5 db/db Exe db/db Control EC 50  -l og [D ru g] (M )  Figure 8-1: A and B-Endothelium-dependent (Ach mediated) and endothelium-independent (SNP mediated) vasorelaxation in coronary arteries of WT mice vs. db/db mice vs. db/db mice exercised with low-intensity. There is a marked decline in ACh induced vasodilation in db/db mice compared to WT mice. Low-intensity exercise preserved endothelium-dependent vasodilation in db/db mice (*p<0.05; n = 6-7; repeated measures ANOVA). C and D-EC50 and Emax of the ACh and SNP response in the coronary arteries. EC50 is not statistically different amongst groups for both ACh and SNP response. Emax is significantly decreased in db/db mice compared to WT group (*p<0.05, n = 8-10 in each group, repeated measures ANOVA). Low-intensity exercise significantly increased Emax in db/db mice. There was no significant difference in Emax in response to SNP amongst groups.  123 A B C D -9 -8 -7 -6 -5 0 20 40 60 80 WT WT with L-NAME Log[U46619] M %  C on st ri ct io n -9 -8 -7 -6 -5 0 20 40 60 80 db/db db/db with L-NAME Log[U46619] M %  C on st ri ct io n -9 -8 -7 -6 -5 0 20 40 60 80 WT Exe WT  Exe with L-NAME Log[U46619] M %  C on st ri ct io n -9 -8 -7 -6 -5 0 20 40 60 80 db/db Exe db/db Exe with L-NAME Log[U46619] M %  C on st ri ct io n  Figure 8-2: U46619-induced constriction curves in the presence or absence of L-NAME in coronary arteries of sedentary and exercised WT and db/db mice. There was no significant difference in EC50 or Emax of the constriction response between db/db and WT mice, and incubation with L-NAME did not change the response.  124

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