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Effects of chronic stress and diabetes on antioxidant status and myocardial susceptibility to ischemia/reperfusion… Toleikis, Philip 1995

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EFFECTS OF CHRONIC STRESS AND DIABETES ON ANTIOXIDANT STATUS ANDMYOCARDIAL SUSCEPTIBILITY TO ISCHEMIA I REPERFUSION INJURYbyPHILIP TOLEIKIS, University of Vermont, 1975M.Sc., University of Michigan, 1985A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of Pharmacology & TherapeuticsFaculty of MedicineWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust, 1995© Philip Toleikis, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature_______________Department of cCçiQThe University of British ColumbiaVancouver, CanadaDateDE-6 (2188)— II —ABSTRACTDiabetes and psychological stress can both be described as states inwhich the actions of glucocorticoids and catecholamines predominate over those ofinsulin. Stress has a permissive, albeit poorly defined, effect on the development ofmyocardial infarction and atherosclerosis, which are major complications of the diabeticstate; reactive oxygen radicals have been implicated in both conditions. Therefore, theeffects of chronic-intermittent variable restraint stress on antioxidant status andmyocardial susceptibility to ischemialreperfusion injury in control, and streptozotocindiabetic rats were investigated. As an initial approach, the influence of chemical and/orsurgical sympathectomy on the, antioxidant status of various tissues was investigated innon-diabetic rats. The effects of acute and chronic stress in diabetic and non-diabeticrats on levels of glucose, corticosterone and catecholamines measured from bloodcollected through in-dwelling catheters were compared. Antioxidant status of blood andtissues from normal and short- and long-term diabetic rats exposed to stress wasassessed in terms of activities of antioxidant enzymes (catalase, superoxide dismutase,g lutathione peroxidase and glutathione reductase), non-enzymatic antioxidants(glutathione, ascorbate and tocopherol) and in vitro peroxide challenge. To determinewhether stress-induced alteration in antioxidant capacity in diabetes was a result of theassociated hyperlipidemia, non-diabetic rats with diet-induced hyperlipidemia weresubjected to stress and resultant effects on plasma lipid profiles and antioxidantcomponents and functional status examined. Finally, the effects of stress onmyocardial susceptibility to ischemialreperfusion injury were investigated in terms ofmyocardial function and antioxidant status. Studies involving sympathectomysuggested differential actions of adrenaline and noradrenaline on antioxidantcomponents. The corticosterone and catecholamine responses to acute and chronicstress are modified by diabetes. While antioxidant systems are normally resistant tostress, their susceptibility to stress-induced modification increases in diabetes, a— III —pathological condition associated with increased oxidative challenge. Stress-inducedchanges in plasma lipids and antioxidant components cannot be solely ascribed to theassociated hyperl ipidemia in diabetes. While some diabetes-associated antioxidantalterations occurred regardless of its duration, other differences related to the longterm effects of the disease. The consequences of stress on diabetes-associatedantioxidant changes and myocardial susceptibility to ischemialreperfusion injury wereto some extent modified by the duration of diabetes.- iv -TABLE OF CONTENTSABSTRACT.iiTABLE OF CONTENTS ivLIST OF TABLES xiiLIST OF FIGURES xviLIST OF ABBREVIATIONS xxiACKNOWLEDGEMENTS xxiiDEDICATION xxiii1. INTRODUCTION I1.1. Behavioral Factors in Cardiovascular Disease I1.2. Considerations in the Experimental Study of Stress 41.3. Effects of High Levels of Catecholamines on the Myocardium:Free Radical Related Processes 81.4. Effects of Elevated Glucocorticoid Levels: Free Radical Related Processes 111.5. Overview of Endogenous Free Radical Scavanging Systems 121.5.1. Antioxidant Enzymes 121.5.2. Metal-Ion Sequestration 131.5.3. Non-Enzymatic Antioxidants 131.6. Pathological Conditions Which Might Be Affected by Stress: Diabetes 141.6.1. Diabetes and Stress: Metabolic Effects and Consequences 15I .6.2. Diabetes as a Sustained Stimulus to Catecholamines and Corticoids 161.6.3. Diabetes and Acute Stress: Effects on Catecholamines and Corticoids 161.7. Evidence for Free Radical Involvement in Diabetes 171.8. Rationale 231.9. Specific Aims 241.9.1. Experimental Approach 25-v -2. MATERIALS AND METHODS .282.1. General Animal Care 282.1 .1. Housing Conditions, Methods of Euthanasia 282.2. Drug Protocols 282.2.1. Chemical and Surgical Sympathectomy 282.2.1.1. 6-hydroxydopamine Treatment and Adrenalectomy 282.2.1.2. Reserpine Treatment and Adrenalectomy 292.2.2. Streptozotocin Treatment 302.2.3. Hypercholesterolemic Diet Procedure 302.3. Description of the Chronic Intermittent Stress Model 302.4. Remote Blood Sampling Procedures 312.4.1. Description of Venous Indwelling Catheter Constructionand Implantation Procedure 312.4.2. Blood Sampling Procedure 322.5. Chemical Assays 332.5.1. Hemoglobin Assay 332.5.2. Glycosylated Hemoglobin Assay 342.5.3. Corticosterone Assay 352.5.4. Catecholamine Assay 362.5.5. Ascorbate Assay 382.5.6. Tocopherol Assay 392.5.7. Glucose, Triglycerides and Cholesterol Assays 392.6. Biochemical Analyses 402.6.1. Tissue Antioxidant Enzyme Measurements 40- vi -2.6.1 .1. Preparation of Tissue Homogenates and Cytosolic Fractions 402.6.1.2. Catalase 402.6.1.3. Cu,Zn-Superoxide Dismutase 412.6.1.4. Glutathione Reductase 422.6.1.5. Glutathione Peroxidase 422.6.1.6. Hemoglobin and Correction of Tissue Enzyme Activityfor the Contribution of Blood 432.6.2. Erythrocyte Antioxidant Enzymes 442.6.2.1. Preparation of Hemolysates 442.6.2.2. Catalase 442.6.2.3. Cu, Zn-Superoxide Dismutase 442.6.2.4. Glutathione Peroxidase 442.7. Functional Antioxidant Measurements 452.7.1. Tissue Susceptibility to In Wtro Peroxide Challenge 452.7.1.1. Preparation of Tissue Homogenates 452.7.1.2. Susceptibility of Tissues to TBHP-Induced SulfhydrylGroup Depletion 452.7.1.3. Susceptibility of Tissues toH20-lnduced SulfhydrylGroup Depletion 462.7.1.4. Susceptibility of Tissues to Time-DependentH20-lnducedSulfhydryl Group Depletion 462.7.1.5. Susceptibility of Tissues to TBHP-lnduced Lipid Peroxidation 472.7.1.6. Susceptibility of Tissues toH20-lnduced Lipid Peroxidation 472.7.2. Erythrocyte Susceptibility to In Vitro Peroxide Challenge 482.7.2.1. Preparation of Erythrocytes 48- VII -2.7.2.2. Susceptibility of Erythrocytes to TBHP-lnducedSulfhydryl Group Depletion 482.7.2.3. Susceptibility of Erythrocytes toH20-lnducedSulfhydryl Group Depletion 492.7.2.4. Susceptibility of Erythrocytes to TBHP-lnduced Lipid Peroxidation 492.7.2.5. Susceptibility of Erythrocytes toH20-lnduced Lipid Peroxidation 502.8. Functional Physiological Measurements: Myocardial Susceptibility tolschemialReperfusion Injury 502.8.1. Preparation of Buffer Solutions 502.8.2. Apparatus for Langendorif Perfusion 502.8.3. Anesthesia, and Surgical Preparation of Heart for Isolation 502.8.4. Methodology of Ischemia/Reperfusion Timing 512.8.5. Description of Myocardial Functional Measurements 512.8.6. Biochemical Measurements 512.9. Statistical Analysis 523. RESULTS 523.1. Effects of Sympathectomy on Antioxidant Status 523.1.1. Adrenalectomy and/or 6-Hydroxydopamine Treatment 523.1.1.1. Catecholamine Levels 533.1.1.2. Antioxidant Enzyme Activities 553.1.1.3. TBHP-lnduced Sulfhydryl Group Depletion 553.1.1.4. TBHP-lnduced Lipid Peroxidation 563.1.2. Adrenalectomy and/or Reserpine Treatment 72- VIII -3.1.2.1. Catecholamine Levels .723.1.2.2. Antioxidant Enzyme Activities 723.1.2.3. TBHP-lnduced SuiThydryl Group Depletion 733.1.2.4. TBHP-lnduced Lipid Peroxidation 733.2. Effects of Acute and Chronic Stress on Corticosterone, Catecholaminesand Metabolic Indices of Diabetes on Days 1, 7 and 14 of Stress 903.2.1. Effects of Diabetes and. Stress on Body Weight 903.2.2. Resting Glucose Levels 903.2.3. Effects of Exposure to Stress on Day I (Corticosterone, Catecholaminesand Glucose Levels) 913.2.4. Effects of Exposure to Stress on Days 7 and 14 (Corticosterone,Catecholamines and Glucose Levels) 923.2.5. Non-Enzymatic Antioxidant Levels 963.2.5.1. Ascorbate Levels in Tissues and Plasma 963.2.5.2. Tocopherol Levels in Tissues and Plasma 963.3. Effects of Chronic Intermittent Stress and Diabetes on Antioxidant Status 1223.3.1. Effects of Stress in Rats With Short-Term (4 Weeks) Diabetes 1223.3.1.1. Body Weights and Plasma Chemical Composition 1223.3.1.2. Corticosterone and Catecholamine Levels 1293.3.1.3. Antioxidant Enzyme Activities 1293.3.1.4.H20-lnduced SuiThydryl Group Depletion 1303.3.1.5.H20-lnduced Lipid Peroxidation 1303.3.2. Effects of Stress in Rats with Long-Term (12 Weeks) Diabetes 155- ix -3.3.2.1. Body Weights and Plasma Chemical Composition 1553.3.2.2. Corticosterone Levels 1563.3.2.3. Antioxidant Enzyme Activities 1563.3.2.4.H20-Induced Sulfhydryl Group Depletion 1573.3.2.5. Non-Enzymatic Antioxidant Levels: Plasma Tocopherol and Ascorbatel 573.3.2.6.H20-lnduced Lipid Peroxidation 1573.4. Effects of Stress on Plasma Lipids and Antioxidant Statusin Rats with Diet-Induced Hyperlipidemia 1863.4.1. Weight Gain and Plasma Lipid Levels 1873.4.2. Antioxidant Components 1873.4.3.H20-lnduced Sulfhydryl Group Depletion and Lipid Peroxidation 1873.5. Effects of Stress and Diabetes on Myocardial Functional Recoveryand Antioxidant Status Following lschemialReperfusion Injury 1903.5.1. Functional Recovery and Antioxidant Status FollowingMyocardial lschemia/Reperfusion Injury: Effects of Stressand Short-Term Diabetes (4 weeks’ duration) 1913.5.1 .1. Body Weights and Glucose and Lipid Status 1913.5.1.2. Myocardial Functional Indices: ±dPldtmax, Developed pressure,End-Diastolic Pressure 1913.5.1.3. Antioxidant Enzyme Activity (Glutathione Reductase) 1923.5.1.4. Levels of Non-Enzymatic Antioxidants: Ascorbate and Glutathione ... 1923.5.1.5.H20-Induced Sulfhydryl Group Depletion 1923.5.1.6.H20-Induced Time-Dependent Sulthydryl Group Depletion 1933.5.1.7.H20-lnduced Lipid Peroxidation 1933.5.2. Functional Recovery and Antioxidant Status Following Myocardiallschemia/Reperfusion Injury: Effects of Stress and Long-Term Diabetes(12 Weeks’ Duration) 207-x -3.5.2.3. Antioxidant Enzyme Activity (Glutathione Reductase) 2083.5.2.4. Levels of Non-Enzymatic Antioxidants: Ascorbate and Glutathione ... 2083.5.2.5.H20-lnduced Sulfhydryl Group Depletion 2093.5.2.6.H20-lnduced Time-Dependent Sulfhydryl Group Depletion 2093.5.2.7.H20-lnduced Lipid Peroxidation 2094. DISCUSSION 2234.1. Effects of Sympathectomy on Plasma Catecholamine Levelsand Antioxidant Status 2234.1 .1. Effects of 6-OH and Adrenalectomy 2234.1.2. Effects of Reserpine and Adrenalectomy 2284.2. Effects of Acute and Chronic Stress on Plasma Levels ofCorticosterone, Catecholamines and Glucose as Measured UsingIndwelling Catheters in Control and Diabetic Rats 2294.2.1. Effects of Acute Stress in Non-Diabetic Rats 2304.2.2. Effects of Repeated Stress in Non-Diabetic Rats 2314.2.3. Effects of Diabetes on Levels of Stress Hormones 2324.2.4. Effects of Acute Stress in Diabetic Rats 2334.2.5. Effects of Repeated Stress in Diabetic Rats 2354.2.6. Effects of Repeated Stress on Levels of the Non-EnzymaticAntioxidants Tocopherol and Ascorbate 2364.3. Antioxidant Status in Diabetes of Short- (4 week) and Long-(12 week) Duration: Effects of Chronic-Intermittent Stress 2374.3.1. Effects of Short-Term (4 Week) Diabetes on Antioxidant Status 2384.3.2. Effects of Long-Term (12 Week) Diabetes on Antioxidant Status 2404.3.3. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (4 Weeks) 2434.3.4. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (12 Weeks) 245- xi -4.3.3. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (4 Weeks) 2434.3.4. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (12 Weeks) 2454.3.5. Stress-Related Alterations in Antioxidant Status 2464.3.5.1. Effects of Chronic-Intermittent Stress on Antioxidant Status in Non-Diabetic Animals 2464.3.5.2. Effects of Chronic-Intermittent Stress on Antioxidant Statusin Diabetic Rats (Short-Term Diabetes) 2474.3.5.3. Effects of Chronic-Intermittent Stress on Antioxidant Statusin Diabetic Rats (Long-Term Diabetes) 2504.3.5.4. Generalizations Regarding Effects of Chronic IntermittentStress on Antioxidant Status in Short- and Long-Term Diabetes 2514.4. Effects of Chronic-Intermittent Stress on Plasma Lipids and AntioxidantStatus in Rats with Diet-Induced as Opposed to Diabetes-InducedHyperlipidemia 2524.5. Effects of Chronic-Intermittent Stress on Myocardial Functional RecoveryFollowing lschemialReperfusion Injury in Short and Long-Term Diabetes 2564.5.1. Diabetes Associated Alterations in Myocardial Functional Recoveryand Antioxidant Status Following lschemia/Reperlusion Injury 2584.5.1.1. Baseline Function Before IschemialReperfusion 2584.5.1.2. Myocardial Function Following lschemialReperlusion 2604.5.2. Stress Related Alterations in Myocardial Functional Recovery andAntioxidant Status Following lschemialReperfusion Injury 2654.5.2.1. Non-DiabeticAnimals 2654.5.2.2. Diabetic Animals 2664.6. Summary and Conclusions 2675. REFERENCES 273- XII -LIST OF TABLESTable 1. Plasma catecholamine levels in control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 54Table 2. Antioxidant status in erythrocytes from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 57Table 3. Antioxidant status in hearts from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 58Table 4. Antioxidant status in liver from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 59Table 5. Antioxidant status in kidney from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 60Table 6. Antioxidant status in lung from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 61Table 7. Plasma catecholamine levels in control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 74Table 8. Antioxidant status in hearts from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 75Table 9. Antioxidant status in liver from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 76Table 10. Antioxidant status in kidney from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 77Table 11. Antioxidant status in erythrocytes from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 78Table 12. Antioxidant status in lung from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 79Table 13. Weights of male Wistar rats before injection of STZ or vehicle andon day I, 7 and 14 of exposure to the stress protocol (stressor-exposed)or control conditions 98Table 14. Plasma glucose levels in control and STZ-treated male Wistar ratson the first, seventh and fourteenth day of exposureto the stress protocol (stressor-exposed) or control conditions 99- XIII -Table 15. Plasma and tissue ascorbate levels in control and STZ-treatedmale Wistar rats on the seventh and fourteenth day of exposureto the stress protocol (stressor-exposed) or control conditions 100Table 16. Plasma and tissue tocopherol levels in control and STZ-treatedmale Wistar rats on the seventh and fourteenth. day of exposureto the stress protocol (stressor-exposed) or control conditions 101Table 17. Weights of male Wistar rats before injection of STZ or vehicle andfollowing exposure to the stress protocol (stressor-exposed) orcontrol conditions following four weeks 132Table 18. Plasma catecholamines and corticosterone from control anddiabetic (four weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 133Table 19. Antioxidant status in hearts from control and diabetic(four weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 134Table 20. Antioxidant status in liver tissue from control and diabetic(four weeks’ duration) male Wistar rats exposedto the stress protocol (stressor-exposed) or control conditions 135Table 21. Antioxidant status in kidney tissue from control and diabetic(four weeks’ duration) male Wistar rats exposedto the stress protocol (stressor-exposed) or control conditions 136Table 22. Antioxidant status in lung tissue from control anddiabetic (four weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 137Table 23. Antioxidant status in packed erythrocytes from control anddiabetic (four weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 138Table 24. Weight and % change in weight of control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 159Table 25. Plasma corticosterone levels in control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 160Table 26. Antioxidant status in hearts from control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 169- xiv -Table 27. Antioxidant status in liver tissue from controll and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 170Table 28. Antioxidant status in kidney tissue from control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 171Table 29. Antioxidant status in packed erythrocytes from control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 172Table 30. Nonenzymatic antioxidant components in plasma from controland diabetic (twelve weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 173Table 31. Plasma lipids and antioxidant status in erythrocytes and tissuesin control and diet-induced hyperlipidemic rats exposed tothe stress protocol (stressor-exposed) or control conditions 188Table 32. Functional antioxidant capacity of erythrocytes and tissues from controland diet-induced hyperlipidemic rats exposed to the stress protocol(stressor-exposed) or control conditions, as assessedby in vitro oxidative challenge with H20 189Table 33. Weight and % change in weight of control and diabetic(four weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 194Table 34. Plasma glucose, % glycosylated hemoglobin and lipid levelsfrom control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions... 195Table 35. Pre-ischemic functional measurements in hearts from control anddiabetic (four weeks’ duration) male Wistar rats exposed to the stressprotocol (stressor-exposed) or control conditions 196Table 36. Comparison of baseline and post-reperfusion functional indicesin hearts from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol (stressor-exposed)or control conditions 197Table 37. Post-reperfusion antioxidant status in hearts from control and diabetic(four weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 198- xv -Table 38. Weights of control and diabetic (twelve weeks’ duration) Wistar ratsfollowing exposure to the stress protocol (stressor-exposed)or control conditions and % change in weight 210Table 39. Plasma glucose, % glycosylated hemoglobin and lipid levelsfrom control and diabetic (twelve weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions... 211Table 40. Pre-ischemic functional measurements in hearts from controland diabetic (twelve weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions... 212Table 41. Comparison of baseline and post-reperfusion functional indicesin hearts from control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol (stressor-exposed)or control conditions 213Table 42. Post-reperfusion antioxidant status in hearts from controland diabetic (twelve weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions... 214- xvi -LIST OF FIGURESFig. 1. TBARS formation following incubation with 0.75 mM t-butylhydroperoxide(TBHP) of liver homogenates from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 62Fig. 2. TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of kidney homogenates from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 64Fig. 3. TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of lung homogenates from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 66Fig. 4 TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of heart homogenates from control and adrenalectomizedmale Wistar rats treated with 6-hydroxydopamine or vehicle 68Fig. 5. Sulfhydryl group depletion following incubation with 0.025 mMt-butylhydroperoxide (TBHP) of heart homogenates from controland adrenalectomized male Wistar rats treated with 6-hydroxydopamineor vehicle 70Fig. 6. Percent sulfhydryl group depletion following incubation with 0.75 mMt-butylhydroperoxide (TBHP) of heart homogenates from control andadrenalectomized male Wistar rats treated with reserpine or vehicle 80Fig. 7. TBARS formation following incubation with 1.50 mM t-butylhydroperoxide(TBHP) of liver homogenates from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 82Fig. 8. TBARS formation following incubation with 1.5 mM t-butylhydroperoxide(TBHP) of kidney homogenates from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 84Fig. 9. TBARS formation following incubation with 1.50 mM t-butylhydroperoxide(TBHP) of lung homogenates from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 86Fig. 10. TBARS formation following incubation with 1.5 mM t-butylhydroperoxide(TBHP) of heart homogenates from control and adrenalectomizedmale Wistar rats treated with reserpine or vehicle 88Fig. 11. Plasma corticosterone levels in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-exposed (B) male Wistar ratssampled during a one-hour-period corresponding to days one, sevenand fourteen of the stress protocol 102-xvii -Fig. 12. Plasma corticosterone levels expressed as area under the curvethrough a one-hour-period corresponding to days one, seven andfourteen of the stress protocol in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-exposed (B) male Wistar rats 105Fig. 13. Plasma adrenaline levels in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-expósed (B) rats sampledduring a one-hour-period corresponding to days one, sevenand fourteen of the stress protocol 108Fig. 14. Plasma adrenaline levels expressed as area under the curvethrough a one-hour-period corresponding to days one, seven andfourteen of the stress protocol in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-exposed (B) rats 111Fig. 15. Plasma noradrenaline levels in control (A), stressor-exposed (A)diabetic (B) and diabetic stressor-exposed (B) rats sampledduring a one-hour-period corresponding to days one, sevenand fourteen of the stress protocol 114Fig. 16. Plasma noradrenaline levels expressed as area under the curvethrough a one-hour-period corresponding to days one, sevenand fourteen of the stress protocol in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-exposed (B) rats 117Fig. 17. Plasma glucose levels in control and stressor-exposed rats collectedduring a one-hour-period corresponding to days one, sevenand fourteen of the stress protocol 120Fig. 18. Plasma glucose levels in control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 123Fig. 19. Plasma cholesterol levels in control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 125Fig. 20. Plasma triglyceride levels in control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 127Fig. 21. Percent sulfhydryl group depletion following incubation with 0.1 mM H20of erythrocytes from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 139Fig. 22. Percent sulfhydryl group depletion following incubation with 0.1 mM H20of myocardial homogenates from control and diabetic (four weeks’duration) male Wistar rats exposed to the stress protocolor control conditions 141-xviii-Fig. 23. Percent sulfhydryl group depletion following incubationwith 0.25 mM H20 of liver homogenates from control anddiabetic (four weeks’ duration) male Wistar rats exposedto the stress protocol or control conditions 143Fig. 24. TBARS formation following incubation with 20.0 mM H20of liver homogenates from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 145Fig. 25. TBARS formation following incubation with 2.5 mM H20 of kidneyhomogenates from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 147Fig. 26. TBARS formation following incubation with 0.75 mM H20 of lunghomogenates from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 149Fig. 27. Malondialdehyde (MDA) formation following incubation with 2.5 mM H20of erythrocytes from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 151Fig. 28. TBARS formation following incubation with 1.5 mM H20 of myocardialhomogenates from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 153Fig. 29. Plasma glucose levels in control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 161Fig. 30. Glycosylated hemoglobin levels in control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocol orcontrol conditions 163Fig. 31. Plasma triglyceride levels in control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions 165Fig. 32. Plasma cholesterol levels in control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control .conditions 167Fig. 33. Percent sulfhydryl group depletion following incubationwith 0.1 mM H20 of erythrocytes from control and diabetic(twelve weeks’ duration) male Wistar rats exposed to the stressprotocol or control conditions 174Fig. 34. Percent sulfhydryl group depletion following incubationwith 0.025 mM H20 of myocardial homogenates from control anddiabetic (twelve weeks’ duration) male Wistar rats exposedto the stress protocol or control conditions 176- xix -Fig. 35. Percent sulfhydryl group depletion following incubationwith 0.25 mM H20 of liver homogenates from control anddiabetic (twelve weeks’ duration) male Wistar rats exposedto the stress protocol or control conditions 178Fig. 36. Percent sulfhydryl group depletion following incubationwith 0.025 mM H20 of kidney homogenates from control anddiabetic (twelve weeks’ duration) male Wistar rats exposedto the stress protocol or control conditions 180Fig. 37. Malondialdehyde (MDA) formation following incubationwith 2.5 mM H20 of erythrocytes from control and diabetic(twelve weeks’ duration) male Wistar rats exposed tothe stress protocol or control conditions 182Fig. 38. TBARS formation following incubation with 1.5 mM H20 ofmyocardial homogenates from control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocolor control conditions 184Fig. 39. Post-lschemic percent developed pressure in hearts from control anddiabetic (four weeks’ duration) male Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 199Fig. 40. Post-ischemic percent +dP/dtm in hearts from control and diabetic(four weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 201Fig. 41. Post-lschemic Percent -dP/dt in hearts from control and diabetic(four weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 203Fig. 42. Post-reperfusion time-dependent peroxide-induced glutathionedepletion in hearts from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol (stressor-exposed)or control conditions 205Fig. 43. Post-lschemic percent developed pressure in hearts from control anddiabetic (twelve weeks’ duration) Wistar rats exposed tothe stress protocol (stressor-exposed) or control conditions 215Fig. 44. Post-ischemic percent +dPIdtm in hearts from control and diabetic(twelve weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 217Fig. 45. Post-lschemic Percent dP/dtm in hearts from control and diabetic(twelve weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions 219- xx -Fig. 46. Post-reperfusion peroxide-induced TBARS formation in heartsfrom control and diabetic (twelve weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions.... 221-xxi-LIST OF ABBREVIATIONSACTH adrenocorticotropin hormoneASC ascorbic acidAUC area under curveCAT catalaseCRH corticotropin-releasing hormoneCu, Zn-SOD Cu, Zn-superoxide dismutaseDTN B 5, 5’-dithio-bis-(2-nitrobenzoic acid)EDTA ethylenediaminetetraacetic acidEGTA ethyleneg Iycol-bis-(beta-amino ethyl ether) N, N’-tetraacetic acidGSH-PX glutathione peroxidaseGSSG-RED glutathione reductaseGSH glutathione, reduced formGSSG glutathione disulfideHPA hypothalamic-pituitary-adrenalHO• hydroxyl radicalhR ischemialreperfusionMDA malondialdehydeNADPH nicotinamide adenine dinucleotide phosphate, reduced formNBT nitroblue tetrazoliumO2 superoxide anion radicalP1 inorganic phosphateRBC red blood cellsRODS reactive oxygen-derived substancesTBARS thiobarbituric acid-reactive substancesTBHP tert-butylhydroperoxideTCA trichioroacetic acidTRIS tris[hydroxymethyl] aminomethane-xxii-ACKNOWLEDGEMENTSI want to thank Dr. David Godin for his continued guidance and committment inthe completion of this project. I am also grateful to him for taking the risk in allowing meto work on a project that changed the focus of research in the laboratory and honourhim for encouraging me to be in control of my research allowing me to gain theconfidence to be a competent independent researcher. Ms. Maureen Garnett’spersistence in maintaining a well organized laboratory and her commitment to the lab inachieving a high level of assay precision enabled me to accomplish this work. I extendmy appreciation to Ms. Nur Shaw whose help during the chronic experiments removedsome of the pressure and allowed me to take the occasional weekend break. Ms.“Dolly” Garnett ensured there was a ready supply of clean glassware and a hot cup oftea. Ms. Elaine Jan and Ms. Wynne Leung contributed their time in the final formattingof the thesis. Ms. Maureen Murphy was instrumental in helping to solve numerouscomputer problems during the writing of the thesis. Dr. Richard Wall contributed hisexpertise in fine tuning the catecholamine assays. Dr. Joanne Weinberg aided incritical discussions involving the design of the stress model and allowed me the use ofher laboratory for the corticosterone analyses. Dr. Michael Walker’s discussionshelped in the design of the myocardial ischemialreperfusion model. In addition I wishto extend my appreciation to the committee members and examiners Dr. James Foulks,Dr. Wolfgang Linden, and Dr. Robert Beamish for their thoughtful comments andstimulating questions during the oral defence.I am indebted to my family who walked along with me through each triumph anddissappointment during the many years of this project. The loving support fromClaudia, my wife, kept my vision focused throughout the myraid of experiments and theconstant optimism and curiosity of my daughters Lauren and Annise provided me withthe drive to reach my goals.Finally, I was able to complete this work through the ongoing scholarship fundingfrom the B.C. and Yukon Heart and Stroke Foundation and Canadian DiabetesAssociation.-xxiii-DEDICATIONThis work is dedicated to my parents Irene and EdwardToleikis who encouraged me with their unwavering supportin all my endeavors to take risks that enabled me to grow.—1—1. INTRODUCTION1.1. Behavioral Factors in Cardiovascular DiseaseCardiovascular disease is a major cause of death and disability in theindustrialized world, with the majority of deaths attributed to atherosclerosis-associatedcoronary artery disease resulting in acute myocardial infarction.2°8 The study ofmyocardial ischemic injury and its clinically relevant consequences, namelyarrhythmias and contractile impairment, has provided much information about themolecular basis of ischemic injury and its exacerbation by post-ischemic reperfusionand in particular the role of endogenously generated reactive oxygen-derivedsubstances (RODS).8° The practical clinical applications of research in this area,however, have been limited to situations in which the timing and conditions of postischemic reperfusion can be controlled, as in revascularization or thrombolyticprocedures and myocardial preservation during cardiopulmonary bypass or prior totransplantation.Additional factors other than improved post-infarction survival appear to beimportant in reducing mortality due to cardiovascular disease. For example, a detailedlong-term (26 year) study of the incidence of first myocardial infarction and associatedfatality rates in a large number of workers from the Dupont Company has demonstrateda gradual decrease in mortality rates, pointing to a reduced incidence of infarctionrather than improved post-infarction survival as the major determining factor.21° Thedecrease in the number of deaths from coronary artery disease over the past several-2-years79 has been attributed to the attention given to the associated risk factorsincluding hyperlipidemia, hypertension, obesity, smoking and high intake of saturatedfat, based on epidemiological data, to predispose to the disease. These findingsseem to underline the importance of preventative measures, given the inadequacy ofcurrently available measures for dealing with the early post-infarction period which isstill associated with a high risk of mortality.In addition to the foregoing “conventional” risk factors, behavioral or emotionalfactors have generally been accepted to play a role in the development ofcardiovascular disease. For example, a strong relationship has been shown betweenphobic anxiety and probability of death due to ischemic heart disease, but not to deathfrom other causes. In addition, measures of life-event stress have been associatedpositively with acute myocardial infarction in a number of studies.1’°7The type-A behavioral pattern, described by Freidman and Rosenman,74 is seen in individualswho are more rushed, highly ambitious and competitive, more intolerant of frustration,impatient and openly-hostile. Interestingly, while some reports suggest that individualsexhibiting Type-A behavior have a higher risk of developing ischemic heartdisease,2’37 a recent Western Collaborative Group Study (involving 257 male patientswith ischemic heart disease) concluded that individuals identified as Type-Apersonalities on the basis of a structured interview showed a lower risk of death fromcoronary artery disease (within 24 hr of the acute ischemic event) than Type Bindividuals,226 suggesting a possible influence of different coping mechanisms. Whilethese studies lead to the suggestion that stress could influence the progression ofcardiovascular disease, it must be cautioned that much of the clinical research in this-3-area has been derived from retrospective studies in which patients with heart diseaseare sampled and compared with a matched group of healthy persons. It has beenshown that patients with pre-existing pathologies often seek social or psychologicalreasons to explain their disease29 and would therefore be expected to cite increasedstress more frequently as a contributing factor. In addition, because in retrospectivestudies data are gathered on the illness and possible risk factors at the same time, itusually cannot be shown that the supposed “cause” preceded the disease.23° Finally,in these associative studies, the consequences of stress cannot be measured in termsof stress-induced pathological changes. Thus, the clinical studies conducted to dateare clearly limited in their ability to provide insights into possible mechanisms by whichstress modifies the course of cardiovascular disease, underscoring the need forexperimental research in this area.In an attempt to investigate the effects of emotional stress on thedevelopment of ischemic heart disease, consideration must be given to the separateeffects of stress on intrinsic myocardial susceptibility to ischemic damage (whetheracute or cumulative) and vascular changes attributable to atherosclerosis. Largecardiovascular reactions to behavioral stimuli have been associated with an increasedpredisposition to atherogenesis.161’2Although the mechanism underlying the proatherogenic effect of stress is incompletely understood, it appears that elevated plasmalevels of catecholamines play an important role.61’97 On the other hand, little is knownabout the effects of stress on intrinsic myocardial susceptiblity to ischemic damage,which is in part the concern of the present study.-4-1.2. Considerations in the Experimental Study of StressStudy of the response of laboratory animals to stress requires carefulconsideration of the “stress hormones” to be monitored as well as the parameters of thestress stimulus. Although catecholamines and corticosteroids are the most extensivelystudied “stress hormones”, the release of numerous other chemical substancesincluding adrenocorticotropic hormone (ACTH), thyrotropin and somatotropin,9betaendorphin,306 interleukin 6,1 prolactin191 and metallothionine11°have been shown tovary in response to acute stressful stimuli. The choice of hormones to be measuredshould be made according to their potential actions on the physiological systems underconsideration. For example, study of stress effects on the cardiovascular system wouldlikely include measurement of catecholamine levels.In addition to the selection of stress hormones to monitor, parameters of thestress stimulus itself must be also be considered. These include: the type of stress,whether physical or psychological, its intensity, duration, frequency and time betweenexposures, and finally whether repeated or variable stressors will be used. Changes inthese parameters appear to influence stress hormone release patterns and the degreeof habituation or sensitization with repeated exposure to stressful stimuli. Theimportance of using more than one stressor and index of stress is shown in a study inwhich the effects of various stressors including noise, restraint or immobilization onserum concentrations of ACTH, thyrotropin and somatotropin, were investigated.Increases in levels of ACTH, paralled by decreases in somatotropin were greatest withimmobilization and least with noise. On the other hand, noise had the greatest effecton levels of thyrotropin, which showed an increase followed by a decrease.9 Thus,-5-measurement of only one index of stress at a fixed time point might lead to misleadingconclusions regarding the complex nature of the stress response.In response to physical or psychological stressors, increased activation of thesympathetic nervous system occurs through release of noradrenaline and adrenalinefrom the adrenal medulla. In addition, the major endocrine mechanism is the activationof the hypothalamic-pituitary-adrenal axis (HPA axis) which results in rapid increases incirculating levels of adrenocorticotropin hormone (ACTH) released from the anteriorpituitary with a subsequent rise in gIucocorticoids61from the adrenal gland. Themechanisms regulating ACTH secretion during stress are multifactorial and include astimulatory effect of hypothalamic-releasing factors, mainly corticotropin-releasinghormone (CRH) and vasopressin released from hypothalamic neurons which reach thepituitary gland through a portal system. An inhibitory influence of gIucocorticoids7can have a direct action on the anterior pituftary to inhibit the formation of mRNA forACTH or inhibit ACTH release; however, this action appears to be impaired in chronicstress. Glucocorticoids stimulate catecholamine synthesis in the adrenal medulla andmodulate metabolic and cardiovascular effects of catecholamines.2115°’34Furthermore,neuronal noradrenaline or adrenaline, released from the adrenal medulla, can stimulateCRH release from the anterior pituitary’1 leading to an increase in ACTH. It has beensuggested that glucocorticoids moderate the normal responses of the organism tostress preventing threats to homeostasis from exagerated reactions.’° While in acutestress ACTH responses are for the most part dependent on the interaction of CRH withreceptors on the pituitary,4’8mechanisms responsible for regulation of ACTH duringchronic stress are not well understood, but may involve changes in the levels of CRH-6-secreted, changes in CRH receptors in the pituitary or alterations in the sensitivity ofthe glucocorticoid feedback mechanism.3As mentioned previously, the most extensively studied indices of experimentalstress are noradrenaline (a neurotransmitter at sympathetic nerve fibers), adrenalineand corticosteroids (released by medullary and cortical components of the adrenals,respectively). Although stress-induced activation of the sympathetic nervous systemcauses alterations in the release of all three components, each may vary independentlyin response to stressful stimuli. For example, in rats subjected to foot shock in theabsence of bedding (a non-coping passive avoidance stress), plasma levels of bothadrenaline and corticosterone increased, while exposure to foot shock in the presenceof bedding (resulting in burying behavior, and hence considered an active avoidancestress) produced increases in both adrenaline and noradrenaline.52 These findingshave led to the suggestion that increases in noradrenaline are likely to accompanystress-induced skeletal muscle activity while increases in adrenaline or corticosteronewould be associated with fear and anxiety arising from the inability to cope.Furthermore, repeated episodes of white noise stress (a total of 20 exposures of 4 mmeach) in rats showed that plasma levels of noradrenaline, adrenaline andcorticosterone had distinct temporal relationships and differed in both the speed anddegree of adaptation as well as in their response to predictibility or unpredictibility ofthe applied stress.In studies of prolonged stress over several days or longer, commonly referred toas chronic intermittent stress, it is clear that repeated stress of the same type can leadto eventual habituation of the stress response measured in terms of corticosterone124’231-7-and catecholamines.’1 Furthermore, adaptation to stress has been shown to haveimportant physiological consequences beyond changes in hormone levels. Forexample, repeated exposure to stressful stimuli whereby the duration of exposure wasincreased over several days in rats was found to reduce the sensitivity of subsequentlyisolated hearts to ischemia/reperfusion-induced arrhythmias (following immobilizationstress) and reduce the sensitivity of isolated rat pacemaker to noradrenaline(following swimming stress).2 Studies in which chronic stress exposure involvedunpredictibility both in the timing of exposure to stress and in the type of stress haveshown either a typical adaptation response,1 or a reduced tendency towardshabituation over time or even sensitization to a novel stressor.222 The physiologicalconsequences of this type of variable chronic stress are not well understood.Stress intensity is an additional factor which can determine the degree ofhabituation or sensitization to repeated stress. This was demonstrated in a study inwhich the effects of swim-stress in rats on habituation and sensitization was found to bedependent on the temperature of the water.137While increased stress intensity appears to reduce the incidence of habituation,studies which employ very high levels of stress such as long periods of inescapablerepeated shock (emotional painful stress170)are of dubious value in our understandingof the functional consequences of chronic psychological stress which are probablyassociated with a relatively more sustained release of lower levels of stress hormones.Such severe acute stress as described above appears to increase free radical relatedprocesses in otherwise normal animals.170 We suggest that study of the pathologicaleffects of these high stress levels is analogous to study of the toxic effects of-8-supranormal doses of pharmacological substances in that the results must be viewed inthe context of the dose.. While one cannot directly equate a dose” in theadministration of stress with the dose of a drug and its consequent pharmacologicalactions, one can differentiate between the severe stress probably associated with acutemyocardial infarction and the chronic but lower levels of psychological stress whichmight have effects on a long-term pre-existing disease state such as diabetes.In summary, in order to gain a better understanding of the complex nature of thestress response, more than one stress index should be measured. In addition, thephysiological effects of repeated stress appear to differ with the degree of predictibilityand intensity of the stimulus. In the study of chronic stress, the choice of thesestimulus factors will determine the degree of adaptation or sensitization to repeatedstress which could have very different pathological implications.1.3. Effects of High Levels of Catecholamines on the Myocardium: FreeRadical Related ProcessesStimulation of the heart with catecholamines possessing beta-agonist activityincreases myocardial oxygen demands.247 In the presence of coronary artery disease,the consequences on supply/demand balance may induce a state of ischemia which, ifprolonged, could result in tissue damage. Acute elevations in blood levels ofcatecholamines can induce damage to the myocardium.2 High doses of exogenouslyadministered isoproterenol,2°adrenaline,9 or noradrenaline can produce similarabnormalities in cardiac ultrastructural features. Endogenous catecholamine releasestimulated by tyramine causes comparable histological changes. Similarly, elevationof catecholamines occurring during the course of myocardial infarction has been-9-implicated in the associated myocardial damage1012 and it has recently been shownthat exogenous noradrenaline at micromolar concentrations contributes to thedeleterious effects of acute regional myocardial ischemia.2 Acute elevations innoradrenaline levels accompanying myocardial infarction may also be associated withpotentially fatal arrhythmogenesis.18 It has been shown that peak catecholamine levelsin humans are reached between 10:00 a.m. and noon, the time corresponding to thepeak incidence of myocardial infarction and sudden death.272Regarding the mechanism of injury, a role for free radical production has beenproposed in such catecholamine-induced myocardial necrosis.5’602 Several lines ofevidence support this suggestion. The myocardium is particularly vulnerable to oxidantattack, in that it has relatively low antioxidant enzyme activities relative to othertissues,3°1 suggesting an important role for free radical-mediated damage inischemiclreperfusion injury. The administration of high doses of catecholamines toexperimental animals can induce myocardial damage which is exacerbated by vitaminE deficiency and prevented by antioxidant supplementation, suggesting theinvolvement of oxidative processes.2 Catecholamines have long been known toundergo autoxidation in vitro to generate oxidation products which, if formed in vivo,could be capable of exerting deleterious effects. However, under physiologicalconditions, spontaneous (i.e. non-enzymatically catalyzed) autoxidation ofnoradrenaline and adrenaline is very slow and of uncertain pathologicalsignificance.1°°’02’21 However, microsomal preparations have been shown to promotethe oxidation of adrenaline by a process involving superoxide radiCals.97’242Catecholamines can also undergo oxidation in the presence of H20/Fe-haem- 10 -proteins123 or transition metal catalysts such as cupric ion.291 Furthermore, theoxidation of catecholamines under certain circumstances may exceed the antioxidantcapacity of the myocardium. This may be potentiated by the inactivation of antioxidantenzymes by reactive oxygen-derived substances themselves.1122 It would follow,therefore, that chronic elevation of catecholamines, such as might occur inpheochromocytoma,1or during sustained emotional stress172 may predispose themyocardium to cumulative RODS-induced damage. Some evidence exists that severeemotional stress produced by inescapable repeated shock can result in peroxidativedamage to myocardial tissues.17017’ Evidence of the permissive role of stress in thedevelopment of myocardial necrosis is shown in a study where cold-restraint stresscaused marked acceleration of myocardial lesion development and ultimately death incardiomyopathic hamsters.2 The stressors used in these studies, however, aresevere and the consequences of more modest but sustained increases incatecholamines, as might be expected with chronic psychological stress, have not beensystematically investigated.One approach to investigating effects of catecholamines on antioxidant statuswould be to study consequences of catecholamine depletion. Although this has notbeen directly investigated, some inferential data do exist; however, experimentalresults to date have been equivocal. For example, reserpine-induced depletion ofcatecholamines prior to ischemia was reported to have a beneficial effect on coronaryflow rate and cardiac muscle metabolism, resulting in prevention of ischemia-inducedmyocardial ATP depletion and subsequent irreversible myocardial damage.114 Incontrast, 6-hydroxydopamine-induced depletion of catecholami nes prior to coronary— 11 —artery ligation-induced ischemia was reported not to decrease infarct size in rabbits1or protect the myocardium against ischemia-induced arrythmias in conscious rats.25More detailed information concerning the biochemical effects of catecholamines onmyocardial antioxidant components and susceptibility to oxidant injury might clarify thisapparent discrepancy.1.4. Effects of Elevated Glucocorticoid Levels: Free Radical RelatedProcessesElevated circulating levels of cortisol have been found in certain pathologicalconditions associated with increased oxidative stress, notably coronary arterydisease28”and diabetes.’°9’2273359 However, the potential contribution of stress-induced enhanced release of adrenal corticosteroids in producing changes in thefunctional integrity of the myocardium has not been examined. Some studies suggestthat glucocorticoids may be capable of inducing oxidant stress.195’9 These studieswere conducted on animals treated with exogenous glucocorticoids which result insuppression of basal hypothalamo-pituitary-adrenalocortical and basaladrenomedullary activity;26°therefore, the effects may not be directly applicable to thesituation of stress-induced elevation of endogenous glucocorticoids. On the otherhand, study of the potential antioxidant properties of steroids using an in vitro peroxyradical generating system has recently shown that cortisol and corticosterone do nothave antioxidant effects and in fact had very mild (probably insignificant) pro-oxidantproperties.182The effects of elevations in endogenous glucocorticoids (as would occur duringstress) on antioxidant status are unknown; however, glucocorticoids could affect- 12 -antioxidant capacity through regulation of antioxidant components. For example,pharmacological suppression of corticosterone production has been associated with adelay in lung antioxidant maturation in neonatal rats2 and induction of metallothionein,a putative free radical scavanger, has been found to occur following restraintstress.108’9 Although these studies did not establish a direct role for glucocorticoids inthis response, steroid induction of metallothionein has been observed in rat primaryhepatocyte cultures,26 Thus, while there is some evidence for alterations ofglucocorticoid levels in pathological conditions associated with increased oxidativestress, conclusions cannot be made regarding a relationship between endogenousglucocorticoid elevation and changes in antioxidant capacity.1.5. Overview of Endogenous Free Radical Scavanging Systems1.5.1. Antioxidant EnzymesThe production of reactive oxygen species is a consequence of normal oxidativecellular metabolism. While these species can lead to deoxyribonucleic acid damage,membrane disruption and cell death, an elaborate series of enzymes has evolved fordetoxification and prevention of damage. These enzymes include copper, zincsuperoxide dismutase and catalase in the cytoplasm, manganese superoxidedismutase in the mitochondria and glutathione peroxidase in the mitochondria andcytoplasm. The formation of superoxide radicals occurs in part when electronswhich leak from electron carriers in the electron transport chain pass directly to oxygenreducing it to the superoxide radicaL Superoxide dismutase removes the superoxideradical by catalysing a dismutation reaction involving the oxidation of one superoxideradical to bimolecular oxygen and the reduction of another to hydrogen peroxide.73- 13 -Hydrogen peroxide itself is toxic to cells, in part through further reaction withsuperoxide radicals in the presence of iron or copper to form highly reactive speciesincluding the hydroxyl radical.3 Therefore, removal of hydrogen peroxide is also ofcritical importance. Superoxide dismutase thus works in conjunction with two additionalenzymes, catalase and glutathione peroxidase, both of which remove hydrogenperoxide. It has been suggested that glutathione peroxidase is the more important ofthe two because it is located in the same subcellular compartments (mitochondria andcytosol) as superoxide dismutase.92 Glutathione peroxidase catalyses the reactionwhich uses hydrogen peroxide to oxidize a cofactor, reduced glutathione, into oxidizedglutathione. Glutathione reductase catalyses the reaction which rejuvinates reducedglutathione from the oxidized form. Catalase catalyses the reduction of hydrogenperoxide to water.1.5.2. Metal-Ion SequestrationMetal ions are believed to play a catalytic role in the generation of RODSYMetal ion sequestration is, therefore, an important antioxidant defence mechanismin vivo.10’9°’ For example, under normal conditions, there are essentially no free ironions in the plasma due to the fact that there is a factor of three times as muchtransferrin iron binding capacity in plasma as iron needing to be transported undernormal conditions. Iron bound to transferrin cannot stimulate lipid peroxidation orformation of hydroxy radicals.1.5.3. Non-Enzymatic AntioxidantsThe major nonenzymatic antioxidant components in vivo are, cc-tocopherol,ascorbic acid, glutathione and uric acid.’9192 Cell membranes and plasma lipoproteins- 14 -contain cL-tocopherol, a lipid soluble molecule, which functions as a chain breakingantioxidant. It is considered the most important lipid-soluble chain breaking antioxidantin vivo in humans116 This hydrophobic molecule contains a hydroxyl group from whichthe hydrogen atom is easily removed, so peroxy and alkoxyl radicals formed duringlipid peroxidation preferentially combine with the antioxidant rather than with a fattyacid side chain, thus terminating a potential chain reaction.92 The c-tocopherolmolecule is converted to a new radical, but one which is unable to attack adjacent fattyacid side chains. The regeneration of c-tocopherol occurs through a reaction withascorbic acid, a chain breaking antioxidant in the aqueous phase.292 Glutathione is inequilibrium with all cellular sulfhydryl groups and is both intra- and extra-cellular.Glutathione can also react with tocopheryl radicals to regenerate tocopherol. In thedetoxification of oxidants, glutathione becomes oxidized and is again reduced usingglutathione reductase with NADPH as a cofactor. Thus, as a cosubstrate of glutathioneperoxidase, glutathione is protective against free radicals and is involved in theprevention of peroxidation of membrane lipids. Furthermore, changes in glutathionestatus provide a sensitive index of oxidative stress.1741.6. Pathological Conditions Which Might Be Affected by Stress:DiabetesStress is thought to have a permissive effect on the development ofcardiovascular disorders including myocardial infarctio&8’72 and atherosclerosis,’27’both being major complications of the long-term diabetic state.125- 15 -1.6.1. Diabetes and Stress: Metabolic Effects and ConsequencesPsychological stress induces a metabolic state in which the actions ofglucocorticoids, catecholamines and other stress hormones predominate over those ofinsulin.27 Similarly, characteristic of uncontrolled diabetes is a decrease in insulinaction, associated with either a reduction in insulin release (Type I) or in the insulinsensitivity of target tissues (Type II). Stress-induced elevation of glucocorticoid levelscan lead to increased tissue insulin resistance in normal subjects6’1631 and thus mightbe expected to exacerbate the metabolic changes in diabetes.4°’’1This reduction ininsulin sensitivity would imply a possible role of stress-related factors in diabetogenesisand/or an increased sensitivity of diabetics to the effects of stress. A sustainedimpairment in insulin sensitivity has recently been reported following acute mentalstress which also elevated plasma levels of adrenaline and cortisol in insulin-dependent diabetic patients.tm Stress has also been associated with increased insulinrequirements in diabetic patients’132 and in fact has been shown to precipitatediabetes in diabetes-prone BB Wistar rats (following exposure to restraint, cagerotation, crowding or resocialization)31 and in predisposed humans following stressfullife events.2 Furthermore, diabetic patients are also reported to be more prone todeveloping neuropsychological disorders and experience more stress relative to thenon-diabetic population.15”24° However, while numerous clinical studies haveattempted to determine the extent that psychological stress affects blood glucoselevels, the control of which has been suggested to result in improved long-term healthoutcomes, many of the same problems have arisen regarding in part the patients- 16 -perception of stress factors as discussed previously. In addition, biochemical markersof stress levels used in clinical studies are frequently not measured.1.6.2. Diabetes as a Sustained Stimulus to Catecholamines andCorticoidsRecent evidence supports the suggestion that diabetes represents a sustainedstimulus to the sympathetic nervous system and the adrenocortical axis. Chemically-induced diabetes has been associated with elevated baseline plasma levels ofadrenaline and noradrenaline’6in rats. Increased basal levels of corticosterone havebeen reported in streptozotocin (STZ)-induced diabetes in rats’227 and geneticdiabetes in ob/ob mice.259 In addition, chronic alterations in plasma corticosteronelevels have been noted in STZ-induced diabetic rats and diabetic mice.169Furthermore, in STZ-induced diabetes, decreased thymus weights, increased urinaryexcretion of corticosterone and increased corticosterone levels averaged over the lighthours of the day have also been reported. In addition, abnormal corticoid circadianrhythms have been observed in diabetic rats2°4 and humans.147 These findings thatdiabetes appears to impose a subtle but persistent sympatho-adrenal stimulationsuggests the possibility that chronic psychological stress may further augment thesehormonal alterations.1.6.3 Diabetes and Acute Stress: Effects on Catecholamines andCorticoidsSensitization to acute stress has been demonstrated in streptozotocin-diabeticrats, as shown by enhanced elevation in plasma corticosterone levels in response tointraperitoneal cold water injections. Likewise, acute foot shock in diabetic rats has- 17 -been shown to produce an exagerated response in plasma adrenaline andnoradrenaline levels1 and diabetic patients under poor glycemic control exposed toacute psychological stressors reportedly show exaggerated elevations in plasmaadrenaline levels.130 Studies of diabetes-associated abnormalities observed underconditions of acute stress, while suggesting an altered reactivity to stress, do notaddress the effects of chronic stress on neuroendocrine function. Furthermore,although documentation of the transient alterations in plasma levels of “stresshormones” is important to provide insight into whether diabetic animals show an alteredsensitivity to stress, it does not provide information on the possibly cumulativefunctional consequences of stress, which may have important pathological implicationson the development of diabetic complications.300 Thus, the molecular basis governingthe interaction between stress and diabetes has yet to be established.17. Evidence for Free Radical Involvement in DiabetesLong-term complications of diabetes mellitus include: kidney failure, heartdisease, haemotological abnormalities, nephropathy, retinopathy and a number ofmetabolic abnormalities. In the kidney, frequent sequelae to both types I and IIdiabetes are increased filtration rate and filtration fraction, excretion of urinary albuminand other proteins,’ synthesis of mesangial actomyosin-like material, hypertrophy,glomerular capillary damage and eventual renal failure.302 In the heart, the presence ofsubtle abnormalities has recently been noted in young patients with insulin-dependentdiabetes mellitus)32 In this study, the cardiovascular findings were associated withincreased creatinine clearance and microalbuminuria, which are considered to be theearliest abnormalities in the pathogenesis of renal dysfunction and ultimate renal failure-18-in diabetic patients. In the adult population, the existence of a diabeticcardiomyopathy, independent of atherosclerotic and hypertensive heart disease, hasbeen firmly established.’’2’31° This cardiomyopathy can be reproduced inexperimental animals made diabetic with STZ.262 Thus, evidence of early diabetes-induced abnormalities has been shown in several organs which appear to progress tomore serious complications with increased duration of the disease.Among other causes, a putative role for reactive oxygen radicals has beensuggested in the development of diabetes and its associated complications.1241 Ingeneral, elevated oxidative stress can arise as a result of increased free radicalproduction, reduced activity of antioxidant defences, or a combination of the two.Oxidative stress may become amplified by an autocatalytic cycle of metabolic stress,tissue damage and cell death, which would in turn lead to an increase in free radicalproduction and compromised scavanger mechanisms, further exacerbating theoxidative stress.Increased oxidative stress has been implicated in the development of type Idiabetes and of spontaneous or chemically-induced diabetes in experimentalanimals.12’52012 4302 Autoimmune processes involving macrophage and lymphocyteinfiltration of the pancreatic islets play a role in the induction of type I diabetes.4Treatment with a synthetic antioxidant prevented diabetes in non—obese diabetic andmultiple low dose streptozotocin-injected diabetic mice, supporting the hypothesis offree radical involvement in this process.103 Cytokines produced by activatedmacrophages and T-lymphocytes may also initiate oxidative processes resulting in thedestruction of pancreatic f3-cells.224 Pretreatment with superoxide dismutase has been- 19 -shown to prevent pancreatic 13-cell damage induced by streptozotocin in vitro or protectagainst the effects of STZ administration to rats.75232Mechanisms contributing to oxidative stress in the progression of diabetes havebeen postulated to include: nonenzymatic glycosylation (glycation) and autoxidativeglycosylation, metabolic stress resulting from changes in energy metabolism, changesin the level of inflammatory mediators and alterations in the status of antioxidantdefense systems.12 While it is difficult to show directly that oxidative stress is increasedin diabetes, diabetes is associated with increased chemical modification of proteins andlipids whose damage appears to be oxidative in origin)2The autoxidation of glucose and of glycated proteins can generate reactiveoxygen species, especially in the presence of transition metals.255’302 Peroxidation andglycosylation of LDL has been shown to occur at the same time in diabetes.115 Anelevation in the glycated form of erythrocyte Cu,Zn-superoxide dismutase was found indiabetic patients.128 Increased glycation of this enzyme was recently shown to beaccompanied by inactivation.202 Glucose can be a source of superoxide ions.271 Itwould be expected, therefore, that the diabetes-associated hyperglycemia couldcontribute to oxidative stress. The mechanism has been suggested to involve the slowenolization of monosaccharides to produce ene-diols, which react nonenzymaticallywith oxygen to yield a semidione radical and superoxide.° The semidione radical canthen decay to a I -hydroxyalkyl radical and hydrogen peroxide at physiological pH. Asa prerequisite, autoxidation reactions frequently require catalysis by excess trace metalions (e.g., Cu, Fe2jY’55 Superoxide can reductively mobilize iron ions from ferritin22’4and its production is known to be increased in polymorphonuclear leukocytes of- 20 -diabetic individuals.”°5 The effectiveness of superoxide dismutases in scavengingsuperoxide radicals73 may be inadequate in the diabetic state, due either to reducedsynthesis or to inactivation of the enzyme by gIycosylation.’28 Increased oxidativestress may result in Fe2 release from mitochondria.175 In addition, an elevation inproduction of hydrogen peroxide in diabetic tissues’5°3could result in release of ironfrom heme proteins, including hemoglobin and myoglobin.22° Indirect evidence thatsuch increased hydrogen peroxide formation could occur in diabetes is suggested fromexperiments in which albumin solutions were incubated under physiological conditions(pH 7.4, 37°C) with varying concentrations of glucose followed by formation ofnanomolar levels of hydrogen peroxide.122 Thus, highly-reactive hydroxyl radicalscould be formed in vivo and mechanistic explanations exist for the increased activityof RODS in diabetes mellitus.1152°2There appears to be an increased sensitivity of tissues and blood to lipidperoxidation in diabetes. For example, erythrocytes from diabetic rats and patientshave increased sensitivity to peroxide-induced lipid peroxidation under in vitroconditions. In addition, increased levels of erythrocyte membrane lipid peroxidationproducts have been measured which correlated with the degree of glycosylatedhemoglobin.U7 Lipid peroxide levels in diabetic patients with micro- and macroangiopathies were elevated in comparison to non-diabetic controls and showedsignificant correlation with the duration of diabetes and presence of ischemic heartdisease. Furthermore, treatment with exogenous antioxidants has been shown toreduce the formation of oxidation products in diabetes.3°7- 21 -Regarding antioxidant defence systems, diabetes is also associated with markedalterations in the activity of enzymatic antioxidants, including catalase, superoxidedismutase, glutathione peroxidase and glutath lone reductase 152, 165.201,241 .270.299 inplasma, erythrocytes and various organs. These changes are tissue-specific, bothquantitatively and qualitatively, and diabetes-associated changes in food intake andweight gain do not account for the observed alterations in antioxidant enzymeactivities.301 Furthermore, alterations in antioxidant activities of liver and heart299 andaortic endothelial cells,261 but not erythrocytes65of STZ-treated rats could be reversedby insulin treatment, indicating that the alterations in antioxidant components are acharacteristic of the uncontrolled diabetic state.Non-enzymatic antioxidant components are also altered in diabetes.119’201Ascorbic acid, for example, which works as an antioxidant by scavanging radicalsdirectly, preventing the propagation of a radical induced chain-reaction such as mayoccur during lipid peroxidation is depleted in patients with diabetes, along withincreased levels of its oxidation product dehydroascorbate.’20’307 Dehydroascorbatelevels remain elevated despite insulin treatment, suggesting the presence of abnormalascorbate metabolism in diabetes.2 Although supplementation of ascorbate may, inaddition to its free radical scavanging activity, also reduce the extent of proteinglycation2which may play a role in the development of diabetic complications, themain oxidation product of ascorbate, dehydroascorbate, may itself directly glycateproteins.47 This pro-oxidant effect appears to occur in the presence of transition metalsand particularly iron.1 Interestingly, increased ferritin levels have been reported inpoorly controlled diabetics” and this could favor the pro-oxidant effects of ascorbate.- 22 -Recently, it has been shown in STZ-induced diabetic rats that while ascorbatesupplementation or iron chelating treatment with desferrioxamine alone failed to reduceoxidative stress, a combination of both interventions restored malondialdehyde,conjugated dienes, antioxidant vitamins, glycated albumin and HbA1 to controllevels°6 Thus, there appears to be a complex relationship between the levels ofascorbate and presence of transition metals which may determine its relative benefit ordetriment in diabetes.In contrast to the status of ascorbate, plasma levels of tocopherol appear to beincreased in chemically-induced and genetic diabetes in experimentalanimals,14’219227°as well as in human diabetic patients.2 Tissue levels oftocopherol were found to be elevated in the liver of STZ-induced diabetic rats27°and inheart, serum, testes and thymus of spontaneously diabetic BB rats.14 In the BB rat,elevated tocopherol levels were normalized with insulin treatment, suggesting that theraised tocopherol concentrations were a consequence of the diabetic state.14 There issome discrepency in the literature regarding diabetes-induced changes in the levels oftocopherol in tissues.1 Alterations in the level of plasma lipoproteins, and in particularof non-HDL cholesterol, have been found to correlate with plasma tocopherol levels intype I and II diabetics.23 This correlation is likely a result of the fact that plasmalipoproteins are the principal carriers of tocopherol in the blood and may account forsome of the discrepancies in the literature. Tocopherol is the only lipophilic antioxidantin blood1and is the principal chain-breaking antioxidant in mammalian membranes.Ascorbate is believed to play an important role in preserving and recyclingtocopherol.2 This action is prevalent at the water/lipid interface since the primary- 23 -actions of ascorbate and tocopherol occur in the aqueous and lipophilic compartments,respectively.In summary, given the similar metabolic consequences of diabetes and stressand the potential involvement of free radical-related processes under both conditions,time-dependent deterioration in antioxidant capacity might occur with an increasedduration of diabetes and the effects of stress on antioxidant alterations in short-termdiabetes might vary with increased duration of the disease.1.8. RationalePsychological stress induces a metabolic state in which the actions ofglucocorticoids, catecholamines and other stress hormones predominate over those ofinsulin.27 Such a metabolic state also characterizes uncontrolled diabetes associatedwith a reduction either in insulin release (Type I) or in the sensitivity of target tissues toinsulin (Type II). In addition, sustained activation of the sympathetic nervous system,as might occur in chronic stress, has been proposed to increase oxidative stress.Furthermore, endogenously generated reactive oxidants and/or alterations inantioxidant systems have been implicated in the pathogenesis of diabetes and itsassociated complications, including myocardial infarction and atherosclerosis. Basedon these considerations, it can be postulated that the diabetic state might beparticularly sensitive to the effects of psychological stress in terms of stress hormoneresponse, exacerbation of the diabetes-associated metabolic abnormalities andantioxidant status. The general aim of this work is, therefore, to develop a model ofchronic-intermittent stress with which to investigate stress hormone release andalterations in endogenous antioxidant components in rats with STZ-induced diabetes of- 24 -relatively short (4 weeks) and long (12 weeks) duration as compared with control rats.Functional consequences of chronic stress would be assessed in terms of alterations intissue susceptibility to ex vivo oxidative challenge and myocardial susceptibility toischemialreperfusion injury. The presence of hyperlipidemia, a characteristic feature ofthe uncontrolled diabetic state, can promote atherogenesis, especially in combinationwith stress.13’266°’61243 Highly resistant to the development of atherosclerosis,1therat will be used as an experimental animal, in order to eliminate atherogenesisassociated effects of stress on antioxidant changes in the myocardium and othertissues studied. Thus, the results should be interpretable with minimal interferencefrom coronary atherosclerotic complications.1.9. Specific Aims1. To investigate the effects of chronic alterations in endogenous catecholamine levelson the antioxidant status of various tissues.2. To develop and characterize a model of chronic intermittent stress.3. To examine the effects of chronic intermittent stress on endogenous enzymatic andnon-enzymatic antioxidant components in several representative tissues, withparallel studies assessing corresponding generalized changes •in tissuesusceptibility to in vitro oxidative challenge and in myocardial susceptibility toischemia/reperfusion injury using the isolated Langendorl heart preparation.4. To examine the effects of streptozotocin-induced diabetes of relatively short(4 weeks) or long (3 months) duration on 3) above.- 25 -1.9.1. ExperimentalApproachAs an initial approach to the study of stress-related factors in the alteration ofantioxidant status, the influence of chronically reduced levels of endogenouscatecholamines on antioxidant status will be examined in erythrocytes and variousorgans, with particular reference to the myocardium. Rats will be subjected to6-hydroxydopamine treatment (or reserpine, in separate experiments), adrenalectomyor a combination of both treatments to reduce plasma levels of adrenaline,noradrenaline or both catecholamhes, respectively. The resulting effects of thesealterations on blood and tissue antioxidant capacity will be examined in terms ofantioxidant enzyme activities and functional antioxidant capacity by peroxide-inducedin vitro oxidative challenge with subsequent measurement of decrease in free acidsoluble sulThydryl group content, an indirect measure of glutathione, and increase inthiobarbituric-acid reactive substances (TBARS), an indirect measure of lipidperoxidation.A model of chronic-intermittent variable restraint stress will then be used toinvestigate alterations in “stress” hormones in plasma of control and STZ- diabetic rats.The associated effects on tissue non-enzymatic antioxidant status will also beinvestigated. Beginning on the fifteenth day, following injection of STZ or vehicle, ratswill be subjected to 1, 7 or 14 days of a stress protocol which will involve exposure, fora one hour period, to one of seven different restraint stressors twice daily for 14 days.Blood samples will be collected from indwelling catheters on the morning prior to andthroughout the stress or control period. Plasma will be analysed for glucose levels andthe “stress” hormones corticosterone, adrenaline and noradrenaline. Tissue and- 26 -plasma tocopherol and ascorbate levels will be measured in samples collectedimmediately following the stress or control period corresponding to days 7 and 14 of thestress protocol.Following exposure of non-diabetic animals and animals with STZ-induceddiabetes of short (4 weeks) and long (12 weeks) duration (in separate experiments) tothe chronic-intermittent stress protocol, erythrocyte and tissue antioxidant capacity willbe examined. Beginning on the fifteenth (short-term diabetes) or seventieth (long-termdiabetes) day following injection of STZ or vehicle, rats will be either exposed to the 14day stress protocol or left undisturbed in their home cages. At 0700 hr on the dayfollowing the 4 or 12 week experimental period, animals will be euthanized and bloodand tissues collected for analysis. Blood chemical and biochemical indices will bedetermined, erythrocyte and tissue antioxidant enzyme activities will be measured andfunctional antioxidant capacity assessed as described above.Experimental results from the previous studies conducted in our laboratory haveshown that the elevation of plasma cholesterol and triglyceride levels in short-termdiabetic rats is attenuated when these animals are subjected to chronic intermittentstress, despite stress-induced increases in the extent of hyperglycemia. In addition,some diabetes-related antioxidant changes were reversed in these chronically stressedanimals.275 The influence of chronic intermittent stress on plasma lipid profiles andtissue antioxidant status in non-diabetic rats with diet-induced hyperlipidemia will beexplored. Two groups of rats will be given access ad libitum to water and food with orwithout added cholesterol (1 %). After 14 days, groups will be subdivided further andsubjected to the two week chronic-intermittent stress protocol or left undisturbed in- 27 -home cages. Animals will be euthanized by decapitation on day 29 at 0700 hr andblood and tissues collected for analysis. Plasma triglyceride and cholesterol levels willbe measured in order to confirm the presence of hyperlipidemia. The activity ofglutathione reductase, previously shown to be altered in diabetes, will be measured inheart, liver and kidney and, in addition, functional antioxidant capacity will be measuredin tissue homogenates and erythrocytes, as previously described.Finally, the interplay of chronic stress and diabetes on myocardial functionalrecovery and antioxidant status following a brief period of ischemia and reperfusion willbe examined. On the day following fourteen or seventy days induction of diabetes withSTZ (corresponding to short and long-term diabetes respectively), the groups will bedivided and either subjected to the chronic-intermittent stress protocol or leftundisturbed in their home cages. On the day following the last stress exposure,animals will be anesthetized and blood collected for measurement of glucose,glycosylated hemoglobin and triglycerides. Hearts will then be isolated, attached to aLangendorff retrograde perfusion apparatus and perfused with Krebs-Henseleit buffer.Heart function (developed pressure and ±dPIdtm) will be measured throughout aninitial stabilization (20 mi, normothermic, no flow global ischemia (2 mm) andreperfusion periods (30 mm) as described in detail in the Methods. At the terminationof reperfusion, myocardial antioxidant components will be assessed in terms ofglutathione reductase activity and levels of ascorbate and glutathione. Functionalantioxidant capacity will be measured in heart homogenates by in vitro oxidativechallenge as previously described.- 28 -2. MATERIALS AND METHODS2.1. General Animal Care2.1.1. Housing Conditions, Methods of EuthanasiaMale Wistar rats weighing 250-300 g were obtained from The University ofBritish Columbia Animal Care Unit and housed in single cages at 22°C, under aconstant light-dark cycle (lights on at 0600 hr and off at 1800 hr). Rats had access toPurina rat chow and water ad libitum. Following transfer from the breeding colony, ratswere stabilized for one week prior to experimental protocols. In experiments involvingchemical and/or surgical sympathectomy, adrenalectomized groups had access to0.9% saline solution ad libitum. One set of experiments required the use of altered ratchow. The method for preparation of this diet is described under thehypercholesterolemia procedure later in this section. All experimental protocols wereapproved by The University of British Columbia Animal Care Committee.2.2. Drug Protocols2.2.1. Chemical and Surgical Sympathectomy2.2.1 .1. 6-hydroxvdoramine Treatment and AdrenalectomyThe rats were randomized into two groups and under halothane anesthesia,adrenalectom?13 was performed on one group while the second received a shamoperation. On the seventh post-operative day, half the adrenalectomized and half thesham-operated animals were lightly anesthetized with halothane and treated with6-hydroxydopamine (20 mg/kg) dissolved under anoxic conditions in 0.5 ml 0.9%- 29 -NaCI-0.001 M HCI deoxygenated by bubbling with high-purity nitrogen (Linde UnionCarbide, Canada) for 10 mm. Trapped air in the 6-hydroxydopamine powder wasreplaced with nitrogen during dissolution and an aliquot was immediately removed fortail vein injection. The 6-hydroxydopamine solution was prepared freshly for eachinjection. A second dose of 40 mg/kg prepared under the same conditions wasadministered on the 14th post-operative day in sham-operated and adrenalectomizedgroups. A one-week separation between doses of 6-OH has been shown to cause amore complete sympathectomy.217 The remaining adrenalectomized and sham-operated groups received vehicle only, on the 7th and 14th post-operative days. Onpost-operative day 20, at 0700 hr, each animal was removed from the animal facilityindividually and killed within 30 sec by decapitation in a room remote from the housingfacility. All antioxidant measurements were performed on post-operative day 20 and, tothis end, a staggered experimental protocol was used in which only one animal waseuthanized each morning.2.2.1.2. Reserpine Treatment and AdrenalectomyRats were divided into two groups and under halothane anesthesia,adrenalectomy213 was performed on one group while the second received a shamoperation. Beginning on the 14th post-operative day, half the adrenalectomized andhalf the sham-operated animals were lightly anesthetized under halothane and treatedwith reserpine (Sigma Chemical Co.) (0.2 mg/kg) dissolved in olive oil (0.2 ml) viaintra-peritoneal injection daily for 6 days. The reserpmne solution was prepared freshlyfor each injection. The remaining adrenalectomized (ADR) and sham-operated (SHAMCON) groups received vehicle (olive oil) only, using the same conditions. Animals- 30 -were randomized with regard to both treatment and day of analysis. All antioxidantmeasurements were conducted on only one animal per day. On post-operative day 20,at 0700 hr, (one hour after lights were turned on) the animal was removed from its cageand, within 30 secs, killed by decapitation in a room remote from the housing facility.2.2.2. Streptozotocin TreatmentDiabetes was induced in halothane anesthetized rats by a single injection ofstreptozotocin (STZ) (60 mg/kg body wt), in 0.3 ml sterile saline, via the tail vein.Control animals received the same volume of saline. Only those STZ-injected animalswhich had blood glucose values above 16.65 mmol/l (300 mg/dl) at the end of thestudies were considered diabetic; results from STZ-injected animals with glucose levelsbelow 16.65 mmol/l were omitted.2.2.3. Hypercholesterolemic Diet ProcedureAnimals were randomly divided into two groups and given access adlibitum towater and Purina rat chow with or without added cholesterol (1 %, by weight). Thecholesterol diet was prepared by dissolving cholesterol in melted lard and thoroughlymixing the solution with the food pellets. The proportion of lard added to the pelletswas 0.5 kg to 10.0 kg commercial chow.2.3. Description of the Chronic Intermittent Stress ModelFor each experiment where stress was used, an equal number of animals waseither exposed to the chronic-intermittent stress protocol or left in their home cages for14 days. The stress protocol involved exposure, for a 1-hr period, to I of 7 differentrestraint stressors twice daily, the first exposure between 0900 hr and 1200 hr, and thesecond between 1300 hr and 1600 hr. In order to minimize habituation, the sequence- 31 -of the stressors was randomized for both the first seven morning and afternoonexposures. The series was repeated during the second week with the exception thatthe morning and afternoon sequences were reversed. The stressors used were:1) towel wrap secured with tape; 2) the latter with animals placed in a supine position;3) restraint in a plastic box with lid; 4) restraint in a polyvinylchloride tube closed ateither end; 5) immobilization on a board with velcro; 6) the latter with animals placedin a supine position; and 7) restraint in a metal bar cage. Each stressor exposure wasconducted in a room remote from the animal facility and animals exposed to stressorswere returned to the animal facility 15 mm following stress exposure to minimizedisturbance to control animals. Restraint stress was used rather than a stress involvingmotion (e.g., swimming) to eliminate a possible training effect due to exercise. Forexperiments involving the collection of blood samples via in-dwelling catheters, theorder of the sequence was adjusted slightly so that blood sampling would always occurduring supine immobilization with velcro during the morning on days 1, 7 and 14 of thestress protocol.2.4. Remote Blood Sampling Procedures2.4.1. Description of Venous Indwelling Catheter Construction andImplantation ProcedureExperiments in which remote blood sampling was conducted required the use ofindwelling catheters. Each catheter was made of PE5O tubing (12.5 cm) into whichthree right angles were formed at 3.5, 6.5, and 10 cm. The angles were formed so thatthe 2.5 cm end of the catheter was placed into the right external jugular vein, followedby a bend, exiting through the muscle, bending again, running along under the skin to- 32 -the skull and bending again to exit the skin. A small lip was formed at 3.0, 7.0 and8.5 cm for use as attachment sites to secure the catheter to the blood vessel and tissueabove the skull. Before placement, each catheter was tested and disinfected in 95%ethanol for several hours. A beveled and sealed needle was used to close the externalportion of the catheter.Catheters were surgically implanted approximately 12 hr before use. Thecatheter was fed through two incisions in the skin, the first above the skull and thesecond above the right external jugular vein on the ventral side of the neck. Followingblunt dissection, the right external jugular vein was located and isolated. The end ofthe vein distal to the tip of the catheter was sutured to the catheter. Following testing ofcatheter patency by injection of sterile isotonic saline, the incisions were sutured andthe animal allowed to recover.2.4.2. Blood Sampling ProcedureThe following morning at 0600 hr, the animal was removed from its cage and a60 cm PE5O tube, surrounded by a teflon sleeve, was connected to the end of thecatheter and flushed with 0.2 ml saline containing (50 U/mI heparin). The animal wasthen returned to its cage and the cage covered to prevent visual disturbance during thecollecting of blood. The syringe and catheter extension were carefully placed on theoutside of the cage to allow free movement of the rat in the cage. The animal was leftundisturbed for three hours. At 0900 hr, a basal sample was collected. The catheterwas first flushed with heparinized saline (0.05 ml) and blood was then withdrawn slowlyuntil it reached the needle of the syringe. The syringe was replaced with a clean I mlsyringe and a dulled 23 guage needle; blood (0.3 ml) was collected and transferred to- 33 -an Eppendorl tube on ice containing reduced glutathione (6 p1, 60 mM/L). The needleand syringe were replaced on the catheter and lactated Ringers solution (0.3 ml) wasflushed to maintain fluid volume. This needle and syringe were replaced with a syringecontaining heparinized saline and 0.1 ml was flushed into the catheter. For animals inthe control condition, this blood collection procedure was repeated at 5, 15, 30 and60 mm following collection of the basal sample. Animals in the stressor-exposedcondition were individually transported to an adjacent room following collection of thebasal sample and blood samples collected at 5, 15, 30 and 60 mm following thebeginning of the stress exposure. Blood samples were collected from animals in boththe stressor-exposed and control conditions on days 1, 7 and 14 of testing. Theexperiment was designed so that each animal was catheterized and subjected to serialblood sampling only once. Blood collected through the catheter was centrifuged at3,000 x g for 5 mm at 4°C. Plasma was removed and separate aliquots frozen at -70°Cfor later determination of corticosterone and catecholamine levels. Following sampling,animals were removed from the experimental room and euthanized by decapitation.2.5. Chemical Assays2.5.1. Hemoglobin AssayHemoglobin (Hb) contents of hemolysates and tissue cytosolic fractions weredetermined using the cyanomethemoglobin method of Drabkin and Austin. Todetermine erythrocyte hemoglobin, a hemolysate was prepared by the addition of aweighed aliquot (50 i.iL) of packed erythrocytes to 1.8 ml double distilled H20, to beused for the oxidative enzyme assays. A 0.2 ml aliquot of this mixture was combinedwith 1.8 ml H20. The resulting diluted hemolysate was centrifuged to remove- 34 -membrane debris. An aliquot of the supernatant (0.5 ml) was mixed with 0.5 ml of1.8 mMK3Fe(CN)6,0.5 ml of 2.5 mM KCN and sufficient H20 to make up a final volumeof 1.5 ml. Absorbance was measured at 540 nm at 30 mm and hemoglobinconcentration (mg/mi) estimated using a standard calibration curve. Hemoglobin wasexpressed as mg Hb/g RBC. Hemoglobin was determined from tissue (heart, liver,kidney, and lung) cytosolic fractions by the addition of an aliquot of 1.0 ml to 0.5 ml of1.8 mMK3Fe(CN)6and 0.5 ml of 2.5 ml of 2.5 mM KCN and the absorbance measuredas above. Cytosolic hemoglobin was expressed as mg Hb/mg wet tissue.2.5.2. Glycosylated Hemoglobin AssayGlycosylated hemoglobin, measured as %HbA1, was determined by aspectrophotometric technique using an adapation of the method developed byWinterhalter.297 Erythrocytes were washed twice with isotonic saline. Double distilledwater (1.2 ml) was added to erythrocytes (0.3 g), and an aliquot (25 jil) of the resultingmixture was clarified by adding sodium dodecyl sulfate (SDS) (0.9 ml, 30 mM).Hemoglobin was measured by adding 2.4mM KCN (1.0 ml) and 1.8mM K3Fe(CN)6(1.0 ml) to the mixture and the absorbance was read at 540 nm after 30 mm. A 1.0 ml50 mg/mI suspension of erythrocytes was made and refrigerated overnight. Thefollowing day, 0.5 M oxalic acid (0.5 ml) was prepared and the mixture boiled for 4.5 hr,followed by cooling to room temperature. 40% TCA (0.5 ml) was added, followed byvortexing and centrifugation for 10 mm using a clinical centrifuge. The resultingsupernatant was decanted into an Eppendorl tube, recentrifuged (10,000 g) and a1.0 ml aliquot of supernatant combined with saturated TBA solution (0.25 ml), followed- 35 -by incubation for 30 mm at 37°C. Following cooling to room temperature, theabsorbance at 443 nm was measured.2.5.3. Corticosterone AssayBlood was collected into a glass centrifuge tube rinsed with heparin solution andcentrifuged at 3,000 x g for 5 mm at 4°C. Plasma was removed and separate aliquotsfrozen at -70°C for later determination of corticosterone levels. Plasma corticosteronewas determined by radioimmunoassay using an adaptation2of the method developedby Kaneko et al.127 Plasma was extracted in absolute ethanol (1:10 vlv). Antiserumwas obtained from lmmunocorp, Montreal, Canada; tracer (1 ,2,6,7-3H+)-corticosteronewas obtained from New England Nuclear and unlabeled corticosterone for standardswas obtained from Sigma, St. Louis, Mo. Dextran-coated charcoal was used to absorb.and precipitate free steroid after incubation, and samples were counted using inAtomlight Formula 989 (New England Nuclear). Total corticosterone (bound plus free),expressed in pg/1 00 ml, is measured by this method. Corticosterone binding globulin(CBG) binding capacity was measured using an adaptation of the method of Martinet aL Plasma was stripped of endogenous corticosterone using dextran-coatedcharcoal. A premix containing plasma and3H-corticosterone (New England Nuclear)was then prepared and added to tubes containing either no unlabeled corticosterone(to estimate total binding) or with an approximately 100-fold excess of unlabeledcorticosterone (to estimate nonspecific binding). Dextran-coated charcoal was used toseparate free and bound steroid after incubation. Bound(3H)-corticosterone wasdetermined by liquid scintillation counting using Scmnti-Verse II (Fisher Scientific). The- 36 -binding capacity of CBG, expressed as jig of corticosterone bound/I 00 ml of plasma,was calculated as the difference between total binding and nonspecific binding.2.5.4. Catecholamine AssayThe following technique was used for blood collected in the studies involvingsympathectomy using 6-OH-dopamine or reserpine. Blood was collected in a glasscentrifuge tube rinsed with heparin solution and centrifuged at 3,000 x g for 5 mm at4°C. Plasma was removed and separate aliquots frozen at -70°C for laterdetermination of adrenaline, noradrenaline and dopamine levels. Catecholamines weremeasured by high performance liquid chromatography (HPLC). The extractionprocedure used was a modification of the method of Davis et aL” Polypropylenemicrocentrifuge tubes (Western Scientific Ltd.) were prepared with 20 mg alumina,250 jil 1.5 M Tris buffer (pH 8.7) and 25 p.1 10 % EDTA. To each tube, 0.5 ml plasmaand 100 p.1 of internal standard, 10 pg/p.l 3,4-dihydroxybenzylamine (DHBA) wereadded. For each assay, 2 spiked samples were also prepared with 0.5 ml 0.1 Msodium phosphate buffer and 50 p.1 each of 100 pg/p.l noradrenaline, adrenaline andDHBA solutions. The tubes were then shaken on a reciprocal shaker for 5 mm followedby 30 sec centrifugation in an Eppendorf centrifuge (Model 3200). The supernatantwas removed by aspiration and the alumina washed twice with double-distilled water.To extract the catecholamines from the alumina, 100 p.1 of 0.1 M HCIO4was added andthe tubes agitated for 5 mm. Following centrifugation, the supernatant was ejectedthrough a disposable filter (Millipore, 0.45 p.m pore size). The filtrate was immediatelyfrozen at -70°C and assayed for catecholamine content the same day. The sampleswere assayed for adrenaline and noradrenaline content by reverse-phase ion pair- 37 -HPLC with electrochemical detection. The HPLC system consisted of a liquidchromatograph (Walters Assoc., Model 590) and a 12.5 cm x 4.6 mm 5 pm columnpacked with ODS Hypersil. The mobile phase was composed of a 0.1 M KH2PO4buffer(pH 3.77) with 50 ml methanol, 100 mg sodium octyl sulphate and 60 mg EDTA addedto each liter of buffer. The flow rate was 1.2 mI/mm. The electrochemical detectionsystem consisted of a carbon paste detector electrode (Bioanalytical Systems Inc.,Model TL-3) packed with a graphite:Nujol paste (Bioanalytical Systems Inc., CP-O).The electrode potential was maintained at +0.60 V versus an Ag-AgCI referenceelectrode (Bioanalytical Systems Inc., Model RE-I). Peak areas were integrated usingan Apple llc computer.The catecholamine assay was modified slightly for the smaller aliquots of bloodcollected in the study involving remote blood sampling using catheters. In addition,dopamine was not assayed. Aliquots of plasma stored at -70°C in the presence ofreduced glutathione were thawed and centrifuged at 3,000 x g for 5 mm at 4°C.Adrenaline and noradrenaline levels were measured by high performance liquidchromatography (HPLC). The extraction procedure used was a modification of themethod of Davis et. aI. The standard in triplicate contained 20 mg alumina, 50 piadrenaline and noradrenaline (10 ng/mI in 0.06 M HCIO4) and 50 pi internal standard(10 ng/ml in 0.06 M HCIO4). To each sample was added 20mg alumina, 100 j.tl plasmaand 50 uI internal standard (10 ng/mI in 0.06 M HCIO4). To each standard and samplewas added 50 u1 10% Na2 EDTA (pH 8.6) and 500 uI 0.15 Tris-0.00I M NaHSO3(pH 8.6). The tubes were then shaken on a reciprocal shaker for 5 mm followed by a30 sec centrifugation in an Eppendorf centrifuge (Model 3200). The supernatant was- 38 -removed by aspiration and the alumina washed twice with 500j.tI 0.01 M Tris (pH 8.6).To extract the catecholamines from the alumina, 200 .tl of 0.06 M HCIO4 was addedand the tubes agitated for 5 mm. Following centrifugation, the supernatant was ejectedthrough a disposable filter (Millipore type HV, 0.45 tm pore size). The filtrate wasimmediately assayed for catecholamine content by reverse-phase ion pair HPLC withelectrochemical detection. The HPLC system consisted of a liquid chromatograph(Walters Assoc., Model 590) and a 12.5 cm x 4.6 mm 5 jim column packed with ODSHypersil. The mobile phase was composed of 0.1 M KH2PO4buffer (pH 3.8), 200 mg/Isodium octyl sulphate and 60 mg/I Na2 EDTA (pH 3.8) in 10% methanol. The flow ratewas 1.0 mI/mm. Preinjection of 75 j.iI of HCIO4 (0.06 M) was followed by injection with75 jil sample and standard. The electrochemical detection system consisted of acarbon paste detector electrode (Bioanalytical Systems Inc., Model TL-3) packed with agraphite:Nujol paste (Bioanalytical Systems Inc., CP-O). The electrode potential wasmaintained at +0.60 V versus an Ag-AgCl reference electrode (Bioanalytical SystemsInc., Model RE-I). The sensitivity was I rA full scale deflection. Peak areas wereintegrated using an Apple llc computer.2.5.5. Ascorbate AssayPlasma and tissue ascorbate levels were determined by a spectrophotometrictechnique using an adaptation of the method developed by Zannoni et aI.2 Freshtissue was weighed (ca. 100 mg) and homogenized in 5% TCA (2.0 ml)(homogenization was complete within 5 mm after tissue was removed from the animal)followed by centrifugation at 12,000 g (15 mm). Ascorbate was assayed as describedfor plasma below. An aliquot of fresh plasma (1.2 ml) was deproteinized with 40% TCA- 39 -(144 p.I)on ice for 10 mm, followed by centrifugation at 12,000 g (15 mm). To 600 il ofsupernatant was added 85% 0-phosphoric acid (40 j.tl), 1.0% 2,2’,dipyridyl (320 .tl,prepared in 95% ethanol) and 3.0% FeCI3•6H20(40 jil), and the absorbance was readafter30 mm at 525 nm.2.5.6. Tocopherol AssayPlasma and tissue cx-tocopherol levels were determined by a spectrophotometrictechnique using an adaptation of the method developed by Sloan et al.25° Previouslyfrozen tissue (0.5 g) was homogenized in KCI (2.5 ml 0.154 M). To the resultinghomogenate (1.8 ml) was added alcohol (1.2 ml 95% ethanol) and ascorbic acid (0.6 ml0.6 g16 ml double distilled water) and the mixture incubated (30 mm 70°C) then cooledin an ice water bath and heptane (1.0 ml) added. The mixture was then vortexed(30 sec) followed by centrifugation in a clinical centrifuge (5 mm). The heptane (0.5 ml)layer was assayed as described below. An aliquot of previously frozen plasma wasdiluted to a volume of 400 tl with double distilled water. Alcohol (0.4 ml 95% ethanol)and heptane (0.4 ml) were added and the mixture vortexed (30 sec), followed bycentrifugation at 12,000 g (5 mm). A 0.25 ml aliquot of the heptane layer was placedinto a glass test tube and combined with 0.25 ml 2,4,6-tripyridyl-s-triazine (1.2 g/l in npropanol) followed by vigorous vortexing. The reaction was started with 50 .tlFeCl3..6H20(1.2 gIl in ethanol) followed by vortexing. The mixture was transferred to a0.5 ml cuvette using a Pasteur pipette and absorbance was read after 2 mm at 593 nm.2.5.7. Glucose, Triglycerides and Cholesterol AssaysPlasma levels of glucose, triglycerides and cholesterol were determined usingstandard kits obtained from Sigma Chemical Co.- 40 -2.6. Biochemical Analyses2.6.1. Tissue Antioxidant Enzyme Measurements2.6.1 .1. Preparation of Tissue Homocjenates and CytosolicFractionsCatalase (CAT), copper, zinc-superoxide dismutase (Cu, Zn-SOD), glutathioneperoxidase (GSH-PX) and glutathione reductase (GSSG-RED) activities were assayedusing cytosolic fractions prepared from heart, liver, kidney and lung tissuehomogenates. To prepare tissue homogenates, tissues (heart, liver, kidney and lung)were removed, blotted dry and homogenized on ice in 50 mM Tris-0.1 mM EDTA,(pH 7.6) (10%, wlv), at 4°C for two 15 sec periods with a Polytron homogenizer(Brinkmann Instruments, Inc., Westbury, N.Y.) at 25% maximum speed. Tissuehomogenates were used in experiments involving tissue susceptibility to in vitroperoxide challenge and were used to make cytosolic fractions for estimation ofantioxidant enzyme activities. To prepare tissue cytosolic fractions, tissuehomogenates were diluted (1:3) with homogenizing buffer and centrifuged for 15 mm at105,000 x g using a Beckman L2-65 ultracentrifuge (lung, liver and kidney), or at16,000 x g in an Eppendorf microcentrifuge (heart). Spectrophotometricmeasurements were performed using a Perkin-Elmer model Lambda6 spectrophotometer at 25°C, which was maintained with a Haake FE2 recirculatingwater bath.2.6.1.2. CatalaseCatalase (CAT) activity was measured by the method described by Aebi.2 Analiquot of diluted ethanol (18 tL) (95% ethanol/H2O, 1:1 (vlv)) was mixed with 0.9 mL- 41 -of the cytosolic fraction and incubated for 30 mm at 4°C. Then 0.1 mL of cold Triton X100 solution (10% v/v) in (50 mM Tris-.01 mM EDTA pH 7.6) was added. From thismixture, 500 p.1 (heart), 100 p1 (liver), 200 p.1 (kidney), or 500 p1 (lung) was diluted to 10ml with 50 mM phosphate buffer, (pH 7.0), immediately prior to assay. In a 3-micuvette, 2.0 ml of this diluted solution was added and the reaction initiated by adding1.0 ml of freshly prepared 30 mM H20 in 50 mM phosphate buffer (pH 7.0) After rapidmixing, the rate of decomposition of H20was determined from the absorbance changeat 15 and 30 sec at 240 nm. Enzyme activity was expressed as K/mg wet tissue,1where K is the first order rate constant.2.6.1.3. Cu.Zn-SuDeroxide DismutaseCu, Zn-Superoxide Dismutase (Cu, Zn-SOD) activity was measured by themethod of Winterbourne et aL2 Cytosolic extracts were prepared by adding an aliquotof cytosolic fraction (heart (1.5 mL), liver (1.5 mL), kidney (1.0 mL), and lung (4.0 mL) tosufficient H20 to make up 4.0mL. Ethanol (0.5 mL for heart and 1.0 mL for liver, kidneyand lung) and chloroform (0.3 rnL for heart and 0.6 mL for liver, kidney and lung) wereadded to the mixture which was then vortexed throughly and centrifuged at 3000 x g for5 mm using a clinical centrifuge. The resulting supernatant was again centrifuged in anEppendorl centrifuge at maximum speed for 5 mm to obtain a clear extract. The assaymixture contained 1.0 mL of 75mM phosphate buffer (pH 7.8), 0.2 mL of 0.1 MNa2EDTA-1.5 mg% NaCN, 0.1 mL of 1.5mM nitro blue tetrazolium (NBT), 0-500 p.L ofthe clear supernatant and H20 to make a final volume of 2.95 mL. Riboflavin (50 p.L,0.12 mM) was added to initiate the reaction and the tubes illuminated twice for 2.5 mm(with vortexing at beginning, during and following each illumination) with fluorescent- 42 -light at a constant distance and protected from any other source of light. The rate ofinhibition of the reduction of NBT by superoxide generated via photoreduction ofriboflavin was determined by measuring the absorbance at 560 nm. Enzyme activitywas expressed in units of SOD per mg wet tissue, where 1.0 U is defined as thatamount of enzyme causing half-maximal inhibition of nitro blUe tetrazolium reduction.2.6.1.4. Glutathione ReductaseGlutathione reductase (GSSG-RD) activity was assayed by the method of Longand Carson.152 The assay system for measuring the enzyme activity contained 0.4 ml0.45 M Tris-90 mM EDTA (pH 7.6) buffer, 0.2 mL of 18 mM glutathione disulfide(GSSG) and aliquots of cytosolic fractions of heart, lung (160 rd), liver or kidney (40 p1)and H20 was added to bring the mixture to a final volume of I ml. The reaction wasstarted by the addition of 100 p1 of 3.0 mM NADPH solution and the absorbancechange at 340 nm recorded spectrophotometrically for 5 mm using an extinctioncoefficient of 6.22 x 106 M/cm. Enzyme activity was expressed as nmoles of NADPHoxidized to NADP/min/mg of wet tissue.2.6.1.5. Glutathione PeroxidaseGlutathione peroxidase (GSH-PX) activity was measured by the method ofPaglia and Valentine2°6as modified by Lawrence and Burke.1” To an aliquot ofcytosolic fraction (heart, kidney and lung (0.5 mL)) was added an equal volume ofdouble-strength Drabkin’s reagent (0.0016 M KCN-0.0012 M K3Fe(CN)6-0.0238 MNaHCO3) and the mixture kept on ice prior to assay. An aliquot of 0.25 mL livercytosolic fraction was diluted with an equal volume of 50 mM Tris-0.1 mM EDTA prior tothe addition of 0.5 mL double strength Drabkin’s reagent. Cytosolic GSH-PX was- 43 -assayed in a I mL cuvette containing a reaction mixture of 800 pL of 75 mM phosphatebuffer (pH 7.0), 20 pL of 60 mM GSH, 40 jiL of glutathione reductase solution(30 units/mI, Sigma Chemical Co.), 20 .tL of 0.12 M sodium azide, 40 iiL of 15 mMNa2EDTA, 40 jiL of 3 mM NADPH, various aliquots of sample (20-120 j.iL) of heart or(50-100 .tL) of liver, kidney or lung and sufficient H20 to make up a volume of 1.16 mL.The reaction was started by the addition of 100 jiL of 7.5 mM H20 solution and theconversion of NADPH to NADP was monitored spectrophotometrically by continuousrecording of the absorbance change at 340 nm for 5 mm. The enzyme activity wasexpressed as nmoles of NADPH oxidized to NADP per minute per milligram of wettissue using an extinction coefficient for NADPH at 340 nm of 6.2 x 106 M/cm.2.6.1.6. Hemoglobin and Correction of Tissue Enzyme Activity forthe Contribution of BloodErythrocytes contain the free radical scavenging systems examined in this study;therefore, cytosolic tissue extract antioxidant enzyme activities were corrected for theamount of erythrocyte contamination estimated from hemoglobin values obtained fromthe cytosolic fractions. Correction was based on the activity of erythrocyte antioxidantenzymes measured in blood samples from the same animal. Depending on the enzymeassayed, blood contamination comprised a major (CAT), intermediate (GSH-PX), or aminor (Cu, Zn-SOD) fraction of the total amount of a given cytosolic antioxidant. Wewere unable to obtain consistent results measuring GSSG-RD using rat blood due toan unknown factor involving the blood. Although it was not possible to correct tissueantioxidant activity levels of GSSG-RD for contamination of blood, from experimentsusing other strains of rats under different experimental conditions, we have- 44 -demonstrated blood GSSG-RD to be a minor contributor of the cytosolic fractionGSSG-RD activities.2.6.2. Erythrocyte Antioxidant Enzymes2.6.2.1. Preparation of HemolysatesCatalase (CAT), copper, zinc-superoxide dismutase (Cu, Zn-SOD), andglutathione peroxidase (GSH-PX) activities were assayed using hemolysates preparedfrom packed erythrocytes.2 Heparinized blood samples were centrifuged at 3,000 x gfor 5 mm at 4°C using a clinical centrifuge (mt. Equip. Co., Needham, Mass.). Thebuffy coat was removed and packed erythrocytes were washed twice with isotonicsaline. Erythrocyte hemolysates were prepared by the addition of weighed aliquots(50 uI) of packed erythrocytes in a 1:10 (v/v) dilution with double distilled water followedby three freeze/thaw cycles using a dry ice/acetone mixture.2.6.2.2. CatalaseAn aliquot (20 uttm) of hemolysate was added to 10 mL phosphate buffer (50 mM,pH 7.0) immediately prior to assay.. This diluted hemolysate was assayed as describedfor the tissue cytosolic fractions. Catalase activity was expressed as K/mg Hb.2.6.2.3. Cu, Zn-Superoxide DismutaseAn aliquot (0.5 ml) of hemolysate was extracted and assayed as described forthe tissue cytosolic fractions. Cu, Zn-SOD activity was expressed as units/mg Hb.2.6.2.4. Glutathione PeroxidaseAn aliquot (0.2 mlL) of freeze-thawed hemolysate was mixed with 1.8 ml H20,and 0.5 ml of this diluted hemolysate combined with an equal volume of double-- 45 -strength Drabkin’s solution and the mixture kept on ice until assayed. Aliquots (20 i.tl60 p1) of sample were assayed as described for the tissue cytosolic fractions.Glutathione peroxidase activity was expressed as nmoles NADPH/min/mg Hb.2.7. Functional Antioxidant Measurements2.7.1. Tissue Susceptibility to In Vitro Peroxide Challenge2.7.1.1. Preparation of Tissue HomopenatesTo prepare tissue homogenates, heart, liver, kidney and lung were removed,blotted dry and homogenized in “homogenizing buffer” (50 mM Tris/0.1 mM EDTA,pH 7.6) (10% w/v) on ice for two 15 sec bursts using a Polytron tissue homogenizer(Brinkmann, Westbury, New York) at 25% maximal speed.2.7.1.2. Susceptibility of Tissues to TBHP-Induced SulthvdrvlGroup DeIetionTo assess tissue susceptibility to peroxide-induced depletion of GSH, 0.2 mLaliquots of tissue homogenate were combined with an equal amount of satine/azide andincreasing amounts of t-butylhydroperoxide (TBHP), resulting in final TBHPconcentrations of between 0 and 0.5 mM for heart and kidney and between 0 and1.0 mM for liver tissues. We have demonstrated that rat lung tissue homogenates have.no detectable GSH (unpubi. data); therefore, lung GSH was not assayed in this study.The mixtures were incubated for 30 mm at 37°C and the reaction terminated with theaddition of 0.15 mM cold 25% (wlv) trichloroacetic acid (TCA) solution. Followingcentrifugation using an Eppendorl centrifuge at maximal speed (4°C), the supernatantswere analysed for free acid- soluble sulfhydryl group content (an indirect measure of- 46 -glutathione) using 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB).2 The assay mixturecontained 0.96 ml of 0.1 M phosphate buffer (pH 8.0) and a 0.2 ml aliquot ofsupernatant. The reaction was initiated by adding 0.40 ml DTNB solution (3 mM)freshly prepared in phosphate buffer. Absorbance of the reaction mixture wasmeasured spectrophotometrically after 10 mm at 412 nm. The GSH content wasdetermined using a standard calibration curve. Tissue GSH content (basal GSH level),expressed as nmoles/mg tissue, was measured in the absence of TBHP. TBHPinduced GSH depletion was expressed as % decrease in GSH content when comparedwith the basal level.2.7.1.3. Susceptibility of Tissues to H2-lnduced SulfhydrylGroup DepletionIn all experiments involving diabetic animals, in vitro functional antioxidant statuswas measured using H20 as the peroxidizing agent rather than TBHP because tissuesfrom diabetic animals were found to be particularly insensitive to TBHP-inducedsulthydryl group depletion. Susceptibility of tissues toH20-induced depletion of GSHwas determined using the same method as for measurement of TBHP-inducedsulfhydryl group depletion described above.2.7.1.4. Susceptibility of Tissues to Time-Dependent H20-Induced SulThydryl Group DepletionIn experiments involving chronic stress and diabetes followed by myocardialischemia/reperfusion injury, time-dependent changes in sulfhydryl group content weremeasured in heart homogenates challenged with a single concentration of hydrogenperoxide. Susceptibility of tissues to time-dependentH20-induced depletion of GSH- 47 -was determined using the same method as that for measurement of TBHP-inducedsulfhydryl group depletion described above. Measurement of sulfhydryl groupdepletion was determined following 0, 1, 3, 5, 7, 10, 20 and 30 mm incubation withH20 at a final concentration of 0.015 mM.2.7.1.5. Susceptibility of Tissues to TBHP-lnduced LipidPeroxidationSusceptibility of heart, liver, kidney and lung tissues to lipid peroxidation wasassessed by incubating 0.4 ml tissue homogenate with an equal volume of TBHP insaline/azide (final peroxide concentrations ranged from 0 to 20 mM) at 37°C for 30 mm.The reaction was terminated by adding 0.4 ml cold 28% (wlv) TCA containing 0.1 Msodium arsenite.. Following centrifugation in an Eppendorf centrifuge at maximalspeed, 0.8 ml of the supernatant was combined with 0.2 ml of thiobarbituric acidsolution (TBA) (0.5% in 0.025 M sodium hydroxide) and boiled for 15 mm. Theformation of thiobarbituric acid-reactive substances (TBARS), an indirect measure oflipid peroxidation, was determined spectrophotometrically at 532 nm, as described byTappel and Zalkin.22.7.1.6. Susceptibility of Tissues to H20-lnduced LipidPeroxidationSusceptibility of tissues toH20-induced lipid peroxidation was determined usingthe same method as for measurement of TBHP-induced lipid peroxidation describedabove.- 48 -2.7.2. Erythrocyte Susceptibility to In Vitro Peroxide Challenge2.7.2.1. Preparation of ErythrocytesThe sensitivity of erythrocytes to GSH depletion and lipid peroxidation followingin vitro incubation with increasing concentrations of TBHP or H20 was used as afunctional measure of antioxidant capacity. To prepare packed erythrocytes,heparinized blood was centrifuged at 3,000 x g for 5 mm at 4°C using a clinicalcentrifuge (International Equipment Company, Needham, Mass.). Plasma and the buffycoat were removed and erythrocytes were washed twice by centrifugation with isotonicsaline.2.7.2.2. Susceptibility of Erythrocvtes to TBHP-lnducedSulfhydryl Group DepletionErythrocyte susceptibility to TBHP-induced depletion of GSH was determined asfollows. Aliquots (50 p1) of erythrocytes were weighed, combined with 0.45 ml ofsaline/azide solution, preincubated for 5 mm at 37°C, and treated as described fortissue homogenates (final TBHP concentrations ranged from 0 to 0.5 mM). The reactionwas terminated by centrifugation for 5 mm using an Eppendorf centrifuge at 4°C. Theresulting pellet was rinsed with saline/azide, then re-centrifuged. To the pellet wasadded 50 p1 water and 325 p.1 5% TCA-1 mM Na2EDTA. Following centrifugation, thesupernatant was assayed for GSH content using DTNB. The reaction mixturecontained 120 p.1 of the supernatant and 1.04 ml of 0.1 M phosphate buffer (pH 8.0).To start the reaction, 40 p.1 DTNB (freshly prepared in phosphate buffer) was addedand absorbance at 412 p.m was measured spectrophotometrically after 5 mm. TheGSH content was determined using a standard calibration curve. Erythrocyte GSH- 49 -content (basal GSH content) was expressed as nmoles/mg packed red cells measuredin the absence of TBHP, and TBHP-induced GSH depletion was expressed as a %decrease in GSH content relative to the basal level.2.7.2.3. Susceptibility of Ervthrocytes toH20-lnduced SuithydrylGroun DepletionSusceptibility of erythrocytes toH20-induced depletion of GSH was determinedusing the same method as for measurement of TBHP-induced sulfhydryl groupdepletion described above.2.7.2.4. Susceptibility of Ervthrocytes to TBHP-Induced LipidPeroxidationTo determine the susceptibility of erythrocytes to TBHP-induced lipidperoxidation, aliquots (50 I) of erythrocytes were weighed, combined with 0.45 ml ofsaline/azide solution (to make a 10% suspension), preincubated for 5 mm at 37°C, andtreated as described above for tissue homogenates (final TBHP concentrations rangedfrom 0.5 to 5.0 mM.) The reaction was terminated by the addition of 0.5 ml cold 28%(wlv) TCA containing 0.1 M sodium arsenite. Following centrifugation, a 1.0 ml aliquotof the supernatant was combined with 0.5 ml TBA solution and boiled for 15 mm. Thedegree of malondialdehyde (MDA) production, expressed as nmoles MDNmg Hb, wasdetermined from absorbances at 532 nm and 453 nm of the reaction mixture using acidhydrolysed malondialdehyde bis(diethylacetyl) (Aldrich) as a standard, according to themethod of Stocks and Dormandy257 and Gilbert et al.78- 50 -2.7.2.5. Suscertibilitv of Ervthrocytes to H,Q2-lnduced LipidPeroxidationSusceptibility of erythrocytes toH20-induced lipid peroxidation was determinedusing the same method as for measurement of TBHPinduced lipid peroxidationdescribed above.2.8. Functional Physiological Measurements: Myocardial Susceptibility tolschemialReperfusion Injury2.8.1. Preparation of Buffer SolutionsThe perfusion solution used was Krebs-Henseleit buffer (NaCI, 118 mM; KCI,4.74 mM; KH2PO4, 0.93 mM; MgSO4.7H20, 1.2 mM; NaHCO3, 25.0 mM; Glucose,10.0 mM; CaCl2, 2.5 mM). The solution was prepared in double distilled water. CaCl2was added after bubbling with 95% 02, 5% CO2 for 10 mm.2.8.2. Apparatus for Langendorif PerfusionThe perfusion method involved the use of a modified Langendorif retrogradepressure-control led perfusion apparatus.2.8.3. Anesthesia, and Surgical Preparation of Heart for IsolationOn the day following the last stress period, hearts were quickly excised underhalothane anesthesia, placed in cold (4°C) Krebs-Henseleit buffer and trimmed,attached to a Langendorff retrograde perfusion apparatus, using an aortic cannula andperfused with Krebs-Henseleit buffer. Temperature (37°C), perfusion pressure(100 mm Hg) and heart rate (375 b/mm) were kept constant.- 51 -2.8.4. Methodology of lschemia/Reperfusion TimingHeart function was measured using a balloon catheter placed in the left ventricleand connected to a pressure transducer and polygraph. The balloon was inflated tomaintain an end-diastolic pressure of 5 mm Hg. Ventricular pressure was measuredand ±dPldtm computed throughout the stabilization (20 mm), normothermic, no flowglobal ischemia (2 mm) (during which pacing continued) and reperfusion (30 mm)periods. Hearts in which ventricular fibrillation occurred were defibrillated manually, ifspontaneous sinus rhythm did not return within 30 sec. Hearts which did not undergoirreversible fibrillation by 25 mm reperfusion were included for functional andbiochemical study.2.8.5. Description of Myocardial Functional MeasurementsThe following functional measurements were recorded: heart rate, perfusionrate, end-diastolic pressure (mm Hg), ventricular pressure (mm Hg) and ±dPldt.2.8.6. Biochemical MeasurementsAt the termination of reperfusion, myocardial antioxidant status was assessed interms of glutathione reductase activity and levels of ascorbate and glutathione.Functional antioxidant capacity was measured in heart homogenates by in vitrooxidative challenge using increasing concentrations of hydrogen peroxide withsubsequent measurement of decreases in free acid soluble sulfhydryl group content(an indirect measure of glutathione), and thiobarbituric-acid reactive substances(TBARS), an indirect measure of lipid peroxidation. In addition, time-dependentchanges in sulfhydryl group content was measured in homogenates challenged with asingle concentration of hydrogen peroxide (0.015 mM).-52-2.9.. Statistical AnalysisResults are presented as the mean ± standard error of the mean. Groups werecompared using one-way analysis of variance followed by application of Duncan’smultiple range test. For the study involving collection of serial blood samples viaremote sampling, data for plasma catecholamines and corticosterone, in addition toanalysis of total levels at each collection period, were also analysed by computing anarea under the plasma catecholamine and corticosterone concentration vs. time curve(AUC, nmol/Llmin) for each animal. These were computed as areas above baselineminus the area below baseline during the 60 mm control or stressor period. The AUCprovides a measure of overall corticosterone and catecholamine response profilesduring stress or control conditions. The analysis of total levels at each of the 5sampling points on days 1, 7 and 14 provides a more detailed description of the effectsof stress throughout the 60-mm-periods. Since the two analyses provide somewhatdifferent, albeit complementary, measures of the stress response, some differences insignificant values were anticipated.3. RESULTS3.1. Effects of Sympathectomy on Antioxidant Status3.1.1. Adrenalectomy and/or 6-Hydroxydopamine TreatmentThe possible influence of altered levels of endogenous catecholamines onantioxidant status in erythrocytes and various organs with particular reference to the- 53 -myocardium has been examined. Rats were subjected to 6-hydroxydopaminetreatment and/or adrenalectomy resulting in chronically altered plasma catecholamineand corticosterone levels. The effects of these treatments were examined onantioxidant status measured by in vitro susceptibility to peroxide-induced glutathionedepletion and lipid peroxidation as well as activities of antioxidant enzymes andglutathione levels. Catecholamines (adrenaline, noradrenaline and dopamine) weremeasured by high performance liquid chromotography (HPLC),42 while plasmacorticosterone was not measured, since non-adrenalectomized animals would beexpected to show basal levels in the morning when the animals were euthanized andsince adrenalectomized animals would be expected to have non-detectable levels.Treatment groups (ADR, 6-OH, 6-OH-ADR) were compared with a sham-operated(sham) control group.3.1.1.1. Catecholamine LevelsAs an index of the effectiveness of sympathectomy, plasma catecholamines(noradrenaline, adrenaline, and dopamine) were analysed in blood specimenscollected following decapitation (Table 1). The levels of catecholamines measuredreflect a stress-induced release stimulated by decapitation. Adrenalectomy wasassociated with significantly reduced plasma adrenaline levels; however, nodifferences were found in plasma noradrenaline when compared to the sham operatedgroup. 6-OH-treatment alone significantly reduced plasma noradrenaline; however,plasma adrenaline was not significantly altered. The combination of adrenalectomyand 6-OH-treatment significantly reduced both plasma adrenaline and noradrenaline.The levels of dopamine were not affected by chemical or surgical sympathectomy.Table1.PlasmacatecholaminelevelsincontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.ControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopamineAdrenaline93.64±16.517.73±3.9662.91±12.131.91±0.34(n=9)(n=7)(n=7)(n=6)Noradrenaline3.38±0.472.24±0.442.03±0.54*0.92±0.16*(n=9)(n=7)(n=7)(n=6)Dopamine1.70±0.500.78±0.251.72±0.571.22±0.39(n=6)(n=6)(n=7)(n=6)Valuesaremean±S.E.M.Adrenalineanddopaminemeasuredinpmol/LandnoradrenalinemeasuredinI0pmol/Lplasmafrombloodcollectedfollowingdecapitation.*p<005p<O.0O1,significantlydifferentfromcontrol.- 55 -3.1.1.2. Antioxidant Enzyme ActivitiesSympathectomy was associated with changes in several antioxidantcomponents. Erythrocyte basal glutathione levels were significantly elevated in theadrenalectomized, 6-OH-treated group relative to the sham operated group (Table 2).The activities of erythrocyte catalase, Cu, Zn-superoxide dismutase, and glutathioneperoxidase were not detectably altered by sympathectomy. Tissue antioxidantcomponents are shown in Tables 3-6. In the heart, glutathione reductase activity waselevated in all treatment groups relative to the sham-operated (control group) (Table 3).In liver, glutathione reductase activity was significantly increased by adrenalectomy andthe combination of 6-OH and adrenalectomy, but not 6-OH-treatment alone (Table 4).No other alterations in antioxidant components were found in liver or heart tissueamong treatment groups and, in addition, no antioxidant components were altered inthe kidney following sympathectomy (Table 5). In the lung, glutathione reductaseactivity was significantly decreased by adrenalectomy and the activities of bothglutathione reductase and glutathione peroxidase were significantly increased with thecombination of adrenalectomy and 6-OH-treatment, while no changes were observedwith 6-OH-treatment alone (Table 6).3.1.1.3. TBHP-lnduced Sulfhydryl Group DeIetion.Functional antioxidant capacity measured in terms of TBHP-induced suithydrylgroup depletion was unaltered by sympathectomy in erythrocytes, liver, kidney andlung (data not shown); however, heart peroxide-induced sulthydryl group depletionwas significantly increased following adrenalectomy alone and in combination with6-OH treatment (Fig. 5).- 56 -3.1.1.4. TBHP-lnduced Lipid PeroxidationSympathectomy was associated with alterations in susceptibility of liver, kidneyand lung to in vitro peroxide-induóed TBARS formation. In the liver and lung, peroxideinduced TBARS formation was significantly decreased by adrenalectomy alone andwith the combination of adrenalectomy and 6-OH-treatment, although no changes wereobserved with 6-OH-treatment alone (Figs. I and 2). Significant reduction in TBARSformation also occurred in kidney tissue homogenates in all sympathectomized groups(Fig. 3). On the other hand, adrenalectomy and/or 6-OH-treatment did not alter thesusceptibility of myocardial tissue to peroxide-induced TBARS formation (Fig. 4).Table2.AntioxidantstatusinerythrocytesfromcontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;(7’ -.4ControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopamine(n10)(n=7)(n=7)(n=7)Catalase0.0431±0.00090.0455±0.00150.0476±0.00150.0472±0.0013(K/mg Hb)Cu,Zn-SOD3.85±0.103.70±0.164.01±0.323.92±0.15(units/mgHb)GSH-PX48.52±1.3446.78±2.0453.37±4.1054.16±5.06(nmolNADPHmin1mg1Hb)GSH1,468.15±90.571,579.30±65.471,582.62±86.801,877.60±72.68*(nmol/mgRBC)GSH=glutathione.*p<0.05significantlydifferentfromcontrol.Table3.AntioxidantstatusinheartsfromcontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione;N/D=notdetectable.*p.<001significantlydifferentfromcontrol.(7’ControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopamine(n=1O)(n=7)(n=7)(n=7)CatalaseNIDNIDNIDNID(K/mgwetwt)Cu,Zn-SOD0.879±0.0360.906±0.0480.992±0.0510.906±0.048(units/mgwetwt)GSH-PX6.17±0.155.82±0.335.97±0.205.70±0.13(nmolNADPHmin1mg1wetwt)GSSG-RD0.437±0.0130.543±0.040*0.521±0.022*0.609±0.01*(nmolNADPHmin1mg1wetwt)GSH1.79±0.041.81±0.041.87±0.071.73±0.02(nmol!mgwetwt)Table4.AntioxidantstatusinliverfromcontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione.*p<OO1p<O.OO1,significantlydifferentfromcontrol.01 CDControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopami ne(n=1O)(n=7)(n=7)(n=7)Catalase0.039±0.0020.036±0.0030.036±0.0020.031±0.002(K/mgwetwt)Cu,Zn-SOD4.27±0.154.45±0.154.65±0.134.65±0.18(units!mgwetwt)GSH-PX15.72±0.7517.39±0.8216.89±0.9118.05±1.07(nmolNADPHmin1mg1wetwt)GSSG-RD3.89±0.104.71±0.15*4.12±0.054.86±0.08(nmolNADPHm1n1mg1wetwt)GSH5.08±0.145.73±0.11*4.86±0.145.63±0.23*(nmol/mgwetwt)Table5.AntioxidantstatusinkidneyfromcontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;0) 0ControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopamine(n=1O)(n=7)(n=7)(n=7)Catalase0.0094±0.0040.0112±0.00050.0109±0.00050.0106±0.0006(K/mgwetwt)Cu,Zn-SOD2.80±0.082.49±0.112.70±0.062.52±0.12(units/mgwetwt)GSH-PX6.42±0.195.89±0.445.56±0.096.41±0.46(nmolNADPHmin1mg1wetwt)GSSG-RD7.49±0.177.08±0.247.90±0.327.37±0.13(nmolNADPHmin1mg1wetwt)GSH2.43±0.042.50±0.132.57±0.082.37±0.08(nmol/gwetwt)GSSG-RD=glutathionereductase;GSH=glutathione.Table6.AntioxidantstatusinlungfromcontrolandadrenalectomizedmaleWistarratstreatedwith6-hydroxydopamineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;0)ControlAdrenalectomy6-HydroxydopamineAdrenalectomyand6-hydroxydopamine(n=1O)(n=7)(n=7)(n=7)CatalaseNIDNIDNIDNID(KImg wetwt)Cu,Zn-SOD0.727±0.0360.755±0.0630.780±0.0760.760±0.043(units/mg wetwt)GSH-PX3.63±0.143.32±0.163.72±0.214.23±0.15*(nmolNADPHmin1mg1wetwt)GSSG-RD1.42±0.041.32±0.06*1.53±0.091.53±0.06*(nmolNADPHmin1mg’1wetwt)GSSG-RD=glutathionereductase;NID=notdetectable.*p<0•01significantlydifferentfromcontrol.- 62 -Fig. 1. TBARS formation following incubation with 0.75 mM t-butylhydroperoxide(TBHP) of liver homogenates from control and adrenalectomized male Wistar ratstreated with 6-hydroxydopamine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 10), adrenalectomized(Adr.) (n = 7), 6-hydroxydopamine treated (6-OH) (n = 7), adrenalectomized and treatedwith 6-hydroxydopamine (6-OH, Adr.) (n = 7). Significant difference relative to shamoperated, *p <0.01.Absorbanceat532nmooopoo0--F’3F)o01001001D) >C)-I 0) 0 I 0)H*II- 64 -Fig. 2. TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of kidney homogenates from control and adrenalectomized male Wistarrats treated with 6-hydroxydopamine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = I 0), adrenalectomized(Adr.) (n = 7), 6-hydroxydopamine treated (6-OH) (n = 7), adrenalectomized and treatedwith 6-hydroxydopamine (6-OH, Adr.) (n = 7). Significant difference relative to shamoperated, *p < 0.05.Absorbanceat532nma) 0 2: a) 0 I 0. 1IIIIpopo00000-0-[‘30)0Cl) D) 3 > 0. -‘Ha) 01jH*_____________________________________*- 66 -Fig. 3. TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of lung homogenates from control and adrenalectomized male Wistar ratstreated with 6-hydroxydopamine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 10), adrenalectomized(Adr.) (n = 7), 6-hydroxydopamine treated (6-OH) (n = 7), adrenalectomized and treatedwith 6-hydroxydopamine (6-OH, Adr.) (n = 7). Significant difference relative to shamoperated, *p <0.01.0 D) 3 -‘p 0 0p 0 010 F’)0Absorbancepnmat532p -op 010 I Gb, 0 I > 0 -I0)- 68 -Fig. 4 TBARS formation following incubation with 1.0 mM t-butylhydroperoxide(TBHP) of heart homogenates from control and adrenalectomized male Wistarrats treated with 6-hydroxydopamine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 10), adrenalectomized(Adr.) (n = 7), 6-hydroxydopamine treated (6-OH) (n = 7), adrenalectomized and treatedwith 6-hydroxydopamine (6-OH, Adr.) (n = 7).0 :i: 0 :i: > -IAbsorbanceat532nmpppppp0000-&0C)0)(0Co0) CD- 70 -Fig. 5. SuiThydryl group depletion following incubation with 0.025 mMt-butylhydroperoxide (TBHP) of heart homogenates from control andadrenalectomized male Wistar rats treated with 6-hydroxydopamine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 10), adrenalectomized(Adr.) (n = 7), 6-hydroxydopamine treated (6-OH) (n = 7), adrenalectomized and treatedwith 6-hydroxyclopamine (6-OH, Adr.) (n = 7). Significant difference relative to shamoperated, *p <0.01.PercentDepletion-‘r\)C)C)0Cli0Cli0(.11.0IIII*-‘ a) 0 IH LU*- 72 -3.1.2. Adrenalectomy and/or Reserpine TreatmentIn order to determine whether the antioxidant changes associated with depletionof endogenous catecholamines are influenced by the nature of the agent used toproduce chemical sympathectomy, a comparison between 6-OH and reserpine wasundertaken. Rats were subjected to chemical and/or surgical sympathectomy usingadrenalectomy and/or reserpine treatment resulting in chronically altered plasmacatecholamine and corticosterone levels.3.1.2.1. Catecholamine LevelsPlasma catecholamines (noradrenaline, adrenaline, and dopamine) are shown inTable 7. Adrenalectomy was associated with significantly reduced plasma adrenalinelevels; however, no differences were found in plasma noradrenaline when compared tothe sham operated group. Following reserpine treatment alone, the associatedreduction in plasma noradrenaline was not significant relative to vehicle-treatedanimals. In addition, reserpine treatment did not significantly alter plasma adrenalinelevels. The combination of adrenalectomy and treatment with reserpine resulted insignificantly reduced plasma adrenaline and noradrenaline levels. The levels ofdopamine were not affected by chemical or surgical sympathectomy.3.1.2.2. Antioxidant Enzyme ActivitiesSympathectomy was associated with changes in several antioxidantcomponents. Heart basal glutathione level was decreased while glutathione reductaseactivity was significantly elevated in the adrenalectomized reserpine treated grouprelative to the sham operated group (Table 8). The activities of Cu, Zn-superoxidedismutase and glutathione peroxidase were not altered by sympathectomy. In liver, Cu,- 73 -Zn-superoxide dismutase activity was increased and in the kidney, glutathionereductase activity was significantly decreased following adrenalectomy and reserpinetreatment; however, no other alterations in antioxidant components were found(Tables 9 and 10). Erythrocytes and lung tissue did not show changes in antioxidantcomponents following sympathectomy (Tables 11 and 12). Sympathectomy wasassociated with alterations in susceptibility of heart liver, kidney and lung, to in vitroperoxide challenge.3.1.2.3. TBHP-lnduced Sulfhydrvl Group DepletionSusceptibility of heart to peroxide-induced sulfhydryl group depletion increasedfollowing adrenalectomy and the combination of adrenalectomy and reserpinetreatment; however, no changes were found following reserpine treatment alone(Fig. 6). Susceptibility to TBHP-induced sulfhydryl group depletion was unaltered inliver, kidney and erythrocytes (data not shown).3.1.2.4. TBHP-lnduced Lipid PeroxidationLiver, kidney and lung, TBHP-induced TBARS formation was significantlydecreased with adrenalectomy and with the combination of adrenalectomy andreserpine treatment, while no changes were observed with reserpine treatment alone(Figs 7, 8 and 9). On the other hand, adrenalectomy and/or reserpine treatment didnot alter the susceptibility of the heart to peroxide-induced TBARS formation (Fig. 10).Table7.PlasmacatecholaminelevelsincontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.ControlAdrenalectomyReserpineAdrenalectomyandreserpine(n=3)(n=4)(n=6)(n=5)Adrenaline81.95±2.821.95±1.15*96.28±16.614.27±1.25*Noradrenaline2.02±0.251.64±0.471.22±0.260.16±0.10*Dopamine1.23±0.521.15±0.600.63±0.210.44±0.34Valuesaremean±S.E.M.Adrenalineanddopaminemeasuredini03pmol/LandnoradrenalinemeasuredinIOpmol/Lplasmafrombloodcollectedfollowingdecapitation.*p<001significantlydifferentfromcontrol.—ITable8.AntioxidantstatusinheartsfromcontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione;N/D=notdetectable.*p.<OOIsignificantlydifferentfromcontrol.0iControlAdrenalectomyReserpineAdrenalectomyandreserpine(n=6)(n=6)(n=6)(n=6)CatalaseNIDNIDNIDNID(KImgwetwt)Cu,Zn-SOD0.901±0.0251.022±0.0500.942±0.0200.977±0.059(U/mgwetwt)GSH-PX7.50±0.396.93±0.277.57±0.697.23±0.44(nmolNADPHmin1mg1wetwt)GSSG-RD0.518±0.0260.568±0.0140.521±0.0210.614±0.019*(nmolNADPHmin1mg1wetwt)GSH1.90±0.041.76±0.0841.74±0.0531.60±0.049*(nmollmgwetwt)Table9.AntioxidantstatusinliverfromcontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione.*p.<001significantlydifferentfromcontrol.-.4 0)ControlAdrenalectomyReserpineAdrenalectomyandreserpine(n=6)(n=6)(n=6)(n=6)Catalase0.028±0.0010.029±0.0030.032±0.0020.032±0.002(K/mgwetwt)Cu,Zn-SOD4.05±0.174.61±0.234.56±0.415.18±0.48*(U/mgwetwt)GSH-PX19.91±1.3417.97±0.962180±1.7022.11±2.14(nmolNADPHmin1mg1wetwt)GSSG-RD3.87±0.154.28±0.244.20±0.234.39±0.25(nmolNADPHmin1mgtwetwt)GSH4.90±0.055.22±0.355.16±0.374.84±0.37(nmol/gwetwt)Table10.AntioxidantstatusinkidneyfromcontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione.*p<001significantlydifferentfromcontrol.—3—IControlAdrenalectomyReserpineAdrenalectomyandreserpineCatalase0.0097±0.00010.0107±0.00060.0097±0.00060.0108±0.0004(K/mgwetwt)(n=6)(n=6)(n=6)(n=6)Cu,Zn-SOD2.88±0.132.54±0.092.52±0.182.44±0.07(U/mgwetwt)(n=5)(n=6)(n=6)(n=6)GSH-PX7.57±0.676.85±0.297.06±0.426.80±0.55(nmolNADPH(n=6)(n=6)(n=6)(n=6)min1mg1wetwt)GSSG-RD7.42±0.206.81±0.237.41±0.356.32±0.25*(nmolNADPH(n=6)(n=6)(n=6)(n=6)min1mg1wetwt)GSH2.43±0.092.42±0.062.56±0.072.44±0.08(nmol/gwetwt)(n=6)(n=6)(n=6)(n=6)Table11.AntioxidantstatusinerythrocytesfromcontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.ControlAdrenalectomyReserpineAdrenalectomyandreserpine(n=6)(n=6)(n=6)(n=6)Catalase0.0438±0.00150.0452±0.00170.0425±0.00110.0426±0.0012(K/mg Hb)Cu,Zn-SOD4.15±0.233.87±0.173.77±0.143.94±0.17(units/mgHb)GSH-PX72.81±5.6967.68*3.4272.55±5.2566.40±2.52(nmolNADPHm1n1mg1Hb)GSH1,599.60±53.811,566.53±89.281,611.07±64.791,466.79±49.77(nmol/mgRBC)o:Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSH=glutathione.Table12.AntioxidantstatusinlungfromcontrolandadrenalectomizedmaleWistarratstreatedwithreserpineorvehicle.Valuesaremean±S.E.M.Cu, Zn-SOD=Cu, Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;NID=notdetectable.CDControlAdrenalectomyReserpineAdrenalectomyandreserpine(n=6)(n=6)(n=6)(n=6)CatalaseNIDNIDN/DNID(K/mg wetwt)Cu, Zn-SOD0.755±0.0310.796±0.150.802±0.0680.933±0.03(U/gwetwt)GSH-PX3.81±0.223.29±0.223.13±0.324.27±0.17(nmolNADPHmin1mg1wetwt)GSSG-RD1.38±0.071.37±0.101.38±0.041.43±0.06(nm0INADPHmin1mg1wetwt)- 80 -Fig. 6. Percent sulfhydryl group depletion following incubation with 0.75 mMt-butylhydroperoxide (TBHP) of heart homogenates from control andadrenalectomized male Wistar rats treated with reserpine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n 6), adrenalectomized(Adr.) (n = 6), reserpine treated (Res.) (n = 6), adrenalectomized and treated withreserpine (Res., Adr.) (n = 6). Significant difference relative to sham operated,*p <0.01.PercentDepletionIIIIHH0L-1’)C)C)010010010011HC,) 3-I CD ci, -I CD Ct)- 82 -Fig. 7. TBARS formation following incubation with 1.50 mM t-butylhydroperoxide(TBHP) of liver homogenates from control and adrenalectomized male Wistar ratstreated with reserpine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 6), adrenalectomized(Adr.) (n = 6), reserpine treated (Res.) (n = 6), adrenalectomized and treated withreserpine (Res, Adr.) (n = 6). Significant difference relative to sham operated,*p <0.01.Absorbanceat532nmopo00-o0000B()-‘ CD Cl) 0. -I CD 0)II- 84 -Fig. 8. TBARS formation following incubation with 1.5 mM t-butylhydroperoxide(TBHP) of kidney homogenates from control and adrenalectomized male Wistarrats treated with reserpine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 6), adrenalectomized(Adr.) (n = 6), reserpine treated (Res.) (n = 6), adrenalectomized and treated withreserpine (Res., Adr.) (n = 6). Significant difference relative to sham operated,p <0.01.Absorbanceat532nmCoppppC)0-r\)001001001IICl) 3 0. CD 0 -I CD 00,C),H- 86 -Fig. 9. TBARS formation following incubation with 1.50 mM t-butylhydroperoxide(TBHP) of lung homogenates from control and adrenalectomized male Wistar ratstreated with reserpine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 6), adrenalectomized(Adr.) (n = 6), reserpine treated (Res.) (n = 6), adrenalectomized and treated withreserpine (Res., Adr.) (n = 6). Significant difference relative to sham operated,*p< 0.01.a) 3 0 -‘ CD CI) > -I CD Cl)Absorbancepat532nmp C oC0 0pF’)c)•00p- 88 -Fig. 10. TBARS formation following incubation with 1.5 mM t-butylhydroperoxide(TBHP) of heart homogenates from control and adrenalectomized male Wistarrats treated with reserpine or vehicle.Values are mean ± SEM. Sham operated (Sham) (n = 6), adrenalectomized(Adr.) (n = 6), reserpine treated (Res.) (n = 6), adrenalectomized and treated withreserpine (Res., Adr.) (n = 6).Absorbanceat532nmpppo00000010010(31IIIC,) 3 1 CD Cl) 0. -‘ CD C’)0, (0H H rH- 90 -3.2. Effects of Acute and Chronic Stress on Corticosterone,Catecholamines and Metabolic Indices of Diabetes on Days 1, 7 and14 of Stress3.2.1. Effects of Diabetes and Stress on Body WeightBody weights and percent change in weight for control, stressor-exposed,diabetic and diabetic/stressor-exposed groups on the first, seventh and fourteenth dayof exposure to the stress protocol or control conditions are shown in Table 13. Rats inthe non-stressed diabetic groups gained weight more slowly than those in thecorresponding control groups at two, three and four weeks following STZ injection,corresponding to the first, seventh and fourteenth day of the experimental protocol.Rats in both stressor-exposed groups (non-diabetic and diabetic) gained weight moreslowly than those in the control groups following the one and two weeks of exposure tothe stress protocol; however, the rate of weight gain in stressor-exposed diabetic ratsdid not differ from that of non-stressed diabetic rats.3.2.2. Resting Glucose LevelsResting glucose levels for control, stressor-exposed, diabetic anddiabetic/stressor-exposed groups on the day 1, 7 and 14 of exposure to the stressprotocol or control conditions are shown in Table 14. As expected, both groups ofdiabetic rats exhibited significant elevations in resting plasma glucose levels.- 91 -3.2.3. Effects of Exposure to Stress on Day I (Corticosterone,Catecholamines and Glucose Levels)Resting levels of corticosterone, adrenaline and noradrenaline measured prior toexposure to the first stressor showed no significant differences between diabetic andcontrol groups.In non-diabetic rats exposed to restraint stress, significantly elevated levels ofcorticosterone (5, 15, 30, 60 mm), adrenaline (15, 30, 60 mm) and noradrenaline (5, 15,60 mm) (Figs. ha, 13a and 15a) were found relative to samples taken fromunstressed controls at the same time intervals. AUC analysis of the data also showedsignificant elevations in the levels of these stress hormones (Figs. 12a, 14a and 16a).The rank order of the magnitude of the increases relative to control was adrenaline>noradrenaline > corticosterone. In addition to the stress-induced changes in stresshormones, plasma glucose levels were also significantly elevated (5, 15, 30, 60 mm)relative to samples taken from unstressed controls at the same time intervals (Fig. 17).Comparisons made within treatments of the control and stressor-exposed groupsshowed no significant differences among samples collected over time in the levels ofcorticosterone, adrenaline, noradrenaline or glucose (Figs. 11 a, I 3a and I 5a).Exposure of diabetic rats to restraint stress also resulted in elevated levels ofcorticosterone (5, 15, 30, 60 mm), adrenaline (60 mm) and noradrenaline (15, 60 mm)(Figs. 11 b, I 3b and I 5b) relative to samples taken from unstressed diabetic animals atcorresponding time intervals. Similarly, AUC analysis of the data showed significantelevations in the levels of these stress hormones (Figs. 12b, 14b and 16b). As in thenon-diabetic group, the rank order of the magnitude of the responses relative to contro[- 92 -was adrenaline> noradrenaline > corticosterone. No differences in plasma glucoselevels were found between diabetic and diabetic stressor-exposed groups at anysampling interval (data not shown). Comparisons made within treatments showed nosignificant change over time (5, 15, 30, 60 mm) for either diabetic-control or diabeticstressor-exposed in the levels of corticosterone, adrenaline or noradrenaline(Figs. 11 b, I 3b and I 5b).Comparisons of the diabetic and non-diabetic stressor-exposed groups weremade to determine the effects of diabetes on the increases in plasma levels ofcorticosterone, adrenaline and noradrenaline found during stress exposure. Thed iabetic/stressor-exposed group had significantly elevated levels of corticosteronerelative to non-diabetic stressor-exposed rats (15, 30 mm) (Figs. lla,b); however, thiselevation did not reach statistical significance in the overall response profile revealedby AUC analysis (Figs. 12a,b). Although differences in adrenaline levels betweengroups did not reach statistical significance at any sampling time, AUC analysisshowed significantly reduced levels of adrenaline in the diabetic stressor-exposedgroup relative to the non-diabetic stressor-exposed group (Figs. 14a,b). Diabetes didnot significantly alter the elevation in plasma noradrenaline levels induced by acuteexposure to stress.3.2.4. Effects of Exposure to Stress on Days 7 and 14 (Corticosterone,Catecholamines and Glucose Levels)Prior to the exposure to stress on day 7 and 14, there were no significantdifferences in resting levels of corticosterone, adrenaline or noradrenaline betweenstressor-exposed and non-stressor-exposed diabetic and control groups.- 93 -Exposure of non-diabetic rats to the stressor on the day 7 of the chronic stressprotocol again produced increases in the levels of corticosterone (5, 15, 30, 60 mm),adrenaline (5, 15 mm) and noradrenaline (15 mm) (Figs. ha, 13a and 15a). Theoverall response profile conducted by AUG analysis of the data showed significantelevations in the levels of corticosterone, but not of adrenaline or noradrenaline(Figs. I 2a, I 4a and I 6a). The rank order of the magnitude of the responses relative tocontrol was adrenaline> corticosterone> noradrenaline. Plasma glucose levels werealso significantly elevated (5, 15, 30, 60 mm) relative to samples taken from unstressedcontrols at the same time intervals (Fig. 17). Comparisons made within treatments ofthe control and stressor-exposed groups showed no significant differences amongsamples collected over time in the levels of corticosterone, adrenaline or noradrenaline(Figs. ha, 13a and 15a).Exposure of diabetic rats to the stressor on day 7 resulted in significantlyincreased levels of corticosterone only at 60 mm relative to the un-stressed controldiabetic group (Fig. 11 b). The increases in the levels of adrenaline and noradrenalinedid not reach statistical significance relative to the control diabetic group (Figs. I 3b and15b). AUG analysis of the data showed no significant differences between stressorexposed and control diabetic rats in the levels of these stress hormones (Figs. 12b,14b, 16b). The rank order of the magnitude of the responses relative to the controldiabetic group was noradrenaline > adrenaline > corticosterone. No differences inplasma glucose levels were found between diabetic control and diabetic stressorexposed groups at any sampling interval (data not shown). Comparisons made withintreatments of the diabetic control and diabetic stressor-exposed groups showed no- 94 -significant differences among samples over time in the levels of corticosterone,adrenaline or noradrenaline (Figs. I Ib, 13b, 15b).Exposure of non-diabetic rats to the stressor on the day 14 of the chronic stressprotocol produced significantly increased levels of corticosterone (5, 15, 30 and60 mm). However, the levels of adrenaline and noradrenaline were not significantlyelevated relative to those of the control group (Figs. 11 a, I 3a and 1 5a). The overallresponse profile obtained by AUC analysis of the data showed significant elevations inthe levels of corticosterone and noradrenaline, but not adrenaline (Figs. 12a, 14a and16a). The rank order of the magnitude of the responses relative to control wascorticosterone > adrenaline> noradrenaline. Plasma glucose levels were significantlyelevated only at the 60 mm sampling interval relative to samples taken from unstressedcontrols (Fig. 17). Comparisons made within treatments of the control and stressorexposed groups showed no significant differences among samples collected over timein the levels of corticosterone, adrenaline or noradrenaline (Figs. 11 a, I 3a and I 5a).Exposure of diabetic rats to the stressor on day 14 produced elevations in thelevels of corticosterone (5,15, 30 and 60 mm), adrenaline (5 and 15 mm) andnoradrenaline (5, 15 and 30 mm) relative to the nonstressed control diabetic group(Figs. 11 b, I 3b and I 5b) AUC analysis of the data showed significant increases in thelevels of corticosterone and noradrenaline; levels of adrenaline did not reach statisticalsignificance relative to control diabetic rats (Figs. 12b, 14b and 16b). The rank order ofthe magnitude of the responses relative to the non-stressed diabetic group wasnoradrenaline > adrenaline > corticosterone. No differences in plasma glucose levelswere found between diabetic control and diabetic stressor-exposed groups at any- 95 -sampling interval (data not shown). Comparisons made within treatments of thediabetic and diabetic stressor-exposed groups showed no significant differencesamong samples collected over time in the levels of corticosterone, adrenaline ornoradrenaline (Figs. lib, 13b and 15b).Comparisons of the diabetic and non-diabetic stressor-exposed groups weremade to determine the effects of diabetes on plasma levels of corticosterone,adrenaline and noradrenaline during the stress exposure on day 7 and 14 of the stressprotocol. On day 7, corticosterone levels were significantly reduced in the diabeticstressor-exposed group relative to the non-diabetic stressor-exposed group (15 and 30mm), while catecholamine levels were unchanged; on day 14, no significantdifferences were found in corticosterone or catecholamine levels (Figs. lla,b; 13a,band 15a,b). AUC analysis of the data paralled the above findings in showing asignificant reduction of corticosterone in the diabetic stressor-exposed group relative tothe non-diabetic stressor-exposed group on day 7 (Figs. 12a,b) and no significantdifferences between stress-exposed diabetic and non-diabetic animals on day 14 of thestress protocol (Figs. 12a,b and 16a,b).To determine whether the total stress response waned during the chronicintermittent stress protocol, comparisons were made within groups of AUC analysesamong day 1, 7 and 14 of the stress protocol. The results showed that whilecorticosterone values did not significantly differ within the control, diabetic or nondiabetic stressor-exposed groups across sampling days, there was a significantincrease in the diabetic stressor-exposed group on day 14 relative to day 7 of stress(Figs. 12a,b). Adrenaline values did not significantly differ within the control, non--96-diabetic and diabetic stressor-exposed groups across sampling days (Figs. 14a,b). Inthe diabetic group, however, adrenaline levels were elevated on day 14 relative to day7 of stress. No significant differences across sampling days were found innoradrenaline values in diabetic, non-diabetic, or diabetic stressor-.exposed groupsacross sampling days (Figs. 16a,b). On the other hand, the day 14 non-diabetic controlvalue was significantly reduced relative to sampling days I and 7 (Fig. 16a).Comparisons of plasma glucose levels within groups on days 1, 7 and 14 of thestress protocol showed significant reductions on day 14 (15, 30, 60 mm) relative to dayI and 7 (Fig. 17).3.2.5. Non-Enzymatic Antioxidant Levels3.2.5.1. Ascorbate Levels in Tissues and PlasmaThe levels of plasma and tissue ascorbate on days 7 and 14 of stress are shownin Table 15. There were no significant differences in the levels of these componentsfollowing chronic stress in non-diabetic rats. In diabetes of 21 and 28 days’ duration,corresponding to days 7 and 14 of the stress protocol, ascorbate levels weresignificantly reduced in plasma, liver and kidney but unchanged in the heart. Thesediabetes-associated changes were not measurably influenced by stress. When thedata from the two stress intervals were pooled, no additional effects were noted.3.2.5.2. Tocopherol Levels in Tissues and PlasmaThe levels of plasma and tissue tocopherol on days 7 and 14 days of stress areshown in Table 16. There were no significant differences in the levels of thesecomponents following chronic stress in non-diabetic rats. In diabetes of 21 and 28days’ duration, corresponding to days 7 and 14 of the stress protocol, tocopherol- 97 -levels were elevated in the heart and liver, but unchanged in the plasma and kidney.These diabetes-associated changes were not measurably influenced by stress. Whenthe data from the two stress intervals were pooled, no additional effects were noted.Table13.WeightsofmaleWistarratsbeforeinjectionofSTZorvehicleandonday1,7and14ofexposuretothestressprotocol(stressor-exposed) orcontrolconditions.ControlStressor-DiabeticDiabeticlstressorexposedexposedDayIInitlalweight293±10267±8302±5293±6(n=7)(n=6)(n=9)(n=7)Finalweight373±9361±113229*322±6*(n=7)(n=6)(n=9)(n=7)%change25±434±274*10.3±10*(n=7)(n=6)(n=9)(n=7)Day7Initialweight270±14274±7284±12276±13(n=5)(n=6)(n=6)(n=5)Finalweight401±10349±6*314±12*301±11*(n=5)(n=6)(n=6)(n=5)%change50±528±2*10j5*10(n=5)(n=6)(n=6)(n=5)Day14Initialweight271±6292±2283±7274±8(n=5)(n=6)(n=8)(n=5)Finalweight435±7376±8*3327*295±14*(n=5)(n=6)(n=8)(n=5)%change61±429±2*14±4*6±8*(n=5)(n=6)(n=8)(n=5)Valuesaremean±S.E.M.*p<005significantlydifferentfromcontrol.Table14.PlasmaglucoselevelsincontrolandSTZ-treatedmaleWistarratsonthefirst,seventhandfourteenthdayofexposuretothestressprotocol(stressor-exposed)orcontrolconditions.Co CDControlStressor-DiabeticDiabeticlstressorexposedexposedGlucose(mmol/L)6.36±0.256.42±0.1224.71±2.09*23.38±1.90*DayI(n=7)(n=6)(n=9)(n=7)Glucose(mmol/L)6.08±0.346.02±0.2527.13±2.52*25.36±2.23*Day7(n=5)(n=5)(n=6)(n=5)Glucose(mmol/L)5.86±0.235.15±0.6024.63±1.03*26.62±3.64*Day14(n=5)(n=5)(n=8)(n=5)Valuesaremean±S.E.M.*p.<005significantlydifferentfromcontrol.Table15.PlasmaandtissueascorbatelevelsincontrolandSTZ-treatedmaleWistarratsontheseventhandfourteenthdayofexposuretothestressprotocol(stressor-exposed)orcontrolconditions.36.34±4.88*(n=6)39.18±795*(n=6)0.45±0.08(n=5)0.51±0.03(n=5)0.79±0.07*(n=6)0.90±0.07*(n=6)0.35±0.05*(n=6)0.36±0.04*(n=6)Diabeticlstressorexposed37.70±3.29*(n=4)34.41±4.20*(n=5)0.67±0.06(n=3)0.61±0.07*(n=5)0.84±0.08*(n=4)0.85±0.09*(n=5)0.41±0.06*(n=4)0.40±0.03*(n=5)ControlStressor-DiabeticexposedPlasma(day7)73.24±3.9265.41±4.88(tmol/L)(n=6)(n=6)Plasma(day14)80.63±11.4170.41±3.18(j.tmol/L)(n=5)(n=4)Heart(day7)0.49±0.090.40±0.05(.tmolIg)(n=5)(n=6)Heart(day14)0.41±0.040.77±0.23(j.imollg)(n=4)(n=3)Liver (day 7)1.30±0.91.26±0.08(imolIg)(n=7)(n=6)Liver (day14)1.27±0.081.52±0.08(imolIg)(n=5)(n=3)Kidney(day7)0.68±0.030.67±0.05(tmoIIg)(n=7)(n6)Kidney(day14)0.52±0.060.83±0.05(imol/g)(n=4)(n=3)Valuesaremean±S.E.M.*p.<005significantlydifferentfromcontrol.- 0 0Table16.PlasmaandtissuetocopherollevelsincontrolandSTZ-treatedmaleWistarratsontheseventhandfourteenthdayof exposuretothestressprotocol(stressor-exposed)orcontrolconditions.ControlStressor-DiabeticDiabeticlstressorexposedexposedPlasma(day7)18.72±1.7513.27±1.4921.48±3.9818.00±1.03(j.imolIL)(n=6)(n=7)(n=5)(n=5)Plasma(day14)13.68±0.9612.94±1.0316.92±1.2023.52±5.69(tmolIL)(n=5)(n=4)(n=8)(n=5)Heart(day7)0.046±0.0020.042±0.0010.060±0.003*0.053±0.002(imolIg)(n=7)(n=6)(n=6)(n=5)Heart(day14)0.043±0.0020.045±0.0030.058±0.002*0.059±0.003*(j.tmollg)(n=5)(n=5)(n=8)(n=5)Liver(day 7)0.024±0.0010.029±0.0010.043±0.004*0.036±0.004*(.tmoI/g)(n=7)(n=6)(n=6)(n=5)Liver(day14)0.026±0.0020.026±0.0020.042±0.003*0.055±0.005*Qimol/g)(n=5)(n=5)(n=7)(n=5)Kidney(day7)0.025±0.0010.025±0.0010.024±0.0010.027±0.002(imolIg)(n=7)(n=7)(n=6)(n=4)Kidney(day14)0.023±0.0050.020±0.0050.027±0.0010.028±0.002(j.imollg)(n=5)(n5)(n=8)(n=5)Valuesaremean±S.E.M.*p.<o.o5,significantlydifferentfromcontrol.-102-Fig. 11. Plasma corticosterone levels in control (A), stressor-exposed (A),diabetic (B) and diabetic stressor-exposed (B) male Wistar rats sampled during aone-hour-period corresponding to days one, seven and fourteen of the stressprotocol.Values are mean ± SEM. Control: day I (—) (n = 7), day 7 (—— ) (n = 5),day 14 (-—) (0, 5, 15 mm (n = 5), 30, 60 mm (n = 4); Stressor-exposed:day I (—) (n = 6), day 7 ( — —) (n = 6), day 14 (•—•) (n = 6). Diabetic:day I (—) (n=9), day 7 (——) (n=6), day 14 (-—) (n=8); Diabeticlstressorexposed:day I ( ) (n = 7), day 7 ( ——) (n = 5), day 14 (-—.) (n = 5).For plot “A” significant difference relative to the control group at the same time interval,<0.05 and in plot “B” significant difference relative to the diabetic group at the sametime interval, *p <0.05 or to the non-diabetic stress-exposed group at the same timeinterval, tp <0.05.A2700-**T2400-T1*I_i_*2100-————1800-*:::300I0IIIIII051015202530354045505560mmB2700-2400-**___T———————900-060:300II0II051015202530354045505560mm-105-Fig. 12. Plasma corticosterone levels expressed as area under the curve througha one-hour-period corresponding to days one, seven and fourteen of the stressprotocol in control (A), stressor-exposed (A), diabetic (B) and diabetic stressorexposed (B) male Wistar rats.Values are mean ± SEM. Control (con): day I (n = 7), day 7 (n = 5), day 14(n = 4); Stressàr-exposed (str): day I (n = 6), day 7 (n = 6), day 14 (n = 6). Diabetic(d): day I (n = 9), day 7 (n = 6), day 14 (n = 8); Diabetic/stressor-exposed (dstr):day I(n = 7), day 7 (n = 5), day 14 (n = 5). For plot “A” significant difference relative to:control, *p < 0.05 and for plot “B” significant difference relative to: diabetic, *p < 0.05 orstress (day 14), tp < 0.05.-106-A1800*1600C1400-I1200**2C1000‘VCo 800e600- 4002000III1IIIFI1___WHUHU___ ___ ___ ___coni stri con7 str7 conl4 strl4-107-B*CE-J02C‘1)C01)0,0C.)0C)1800160014001200100080060040020001dl dstrl dstr7 d14 dstrl4d7-108-Fig. 13. Plasma adrenaline levels in control (A), stressor-exposed (A), diabetic(B) and diabetic stressor-exposed (B) rats sampled during a one-hour-periodcorresponding to days one, seven and fourteen of the stress protocol.Values are mean ± SEM. Control: day I (— ) (n = 7), day 7 ( ——) (n = 5),day 14 (-—-) (0, 5, 15 mm (n = 5), 30, 60 mm (n = 4); Stressor-exposed:day I (—) (n 6), day 7 ( —...) (n = 6), day 14 (.—.) (n = 6) Diabetic:day I ( ) (n = 9), day 7 (—— ) (n = 6), day 14 (-—-) (n = 8); Diabetic/stressorexposed: day I ( ) (n = 7), day 7 (——) (n = 5), day 14 (.—.) (n = 5).For plot “A” significant difference relative to the controlgroup at the same time interval,<0.05 and for plot “B” significant difference relative to the diabetic group at thesame time interval, *p < 0.05.A8000-7000-**6000-I5000-*4000-VI,3000-I,‘I,,,,*2000-I/I—.—.——.—.—.—.—.—.—.—11000:rmmB8000-7000-6000-5000-40003000--*-12000/./aa-‘//I1000-.T————————0051015202530354045505560mm—111—Fig. 14. Plasma adrenaline levels expressed as area under the curve through aone-hour-period corresponding to days one, seven and fourteen of the stressprotocol in control (A), stressor-exposed (A), diabetic (B) and diabetic stressorexposed (B) rats.Values are mean ± SEM. Control (con): day 1 (n = 7), day 7 (n = 5), day 14(n = 4); Stressor-exposed (str): day I (n = 6), day 7 (n = 6), day 14 (n = 6). Diabetic(d): day 1 (n = 9), day 7 (n = 6), day 14 (n = 8); Diabetic/stressor-exposed (dstr):day I(n = 7), day 7 (n = 5), day 14 (n = 5). For plot “A” significant difference relative to:control, *p < 0.01 and for plot “B” significant difference relative to: diabetic, *p < 0.05 orto the non-diabetic stress-exposed group at the same time interval, tp < 0.05.-112-A7600673058604990EC4120*1)3250C2380-o1510E0 640III II I-230-1100coni stri con7 str7 conl4 strl4-113-B760067305860- 4990EC4120a) *t3250CIIC___2380__a’1510CII____E(1) 640a’_ __-a-230_____-1100dl dstrl d7 dstr7 dl 4 dstrl 4I I-114-Fig. 15. Plasma noradrenaline levels in control (A), stressor-exposed (A) diabetic(B) and diabetic stressor-exposed (B) rats sampled during a one-hour-periodcorresponding to days one, seven and fourteen of the stress protocol.Values are mean ± SEM. Control: day I ( ) (n = 7), day 7 (—— ) (n = 5),day 14 (-_-) (0, 5, 15 mm (n = 5), 30, 60 mm (n = 4); Stressor-exposed:day I ( ) (n = 6), day 7 ( ——) (n = 6), day 14 (.—.) (n = 6). Diabetic: day I( —) (n = 9), day 7 (——) (n = 6), day 14 (-----.) (n = 8); Diabeticlstressorexposed:day I ( —) (n=7), day 7 (———) (n=5), day 14 (—) (n=5).For plot “A” significant difference relative to the control group at the same time interval,<0.05 and for plot “B” significant difference ielative to the diabetic group at thesame time interval, *p < 0.05.-115-001001F’)0F’)013 CA)0CA)0101(71001010)00plasma noradrenaline (nmol/L)1’)0-01 0 (71***B20-I*_J-02*U)1105,ILI-----I----O:1mn-117-Fig. 16. Plasma noradrenaline levels expressed as area under the curve througha one-hour-period corresponding to days one, seven and fourteen of the stressprotocol in control (A), stressor-exposed (A), diabetic (B) and diabetic stressorexposed (B) rats.Values are mean ± SEM. Control (con): day I (n = 7), day 7 (n = 5), day 14(n = 4); Stressor-exposed (str): day I (n = 6), day 7 (n = 6), day 14 (n = 6). Diabetic(d): day I (n = 9), day 7 (n = 6), day 14 (n = 8); Diabetic/stressor-exposed (dstr):day I(n = 7), day 7 (n = 5), day 14 (n = 5). For plot “A” significant difference relative to:control at the same time interval, *p <005. or to the control groups at days I and 7,tp <0.05 and for plot “B” significant difference relative to: diabetic, *p <0.05.CE-J0ECCCa,I0C-118-A131211109876543210-1-2-3-4ECl,coni stri con7 str7 coni 4 stri 4plasmanoradrenaline(nmol/L/min)iei-L•_Ci)I\)L0-F’.3Ci).(flO)40)(00-L))IIIIIIIIIIIIII1*Q. - 0) b4 0 CI) -‘ bJ 0 -‘H Fl(0-120-Fig. 17. Plasma glucose levels in control and stressor-exposed rats collectedduring a one-hour-period corresponding to days one, seven and fourteen of thestress protocol.Values are mean ± SEM. Control: day I (—) (n = 7), day 7 (—--—) (n = 5),day 14 (-—-) (0, 5, 15 mm (n = 5), 30, 60 mm (n = 4)); Stressor-exposed: day I( ) (n = 6), day 7 ( ——) (n = 6), day 14 (.—.) (n = 6). Significant differencerelative to the control group at the same time interval, *p <0.05. Significant differencerelative to days I and 7 of stress at the same time interval, tp < 0.05.110/rZ*C--r9*——————8——,ft7I‘i.’iT2f”,‘•i———.—._L........j.4IIIIII051015202530354045505560mm-122-3.3. Effects of Chronic Intermittent Stress and Diabetes on AntioxidantStatus3.3.1. Effects of Stress in Rats With Short-Term (4 Weeks) DiabetesGiven the putative role of reactive oxygen radicals in the development ofdiabetes and its associated cardiovascular complications,11 stress could influence thecourse of their development through effects on antioxidant systems. The aim of thepresent study was, therefore, to examine the effects of chronic stress on tissueantioxidant status in normal and diabetic animals.3.3.1.1. Body WeiQhts and Plasma Chemical ComiositionBody weights and plasma biochemical composition for control, stressor-exposed,diabetic, and diabetic/stressor-exposed groups are shown in Table 17 and Figs. 18-20.As expected, both groups of diabetic rats exhibited significant elevations of plasmaglucose and gained weight more slowly than the control and stress-exposed groups.Diabetic animals also showed significant increases in plasma cholesterol andtriglycerides. Stress alone did not alter the plasma levels of glucose, triglycerides orcholesterol; however, diabetic rats exposed to the stress protocol exhibited significantincreases in plasma glucose levels and significant decreases in plasma cholesterol andtriglyceride levels. In fact, the cholesterol and triglyceride values in thediabetic/stressor-exposed group decreased to levels that were not significantly differentfrom control values.-123-Fig. 18. Plasma glucose levels in control and diabetic (four weeks’ duration) maleWistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5 forthe diabetic/stress group. Significant difference relative to: control, *p < 0.01, ordiabetic tp < 0.01.PlasmaGlucose(mmol/L)C01L0).0010010.010IIIIIC) 0 -I 0 CI, -I CD Co Co 0 CD 0-0CDCDCD—- N)*IIIII_ITIFIIIILI-125-Fig. 19. Plasma cholesterol levels in control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5 forthe diabetic/stress group. Significant difference relative to: control, *p <0.01, ordiabetic tp < 0.01.CPlasmaCholesterol(mmol/L)-F’)C,)HC, 0 -‘ 0 Co CD Co CI) ci CD 0 ci-0CDCDH- 0)H-127-Fig. 20. Plasma triglyceride levels in control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5 forthe diabetic/stress group. Significant difference relative to: control, *p <0.01, ordiabeticp <0.01.-128-*-J0ECl,0>1IcIECl,0876543210tControl Stress Diabetic Diabetic?Stress-129-3.3.1.2. Corticosterone and Catecholamine LevelsBasal corticosterone and catecholamine levels are shown in Table 18. Therewere no differences in corticosterone levels within treatment groups in blood samplescollected on the day following the last stress. There were no differences in adrenalineor dopamine within treatment groups. Plasma norepinephrine levels, however, weresignificantly reduced in both stressed and unstressed diabetic groups.3.3.1.3. Antioxidant Enzyme ActivitiesThe diabetic state was associated with changes in several antioxidantcomponents in heart, liver, kidney and lung tissue, but not erythrocytes. In the heart,glutathione reductase activity and basal glutathione level were elevated; however,glutathione peroxidase and Cu, Zn-superoxide dismutase activities were not altered(Table 19). In liver tissue, glutathione reductase activity was elevated, catalase andglutathione peroxidase activities as well as glutathione levels were reduced and Cu,Zn-superoxide dismutase activity was unchanged (Table 20). Kidney tissue showed adecrease in catalase activity (Table 21) and lung tissue had reduced glutathioneperoxidase activity (Table 22). Exposure of non-diabetic rats to the stressor protocolhad minimal effects on antioxidant components. The only significant change noted wasa reduction in kidney basal glutathione levels (Table 21). In diabetic rats exposed tothe stressor protocol, the increase in liver glutathione reductase (Table 20) anddecreases in liver and lung glutathione peroxidase activities (Tables 20 and 22)observed with diabetes alone were not present. In addition, the diabetes-inducedincrease in heart basal glutathione level was no longer significant following exposure tothe stress protocol. Other diabetes-associated changes, notably the elevated-130-glutathione reductase activity in heart (Table 19), and reduced catalase activity in liver(Table 20) and kidney (Table 21) were not altered by repeated exposure to thestressors. Erythrocyte catalase, although not changed by either diabetes or stressalone, was increased in diabetic rats exposed to the stress protocol (Table 23).3.3.1.4. H20-lnduced Sulfhydrvl Grouo DerletionThe diabetic state was associated with a reduction in susceptibility oferythrocytes (Fig. 21), heart (Fig. 22) and liver (Fig. 23) but not kidney or lung (data notshown) to in vitro sulfhydryl group depletion. Exposure of non-diabetic rats to thestressor protocol had minimal effects on antioxidant status. The only significant changenoted was an increase in the susceptibility of erythrocytes toH20-induced sulfhydrylgroup oxidation (Fig. 21). In diabetic rats exposed to the stressor protocol, thereduction in H20-induced sulThydryl group depletion in heart (Fig. 22) and liver(Fig. 23) found in non-stressed diabetic rats was not present in the diabetic stressorexposed group; however, the diabetes-associated reduction in erythrocyte sulfhydrylgroup susceptibility to oxidation (Fig. 21) was not altered by stress.3.3.1.5. H20-lnduced Lipid PeroxidationIn diabetic rats,H20-induced lipid peroxide formation in liver (Fig. 24), kidney(Fig. 25), lung (Fig. 26) and erythrocytes (Fig. 27) was significantly increased, whileperoxide formation was significantly reduced in myocardial tissue (Fig. 28). Exposureof non-diabetic rats to the stressor protocol did not alter peroxide-induced TBARSformation. In diabetic rats exposed to the stressor protocol, the elevation in H20-induced lipid peroxidation in both kidney (Fig. 25) and lung (Fig. 26) found in nonstressed diabetic rats was not present in the diabetic stressor-exposed group; however,-131-the diabetes-induced increases in MDA formation in erythrocytes (Fig. 27) and TBARSformation in liver (Fig. 24) and the reduction in myocardial TBARS formation (Fig. 28)were not altered by stress.Table17.WeightsofmaleWistarratsbeforeinjectionofSTZorvehicleandfollowingexposuretothestressprotocol(stressor-exposed)orcontrolconditionsfollowingfourweeks.-C)ControlStressor-DiabeticDiabeticlstressorexposedexposedlnitialweight317±16300±9333±19302±6(n=6)(n=6)(n=6)(n=5)Finalweight467±18400±10*368±19*345±8*(n=6)(n=6)(n=6)(n=5)%change44±533±211±6*14±2*(n=6)(n6)(n=6)(n=5)Valuesaremean±S.E.M.*p<005significantlydifferentfromcontrol.Table18.Plasmacatecholaminesandcorticosteronefromcontrolanddiabetic(fourweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.AdrenalineanddopaminemeasuredinI02pmol/Lplasma.Corticosteronemeasuredfromcontrol.measuredinpmol/Lplasma,noradrenalineinnmol/Lplasma.*p.<001significantlydifferentControlStressor-DiabeticDiabeticlstressorexposedexposedAdrenaline106.54±21.0392.50±20.3065.82±9.5569.48±15.61(n=6)(n=5)(n=6)(n=5)Noradrenaline4.47±1.363.81±0.602.12±0.50*1.75±0.40*(n=5)(n=6)(n=6)(n=5)Dopamine2.61±1.304.24±1.702.35±0.852.68±1.70(n=6)(n=6)(n=6)(n=5)Corticosterone70.42±14.14138.82±66.38196.25±63.78162.48±74.17(n=6)(n=5)(n=5)(n=5)- c) C)Table19.Antioxidantstatusinheartsfromcontrolanddiabetic(fourweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.Cu, Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione;N/A=enzymeactivitybelowmeasurablelevels.*p.<OO1significantlydifferentfromcontrol.- .ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=6)(n=5)CatalaseN/AN/AN/AN/A(K/mg wetwt)Cu,Zn-SOD1.09±0.050.97±0.041.14±0.051.23±0.20(U/mgwetwt)GSH-PX6.65±0.786.04±0.555.54±0.095.23±0.41(nmolNADPHmin1mgwetwt)GSSG-RD0.493±0.0100.525±0.0210.611±0.020*0.574±0.039*(nmolNADPHmin1mg1wetwt)GSH1.82±0.121.78±0.122.14±0.11*2.02±0.08(nmol/mgwetwt)Table20.Antioxidantstatusinlivertissuefromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.c) 01Valuesaremean±S.E.M.Cu,Zn-SOD=GSSG-RD=glutathionereductase;GSH=significantlydifferentfromdiabetic.Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;glutathione.*p<O.01significantlydifferentfromcontrol;tp<0.01,ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=6)(n=5)Catalase0.031±0.0050.028±0.0030.020±0.003*0.020±0.004*(K/mgwetwt)Cu,Zn-SOD4.75±0.254.89±0.405.24±0.425.56±0.38(UImgwetwt)GSH-PX17.13±1.5319.03±0.8111.46±1.18*17.28±2.21t(nmolNADPHmin1mg1wetwt)GSSG-RD4.13±0.134.03±0.125.14±0.54*4.57±0.26t(nmolNADPHmin1mg1wetwt)GSH4.82±0.244.18±0.243.81±0.24*3.90±0.28*(nmol/mgwetwt)Table21.Antioxidantstatusinkidneytissuefromcontrolanddiabetic(fourweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.Cu, Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione.*p<001significantlydifferentfromcontrol.() 0)ControlStressor-DiabeticDiabetic/stressorexposedexposed(n=6)(n=6)(n=6)(n=5)Catalase0.0087±0.00080.0080±0.00100.0044±0.0006*0.0051±0.0010*(K/mgwetwt)Cu, Zn-SOD2.80±0.222.66±0.142.94±0.192.45±0.18(U/mg wetwt)GSH-PX6.25±0.685.73±0.527.70±0.657.37±0.60(nmolNADPHmin1mg1wetwt)GSSG-RD7.54±0.347.70±0.337.60±0.867.02±0.45(nmolNADPHmin1mg1wetwt)GSH2.48±0.182.13±0.12*2.55±0.092.37±0.08(nmol/g wetwt)Table22.Antioxidantstatusinlungtissuefromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu, Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;N/A=enzymeactivitybelowmeasurablelevels.*p<001significantlydifferentfromcontrol.tp<0.01significantlydifferentfromdiabetic.() •-1ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=6)(n5)CatalaseN/AN/AN/AN/A(K/mgwetwt)Cu, Zn-SOD0.95±0.130.72±0.060.72±0.040.72±0.08(U/mgwetwt)GSH-PX3.27±0.383.17±0.31.78±0.17*2.68±0.14t(nmolNADPHmin1mg1wetwt)GSSG-RD1.62±0.061.56±0.051.51±0.061.58±0.09(nmolNADPHmin1mg1wetwt)Table23.Antioxidantstatusinpackederythrocytesfromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.c) 0,Valuesaremean±GSH=glutathione.S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;tP<0.01,significantlydifferentfromdiabetic.ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=6)(n=5)Catalase0.0492±0.00200.0493±0.00280.0447±0.00160.0536±0.0027t(K/mgHb)Cu,Zn-SOD4.83±0.314.72±0.265.18±0.564.68±0.42(U/mgHb)GSH-PX70.39±6.9066.39±5.5668.70±8.9075.02±3.03(nmolNADPHmin1mg1Hb)GSH1,594.80±55.281,592.19±95.461,371.51±69.161,365.42±128.24(nmol/mgRBC)-139-Fig. 21. Percent sulfhydryl group depletion following incubation with 0.1 mMH20 of erythrocytes from control and diabetic (four weeks’ duration) male Wistarrats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p < 0.01.PercentDepletion-0)0000000C, 0 -‘ 2. Cl)___HCD Cl, Cl)____________________CD I-I.0co.-o.__CDCD0 ø.II-141 -Fig. 22. Percent sulfhydryl group depletion following incubation with 0.1 mMH20of myocardial homogenates from control and diabetic (four weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p < 0.01 ordiabetic tp <0.01.PercentDepletionC-F’)C,)Cii0)0000000IIIIC) 0 -I 0 ci) -‘ CD ci) ci) 0 CD 0-143-Fig. 23. Percent sulfhydryl group depletion following incubation with 0.25 mMH20 of liver homogenates from control and diabetic (four weeks’ duration) maleWistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p <0.01.PercentDepletionL0)00000000C) 0 P. -‘ 0 Cl) - CD CI) 0) CD P. 0 ciP.CDCDH-I1IIIIIIIIII_I_I_Ii_IIIIII-145-Fig. 24. TBARS formation following incubation with 20.0 mM H20 of liverhomogenates from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p < 0.01.Absorbanceat532nmoopo--L-0o-ooi0010010010010IIIIIIIIC) 0 0 Co CD ci, crj CD 009-I- 0)IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-147-Fig. 25. TBARS formation following incubation with 2.5 mM H20 of kidneyhomogenates from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p < 0.01 ordiabetic tp <0.01.D) 0• CD C)0 0 0Absorbanceat532nmp0 F’)000 CA)0C, 0 0 Cl) -‘ CD Cl) Cl)IIIIIIII111111IIIIII11111II-149-Fig. 26. TBARS formation following incubation with 0.75 mM H20 of lunghomogenates from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p < 0.01 ordiabetic tp 0.01.Absorbanceat532nmp000000-LC)000000IIC, 0 0 cr -I CD CO Cl) ci CD ‘-I. 0 ciCl)0H- 01 0H-151-Fig. 27. Malondialdehyde (MDA) formation following incubation with 2.5 mM H20of erythrocytes from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5 forthe diabetic/stress group. Significant difference relative to control, *p <0.01.nmolesMDA/gRBCL 0000000000000C) 0 0 C’) CD Cl) Cl) CD F. 0CO9 CDCDH0i-153-Fig. 28. TBARS formation following incubation with 1.5 mM H20 of myocardialhomogenates from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. n = 6 for control, stress, and diabetic groups; n = 5for the diabetic/stress group. Significant difference relative to: control, *p <0.01.Absorbanceat532nmp0000-))0000C, 0 -I 0 Cl) CD C’) CI, CD 0‘0CDCD0 Co.H01H H-155-3.3.2. Effects of Stress in Rats with Long-Term (12 Weeks) DiabetesWe have reported that chronic-intermittent stress can modify antioxidant alterationsassociated with streptozotocin-induced diabetes in animals with diabetes of relativelyshort duration (i.e., 4 weeks). However, the incidence of complications increase withthe duration of the disease and stress is suspected to exacerbate their development.It, therefore, follows that time-dependent deterioration in antioxidant capacity mightoccur with an increased duration of diabetes and that the effects of stress onantioxidant alterations in short-term diabetes might vary with increased duration of thedisease. In view of these considerations, the present study was undertaken to examinethe effects of chronic-intermittent stress on tissue antioxidant status in long-termdiabetes.3.3.2.1. Body Weicihts and Plasma Chemical CompositionBody weights and plasma chemical composition for control, stressor-exposed,diabetic, and diabetic/stressor-exposed groups are shown in Table 24 and Figs. 29-32.Rats in the non-stressed diabetic group and in both stressor-exposed groups (non-diabetic and diabetic) gained weight more slowly than those in the control group. Asexpected, both groups of diabetic rats exhibited significant elevations in plasmaglucose and glycosylated hemoglobin levels. Non-stressed diabetic animals showedsignificant increases in plasma triglyceride, but not cholesterol levels. Stressorexposure in non-diabetic rats reduced resting plasma triglyceride levels; however, thelevels of glucose, glycosylated hemoglobin and cholesterol were not significantlyaltered by the stress protocol. Stressor exposure in diabetic rats was not associatedwith an increase in the extent of hyperglycemia or glycosylated hemoglobin, but did-156-lead to a reduction in the elevated plasma triglyceride levels found in diabetes alone.In fact, the triglyceride values in the diabetic/stressor-exposed group decreased tolevels that were not significantly different from control.3.3.2.2. Corticosterone LevelsResting levels of plasma corticosterone levels were significantly elevated in thediabetic group alone (Table 25).3.3.2.3. Antioxidant Enzyme ActivitiesThe long-term diabetic state was associated with changes in several antioxidantcomponents in heart and liver, but not kidney or erythrocytes (Tables 26-29). In heart,basal glutathione levels and glutathione reductase activity were elevated, althoughglutathione peroxidase and Cu, Zn-superoxide dismutase activities were not altered(Table 26). In liver, on the other hand, basal glutathione levels were decreased but theactivities of catalase, Cu, Zn-superoxide dismutase, glutathione peroxidase andglutathione reductase were all unchanged (Table 27). Exposure of non-diabetic rats tothe stressor protocol had minimal effects on antioxidant status. The only change notedwas a small, but significant reduction in liver glutathione reductase activity (Table 27).In diabetic rats exposed to the stressor protocol, the increase in heart glutathionereductase activity (Table 26), as well as the decrease in liver basal glutathione level(Table 27) observed with diabetes alone, were not present. Some antioxidantcomponents that had been unaffected by diabetes alone were altered in the diabeticstressor-exposed group, including elevations in erythrocyte and kidney glutathioneperoxidase activities (Tables 29 and 28) and a reduction in kidney glutathionereductase activity (Table 28). Other diabetes-associated changes were not influenced-157-by repeated exposure to stress, including the elevation in heart basal glutathione level(Table 26).3.3.2.4. H20-lnduced Sulfhydrvl Group DepletionThe long-term diabetic state was associated with reduced in vitro peroxide-induced susceptibility to sulThydryl group depletion in erythrocytes (Fig. 33), heart(Fig. 34), liver (Fig. 35) and kidney (Fig. 36). Exposure of non-diabetic rats to thestressor protocol did not significantly alterH20-induced sulThydryl group depletion intissues or erythrocytes. Other diabetes-associated changes, including reductions inerythrocyte (Fig. 33), heart (Fig. 34), liver (Fig. 35) and kidney (Fig. 36) peroxide-induced sulfhydryl group depletion, were not influenced by repeated exposure tostress.3.3.2.5. Non-Enzymatic Antioxidant Levels: Plasma Tocoheroland AscorbateIn animals with long-term diabetes, plasma ascorbate was reduced, while thelevel of tocopherol was increased (Table 30). Exposure of non-diabetic rats to thestressor protocol did not significantly alter plasma tocopherol or ascorbate levels. Indiabetic rats exposed to the stressor protocol, the increase in plasma tocopherol level(Table 2) observed with diabetes alone was abolished; however, the diabetesassociated reduction in plasma ascorbate was not influenced by repeated exposure tostress (Table 2).3.3.2.6. H20-lnduced Lipid PeroxidationPeroxide-induced TBARS formation following long-term diabetes was increasedin erythrocytes (Fig. 37), reduced in myocardial tissue (Fig. 38), and unchanged in the-158-liver and kidney (data not shown). Exposure of non-diabetic rats to the stressorprotocol did not significantly alter H20-induced TBARS formation in tissues orerythrocytes. Other diabetes-associated changes were not influenced by repeatedexposure to stress, including the elevation in erythrocyte peroxide-induced MDAformation (Fig. 37) and the reduction in heart TBARS formation (Fig. 38).Table24.Weightand%changeinweightofcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=8)(n=6)(n=7)Initialweight261±15275±13274±12280±6Finalweight545±20490±9*381±31*360±18*%change112±1381±8*41±15*29±9*- (71 CDValuesaremean±S.E.M.*p.<OOIsignificantlydifferentfromcontrol.Table25.Plasmacorticosteronelevelsincontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.ControlStressor-DiabeticDiabeticlstressorexposedexposedCorticosterone58.87±9.23101.0±19.6240.4±98.11*179.8±59.7(n=7)(n=8)(n=6)(n=7)Valuesaremean±S.E.M.CorticosteronemeasuredinnmolIL.*p.<001significantlydifferentfromcontrol.0) 0-161-Fig. 29. Plasma glucose levels in control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p <0.01.PlasmaGlucose(mmol/L)--C.)C)CC01001001C,I1ItIHHIH1IIoiIIII[IIIIIIIII____ll1h—lilIllillIllIll-‘IIIIIIII!IIII2.. C,, -‘ CDF’3Co C’)________CD ‘-I 0 ci_________________________________c1)•1111111III1111111111111ii1111111111111111111II 111111IIII‘I’IIIIICDCDIIllIIiI III1111I11111II111111111111111111111111I[I*Cl)..—..IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIJIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIICo.IIII1111111111111111111IliiiIII1111111111111111IIIIIIIIII-163-Fig. 30. Glycosylated hemoglobin levels in control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p < 0.01.%HbAlc00)111111liii11111111111111111IliiiI0IIIIIIII‘IilliIII1111111ilIliillIl1111111iiiIIII—..IIIIIIIII!IIIIIIIIIIIIIIIII2..IlIllilIlIllCl) — -I CD0)Cs) Co____________________9__________[H*CDci_______________________________________________________________________________________________1H*IHHHco.-165-Fig.. 31. Plasma triglyceride levels in control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 6), stress (n = 7), diabetic (n = 6),diabetic/stress (n = 6). Significant difference relative to: control, *p <0.01, or diabetictp< 0.01.11I.niiiHHiH1NWH[UJi}U+A—0PlasmaTriglycerides(mmol/L)roC, 0 0 Cl, .-I. 1 CD Cl) Co 0 CD ‘-I. 0- a) 0)Fl_IIllIIIIIII1FlH-167-Fig. 32. - Plasma cholesterol levels in control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7).0 CD 0PlasmaCholesterol(mmol/L)00) co0 001001000000C) 0 0 C,) -I CD Co C’)ui H-i0CDCD0 CO111111111111 1111111111111111IIIIII11111liiiIIIliiiII IIIIIIIII111111Table26.Antioxidantstatusinheartsfromcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione;N.D.=notdetectable.*p<001significantlydifferentfromcontrol.0) (0ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=8)(n=6)(n=7)CatalaseN.D.N.D.N.D.N.D.(K/mgwetwt)Cu,Zn-SOD1.19±0.041.03±0.081.24±0.131.07±0.10(U/mgwetwt)GSH-PX5.14±0.405.10±0.284.61±0.205.11±0.34(nmolNADPHmin1mg1wetwt)GSSG-RD0.528±0.0440.480±0.0120.688±0.036*0.610±0.031(nmolNADPHm1n1mg’wetwt)GSH1.90±0.091.66±0.072.34±0.12*2.22±0.06*(nmol/mgwetwt)Table27.Antioxidantstatusinlivertissuefromcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSHglutathione.*p<O01significantlydifferentfromcontrol.0ControlStressor-DiabeticDiabetic!stressorexposedexposed(n7)(n=8)(n=6)(n=7)Catalase0.034±0.0040.037±0.0030.027±0.0070.025±0.002(K!mg wetwt)Cu,Zn-SOD5.71±0.595.00±0.355.93±0.325.07±0.35(U/mg wetwt)GSH-PX18.25±1.2520.12±1.0815.38±1.5615.40±2.83(nmolNADPHmin1mg1wetwt)GSSG-RD4.74±0.084.30±0.09*4.89±0.054.58±0.15(nmolNADPHmin1mg1wetwt)GSH5.71±0.285.51±0.144.28±0.26*4.93±0.61(nmol/mgwetwt)Table28.Antioxidantstatusinkidneytissuefromcontrolanddiabetic(twelveweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.Valuesaremean±S.E.M.Cu,Zn-SOD=Cu,Zn-superoxidedismutase;GSH-PX=glutathioneperoxidase;GSSG-RD=glutathionereductase;GSH=glutathione.*p.<001significantlydifferentfromcontrol.- -ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=8)(n=6)(n=7)Catalase0.007±0.0010.008±0.0010.006±0.0010.006±0.003(K/mgwetwt)Cu,Zn-SOD3.08±0.212.57±0.193.05±0.362.49±0.14(U/mgwetwt)GSH-PX6.66±0.366.76±0.317.18±0.398.29±0.38*(nmolNADPHmin1mg1wetwt)GSSG-RD7.67±0.237.67±0.197.13±0.216.74±0.26*(nmolNADPHmin1mg1wetwt)GSH2.68±0.122.56±0.122.66±0.122.77±0.16(nmol/gwetwt)Table29.Antioxidantstatusinpackederythrocytesfromcontrolanddiabetic(twelve weeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.-‘Ir,)Valuesaremean±GSH=glutathione.S.E.M.Cu,Zn-SOD=Cu,Zn-superoxide*p<0O1significantlydifferentfromcontrol.dismutase;GSH-PX=glutathioneperoxidase;ControlStressor-DiabeticDiabetic!stressorexposedexposed(n=7)(n=8)(n=6)(n7)Catalase0.041±0.0040.041±0.0020.041±0.0050.042±0.001(K/mgHb)Cu,Zn-SOD4.94±0.204.80±0.175.31±0.424.73±0.25(U/mgHb)GSH-PX75.77±3.074.83±1.6278.07±2.8183.69±3.02*(nmolNADPHmin1mg1Hb)GSH1,408.49±64.741,468.12±95.881,668.65±147.191,650.33±101.88(nmol/mgRBC)Table30.Nonenzymaticantioxidantcomponentsinplasmafromcontrolanddiabetic(twelveweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=8)(n=6)(n=7)Ascorbate55.13±2.6151.38±2.3832.76±4.09*24.98±2.38*(pm/L)Tocopherol17.38±1.5113.56±1.3924.41±3.02*22.70±2.64(pm/L)Valuesaremean±S.E.M.*p.<OO1significantlydifferentfromcontrol.- —.1C)-174-Fig. 33. Percent sulfhydryl group depletion following incubation with 0.1 mMH20 of erythrocytes from control and diabetic (twelve weeks’ duration) maleWistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p < 0.01.PercentDepletionC)010)00000000________ICl)— -I- (7’CI)______________0•I*CD 0-I CDCD-176-Fig. 34. Percent sulfhydryl group depletion following incubation with 0.025 mMH20 of myocardial homogenates from control and diabetic (twelve weeks’duration) male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p < 0.01.C) 0 0 Cl, -‘ CD CI) C,)cO;.CDCDCO0PercentDepletion00010C)1001IIIIci C- CD 0H-.4-178-Fig. 35. Percent sulfhydryl group depletion following incubation with 0.25 mMH20 of liver homogenates from control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p <0.01.PercentDepletion-&F’)010)00000000IIIIC)I11111111111111111111111I1111111I1111111111111I1111111111111110liiiI IIliiiiii11111liiiIIIIIII Iii 1111111111II11111111111[I 11111IIIHI111111111II11111111111liii1111111111111111111liii1111111111ilIl 111111111III_....1....‘IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII11111II11111liii11111111111111111111111111111111111111111111111111111112..liiiIII111111111111111111 11111111111 liiiillIIIliiiCl)I—Ii-‘II-CoCDflci) C,,____________________-H*CD 9CS,(DCD-180-Fig. 36. Percent sulfhydryl group depletion following incubation with 0.025 mMH20 of kidney homogenates from control and diabetic (twelve weeks’ duration)male Wistar rats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to: control, *p <0.01.PercentDepletion010010C) 0 -I 0 Cr) -‘ CD Ci, Cl) D) CD 0CDCD0,-H-182-Fig. 37. Malondialdehyde (MDA) formation following incubation with 2.5 mM H20of erythrocytes from control and diabetic (twelve weeks’ duration) male Wistarrats exposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to control, *p < 0.01.)NmolesMDA/gRBCC-kC,)(000—.100000IHC, 0 0 Cl) CD Cl) C’) 0• CD 0o.CDH H I-H*()-184-Fig. 38. TBARS formation following incubation with 1.5 mM H20 of myocardialhomogenates from control and diabetic (twelve weeks’ duration) male Wistar ratsexposed to the stress protocol or control conditions.Values are mean ± S.E.M. Control (n = 7), stress (n = 8), diabetic (n = 6),diabetic/stress (n = 7). Significant difference relative to control, *p <0.01.Absorbanceat532nmpp000r’30000C) 0 -I 0 Cl) - CD 0) C,) 0 CD 0CDCD0..H(7’-186-3.4. Effects of Stress on Plasma Lipids and Antioxidant Status in Ratswith Diet-Induced HyperlipidemiaIn diabetes of four weeks’ duration, the elevation of plasma cholesterol andtriglyceride levels associated with diabetes was attenuated when these animals weresubjected to the chronic intermittent stress protocol, despite stress-induced increasesin the extent of hyperglycemia. In addition, some diabetes-associated antioxidantchanges were reversed in these chronically stressed animals. These findings raisequestions concerning the influence of stress in conditions of hyperlipidemia generallyand the interplay of stress and hyperlipidemia in relation to antioxidant status. Thepresent study explored the influence of chronic intermittent stress on plasma lipidprofiles and tissue antioxidant status in non-diabetic rats with diet inducedhyperlipidemia. Plasma levels of cholesterol and triglycerides were measured in orderto confirm the presence of hyperlipidemia, and to determine the extent to which stressaffects these levels. The activity of glutathione reductase, previously shown to bealtered in diabetes2 was measured and functional antioxidant capacity assessed byin vitro oxidative challenge with increasing concentrations of H20 followed bymeasurement of acid-soluble sulfhydryl group depletion and thiobarbituric acid-reactivesubstance (TBARS) formation in tissues or malondialdehyde in erythrocytes (bothbeing indirect indices of lipid peroxidation).21 The results suggest that the effects ofstress on diabetes induced changes in plasma lipids and antioxidant componentscannot be ascribed solely to the associated hyperlipidemia.-187-3.4.1. Weight Gain and Plasma Lipid LevelsAs expected, plasma cholesterol and triglyceride levels were significantlyelevated in the groups fed a diet supplemented with 1% cholesterol (Table 1).Although the cholesterol-supplemented diet had no observable effect on weight gain(control: 155.3 ± 3.9 g , cholesterol-fed: 164.7 ± 7.5 g), animals exposed to the stressprotocol gained weight more slowly regardless of diet (stress: 75.3 ± 7.8 g, cholesterol-fed/stress: 83.7 ± 6.6 g). Exposure to the stress protocol elevated plasma levels ofcholesterol but not triglycerides in animals fed a control diet (Table 31). However,stress did not alter the extent of hyperlipidemia in cholesterol-fed animals (Table 32).3.4.2. Antioxidant ComponentsExposure to the stress protocol did not alter glutathione reductase activities orbasal glutathione levels in heart, liver and kidney in rats fed either a control orcholesterol-supplemented diet (Table 31).3.4.3. H2Orlnduced SuiThydryl Group Depletion and Lipid PeroxidationAssessment of functional antioxidant capacity indicated that peroxide-inducedsulfhydryl group depletion was not affected by hypercholesterolemia or stress(Table 32). Exposure of normolipidemic animals to the stress protocol reducedperoxide-induced MDA production in erythrocytes and increased TBARS formation inheart and liver, but not kidney (Table 2). Hyperlipidemia resulted in a preferentialincrease in TBARS formation in the heart, but chronic intermittent stress had no effecton TBARS formation in any of the tissues examined (Table 2).Table31.Plasmalipidsandantioxidantstatusinerythrocytesandtissuesincontrolanddiet-inducedhyperlipidemicratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.o o:Valuesaremean±S.E.M.n=6forcontrol;stress(str.);cholesterol-fed(chol.);n=7forcholesterol-fed/stress(stress/chol.).BasalGSH(acidsolublesulfhydrylgroups(nmol/mgRBCortissue);GSSG-RD(glutathionereductase(nmol/min/mgtissue)).Forcholesterol(mg/dl)andtriglycerides(mg/dl)measurements,comparisonsweremadeofallgroupswithcontrolandforbasalGSHandGRd,ofstressandchol.withcontrolandbetweenstress/chol.andchol.*p.<005significantlydifferentfromcorrespondingcontrol.PlasmaErythrocytesHeartLiverKidneyCholes-Trigly-BasalGSHBasalGSSG-BasalGSSG-BasalGSSGterolceridesGSHRDGSHRDGSHRDControl53.4150.31,608.21.590.5054.994.892.368.34±2.5±4.5±91.0±0.06±0.028±0.33±0.12±0.23±0.22Stress66.4*141.11,409.41.370.5265.234.872.388.37±3.4±6.1±57.3±0.13±0.034±0.10±0.20±0.13±0.24Chol.91.8*233.8*1,476.61.600.5025.215.482.447.63±3.0±10.4±47.85±0.08±0.021±0.30±0.28±0.11±0.21Stress/chol.90.3*211.2*1,482.81.520.5314.485.272.098.21±3.3±7.8±38.9±0.09±0.019±0.34±0.13±0.14±0.19Table32.Functionalantioxidantcapacityoferythrocytesandtissuesfromcontrolanddiet-inducedhyperlipidemicratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions,asassessedbyJvitrooxidativechallengewithH202.Valuesaremean±S.E.M.Measurementsofacidsolublesulfhydrylgroupdepletion(%GSHdepl)usedfinalconcentrationsofH202of0.1mMforredcells,heartandkidneyand0.25mMforliver.Formalondialdehydeformation(MDA)2.5mMH202wasusedforerythrocytes(RBC).Forthiobarbituricreactivesubstances(TBARS)formation;1.5mMH202wasusedforheart;2.5mMforkidneyand20.0mMforliver.Comparisonsweremadeofstressandchol.withcontrolgroupsandbetweenstress/chol.andchol.groups.*p.<005significantlydifferentfromcorrespondingcontrol.(0ErythrocytesHeartLiverKidney%GSHMDA(nmotl%GSHTBARS%GSHTBARS%GSHTBARSdept.mgRBC)depl.(0D532)depl.(0D532)depi.(0D532)Control66.91385.3653.280.24857.580.77650.060.186±5.05±22.47±5.90±0.037±3.43±0.102±1.91±0.028(n=6)(n=6)(n=5)(n=5)(n=6)(n=6)(n=6)(n=6)Stress64.68300.11*53.040.338*55.911.22*50.170.189±4.77±25.23±4.26±0.031±5.51±0.18±3.46±0.010(n=6)(n=6)(n=5)(n=6)(n=6)(n=6)(n=6)(n=6)Chol.70.74331.9655.100.342*65.530.86152.260.211±6.06±18.95±3.88±0.018±3.92±0.127±5.09±0.021(n=6)(n=6)(n=6)(n=6)(n=6)(n=6)(n=6)(n=6)Stress/chol.73.44334.2656.220.35770.051.1249.820.240±5.48±42.62±3.51±0.019±4.84±0.98±4.17±0.029(n=5)(n=5)(n=7)(n=6)(n=7)(n=6)(n=7)(n=6)-190-3.5. Effects of Stress and Diabetes on Myocardial Functional Recoveryand Antioxidant Status Following lschemialReperfusion InjuryWe have previously demonstrated that chronic stress can modulate diabetes-associated alterations in antioxidant status, including enzymatic, non-enzymatic and afunctional in vitro measure of tissue susceptibility to peroxide challenge. Thesemolecular changes induced by stress are consistent with the long-held clinicalimpression that stress can promote or aggravate the severity of both diabetes and amajor associated complication, ischemic heart disease. The aim of the following twostudies was, therefore, to examine the interplay of chronic stress and diabetes of shortduration (4 weeks), and long duration (12 weeks) on myocardial functional recoveryand antioxidant status following ischemia/reperfusion injury in isolated perfusedLangendorff hearts. At the termination of reperfusion, myocardial antioxidantcomponents were assessed in terms of glutathione reductase activity and levels ofascorbate and glutathione. Functional antioxidant capacity was measured in hearthomogenates by in vitro oxidative challenge with increasing concentrations of hydrogenperoxide with subsequent measurement of decreases in free acid soluble sulfhydrylgroup content (an indirect measure of glutathione), and thiobarbituric-acid reactivesubstances (TBARS) an indirect measure of lipid peroxidation. In addition, timedependent change in sulfhydryl group content was measured in homogenateschallenged with a single concentration of hydrogen peroxide.-191-3.5.1. Functional Recovery and Antioxidant Status Following MyocardialIschemialReperfusion Injury: Effects of Stress and Short-TermDiabetes (4 weeks’ duration)3.5.1.1. Body Weights and Glucose and Lipid StatusBody weights were significantly reduced in both diabetic groups relative tocontrol and in the diabetic stress-exposed relative to the diabetic group (Table 33).Percent weight change through the stress protocol was significantly reduced in thestressor-exposed groups relative to control, and in the diabetic stressor-exposedrelative to the diabetic group.In blood collected from animals anesthetized with halothane, plasma glucose,glycosylated hemoglobin and triglyceride levels were significantly elevated in bothdiabetic groups relative to control (Table 34); however, no significant differences werefound between groups in plasma cholesterol levels. In addition no differences in theseindices were found between diabetic groups.3.5.1.2. Myocardial Functional Indices: ±dP/dt,, Developedpressure, End-Diastolic PressureThere were no significant differences in baseline heart function measured interms of developed pressure and ±dPldt in hearts from normal and diabetic ratsexposed to the stress protocol or control conditions (Table 35). Post-ischemicmyocardial function measured in terms of developed pressure and ±dP/dt weresignificantly reduced relative to baseline measurements in all groups following globalischemia and reperfusion. (Table 36).-192-Percent changes in developed pressure and ±dPldtmax were significantlyimproved in the non-stressed diabetic group relative to control, and significantlyreduced in the diabetic stress-exposed relative to the diabetic group; however, theseeffects were lost after one minute of reperfusion (Figs. 39-41).3.5.1.3. Antioxidant Enzyme Activity (Glutathione Reductase)Post-reperfusion myocardial antioxidant status measured in terms glutathionereductase activity was elevated only in the non-stressed diabetic group alone. Therewere no significant differences in glutathione reductase activity in the non-diabetic ordiabetic stress-exposed groups relative to control (Table 37).3.5.1.4. Levels of Non-Enzymatic Antioxidants: Ascorbate andGlutathionePost-reperfusion myocardial antioxidant status measured in terms of basalglutathione levels were increased in non-stressed and stress-exposed diabetic groupsrelative to control. There were no significant differences in basal glutathione in thestress-exposed group relative to control. The level of ascorbate was elevated in thestress-exposed group relative to control. The level of glutathione was reduced in thediabetic stressor-exposed relative to the diabetic group (Table 37).3.5.1.5. H2Q-lnduced Sulfhydryl Grour DeiletionNo significant differences occurred between groups in concentration dependentperoxide-induced sulfhydryl group depletion (data not shown).-193-3.5.1.6. H20-lnduced Time-Dependent SuiThydryl GroupDepletionTime-dependent sulThydryl group depletion was significantly increased in thestress-exposed group relative to the control group (Fig. 42).3.5.1.7. H20-lnduced Lipid PeroxidationNo significant differences between groups were found in peroxide-inducedTBARS formation (data not shown).Table33.Weightand%changeinweightofcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.ControlStressor-DiabeticDiabetic!stressorexposedexposed(n=7)(n=6)(n=7)(n=7)Finalweight429±11401±7380±14*332±5*t(g) %weightchangea11.8±1.22.5±3.0*10.5±1.91.7±1.5jValuesaremeans±S.E.M.Significantdifferencerelativetocontrol,*anddiabetic, tp<0.01.aRelativetopre-stressweighttakenonday1ofthestressprotocol.Table34.Plasmaglucose,%glycosylatedhemoglobinandlipidlevelsfromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.Control(n=7)12.3±0.63.01±0.040.86±0.041.50±0.12Stressorexposed(n=6)11.8±0.83.19±0.140.99±0.121.47±0.05Diabetic(n=7)30.6±1.8*5.51±0.20*1.87±0.29*2.08±0.16Diabeticlstressorexposed(n=7)28.9±1.3*5.78±0.13*1.99±0.59*1.87±0.15Glucose(mmoIIL)GlycosylatedHb(%)Triglycerides(mmolIL)Cholesterol(mmolIL)Valuesaremean±S.E.M.significantlydifferentfromcontrol.-CD 01Table35.Pre-ischemicfunctionalmeasurementsinheartsfromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.Valuesaremean±S.E.M.Comparisonsmadeofstress,diabeticanddiabeticlstressor-exposedwithcontrolanddiabetic/stressor-exposedwithdiabetic.(0 0)ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=6)(n=7)(n=7)Developed118.1±8.8124.8±4.1118.1±4.9116.9±4.4pressure(mmHg)+dP/dTmax4,952±4175,472±3784,813±2985,050±244(mmHg/sec)dP/dTmax3,657±2534,397±2183,632±1943,793±216(mmHg/sec)Enddiastolic5.7±0.45.8±0.57.0±0.66.3±0.4pressure(mmHg)Table36.Comparisonofbaselineandpost-reperfusionfunctionalindicesinheartsfromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.Valuesaremean±S.E.M.Pairedcomparisonsmadeofstress,diabeticanddiabeticlstressor-exposedatbaseline(20mmstabilization)withthesamegroupfollowing20mmreperfusion.Significantdifferencerelativetobaseline*p<0.01.Co —.1ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=7)(n=6)(n=7)(n=7)DevelopedBaseline118±9125±4118±5117±4pressure(mmHg)Reperfusion77±*85±9*915*787*+dP/dtmaxBaseline4,950±4005,500±4004,800±3005,050±250(mmHg/sec)Reperfusion3,200±500*3,650±55Q*3,600±250*3,250±350*dP/dtmaxBaseline3,650±2504,400±2003,650±2003,800±200(mmHg/sec)Reperfusion2,350±300*2,950±350*2,750±200*2,300±200*EnddiastolicBaseline6±0.46±0.57±0.66±0.4pressure(mmHg)Reperfusion12±49±210±210±2Table37.Post-reperfusionantioxidantstatusinheartsfromcontrolanddiabetic(fourweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.Valuesaremean±S.E.M.GSSG-RD=glutathionereductase;GSH=glutathione.*p<OO1significantlydifferentfromcontrol.tp<O.O1,significantlydifferentfromdiabetic.Co oControlStressor-DiabeticDiabetic!stressorexposedexposed(n=7)(n=6)(n=7)(n=7)GSH1.03±0.021.03±0.041.55±0.08*1.22±0.08*t(nmol/mgwetwt)GSSG-RD0.298±0.0120.293±0.020.350±0.02*0.314±0.02(nmolNADPHmin1mg1wetwt)Ascorbate0.22±0.040.44±0.11*0.22±0.030.17±0.03(p.mol/gtissue)-199-Fig. 39. Post-lschemic percent developed pressure in hearts from control anddiabetic (four weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions.Values are mean ± S.E.M. n = 7 for Control, Diabetic, Diabetic Stressorexposed and n = 6 for Stress-exposed. Comparisons made of Stress, Diabetic andDiabetic/Stress with control and Diabetic/Stress with Diabetic. Significant differencerelative to control, or *diabetic, p < 0.01.CON-STR——-DIAB——-DIAB/STR10009080I0—————————Post-IschemicTime(mm)-201 -Fig. 40. Post-ischemic percent +dPIdt, in hearts from control and diabetic (fourweeks’ duration) male Wistar rats exposed to the stress protocol (stressorexposed) or control conditions.Values are mean ± S.E.M. n = 7 for Control, Diabetic, Diabetic Stressorexposed and n = 6 for Stress-exposed. Comparisons made of Stress, Diabetic andDiabetic/Stress with control and Diabetic/Stress with Diabetic. Significant differencerelative to diabetic, p <0.01.CON-STR——-DIAB——-DIAB/STR10090jjI0Post-IschemicTime(mm)-203-Fig. 41. Post-Ischemic Percent -dP!dt in hearts from control and diabetic (fourweeks’ duration) male Wistar rats exposed to the stress protocol (stressorexposed) or control conditions.Values are mean ± S.E.M. n = 7 for Control, Diabetic, Diabetic Stressorexposed and n = 6 for Stress-exposed. Comparisons made of Stress, Diabetic andDiabetic/Stress with control and Diabetic/Stress with Diabetic. Significant differencerelative to control, or *diabetic p < 0.01.CON-STRDIAB—-DIAB/STR100-x90-asET480-•.0—.———----——oW——————I••r.“..————————I.—‘T_—I_———————70-Ja)-C)—F1%U)—60-t————————**50II012345Post-IschemicTime(mm)-205-Fig. 42. Post-reperIusion time-dependent peroxide-induced glutathione depletionin hearts from control and diabetic (four weeks’ duration) male Wistar ratsexposed to the stress protocol (stressor-exposed) or control conditions.Concentration of H20 used is 0.015 mM. Values are mean ± S.E.M. n = 7 forControl, Diabetic, Diabetic/Stressor-exposed and n = 6 for Stress-exposed.Comparisons made of Stress, Diabetic and Diabetic/Stress with control andDiabetic/Stress with Diabetic. Significant difference relative to control, <0.01.CON-STRDIAB——-DIAB/STR60*50-*———0—————U)40o(N•U%%J*30ci)02010IIIIIIIIII024681012141618202224262830IncubationTime(mm)-207-a 5.2. Functional Recovery and Antioxidant Status Following Myocardiallschemia/Reperfusion Injury: Effects of Stress and Long-TermDiabetes (12 Weeks’ Duration)We have previously demonstrated that chronic stress can modulate diabetes-associated alterations in antioxidant status, including enzymatic, non-enzymatic and afunctional in vitro measure of tissue susceptibility to peroxide challenge in long-termdiabetes. In addition, following a short period of ischemia, we have also shown that inchronically stressed four week diabetic rats, post-ischemic myocardial functionalrecovery during the early reperfusion phase is impaired to an extent that parallels areduction in myocardial antioxidant capacity. The aim of the present study was,therefore, to examine the interplay of chronic stress and long-term diabetes onmyocardial functional recovery and antioxidant status following ischemialreperfusioninjury.3.5.2.1. Body Weicihts and Glucose and Lipid StatusThe final weights and percent weight change through the stress protocol weresignificantly reduced in stressor-exposed and diabetic groups relative to control(Table 38). In blood collected from animals anesthetized with halothane, plasmaglucose and glycosylated hemoglobin were significantly elevated in both diabeticgroups (Table 39). Plasma triglycerides were significantly elevated in both diabeticgroups relative to control. In the diabetic, stress-exposed group, plasma triglycerideswere significantly reduced relative to the diabetic group alone (Table 39). Nosignificant differences occurred between groups in plasma cholesterol levels.-208-3.5.2.2. Functional Indices: ±dP/dt. Developed Pressure. End-Diastolic PressureBaseline myocardial function measured in terms of developed pressure, and±dP/dtmax were significantly reduced in both diabetic groups relative to control. Therewere no differences in myocardial function in the stress-exposed group relative tocontrol or in the diabetic, stress-exposed group relative to the diabetic group(Table 40). Following global ischemia and reperfusion, developed pressure, and ±dP/dtmax were significantly reduced relative to baseline measurements in all groups(Table 41). Percent changes in developed pressure and ±dP/dtmax were significantlyimproved in both diabetic groups relative to control; however, this effect was lost bythree minutes of reperfusion (Figs. 43-45).3.5.2.3. Antioxidant Enzyme Activity (Glutathione Reductase)Giutathione reductase activity was increased in both diabetic groups relative tocontrol. There were no significant differences in glutathione reductase activity in thestress-exposed group relative to control or in the diabetic stress-exposed group relativeto the diabetic group (Table 42).3.5.2.4. Levels of Non-Enzymatic Antioxidants: Ascorbate andGlutathioneFollowing the ischemia and reperfusion periods, basal glutathione levels wereincreased in both diabetic groups relative to control. There were no significantdifferences in basal glutathione levels in the stress-exposed group relative to control orin the diabetic stress-exposed group relative to the diabetic group. No significantdifferences in ascorbate level occurred between groups (Table 42).-209-3.5.2.5. H,02-lnduced Sulfhydrvl Group DepletionFollowing the ischemia and reperfusion periods, no significant differences inconcentration dependent peroxide-induced sulfhydryl group depletion occurredbetween groups (data not shown).3.5.2.6. H20-lnduced Time-Dependent Sulthydrvl GroupDepletionFollowing the ischemia and reperfusion periods, no significant differences intime dependent peroxide-induced sulfhydryl group depletion occurred between groups.3.5.2.7. H20-lnduced Lipid PeroxidationFollowing the ischemia and reperfusion periods, peroxide-induced TBARSformation was significantly reduced in the non-stressed diabetic group relative tocontrol (Fig. 46). TBARS formation was significantly elevated in the diabetic stressexposed group relative the the diabetic group alone, but not significantly different fromcontrol at higher peroxide concentrations.Table38.Weightsofcontrolanddiabetic(twelveweeks’duration)Wistarratsfollowingexposuretothestressprotocol(stressor-exposed)orcontrolconditionsand%changeinweight.ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=7)(n=5)Finalweight(g)565±16500±14*393±8*376*±11%changea4.99±1.03-9.84±0.78*5.04±0.74-6.27±I.94*tValuesaremean±S.E.M.Significantdifferencerelativetocontrol,*anddiabetictp<0.01.aRelativetopre-stressweighttakenonday1’ofthestressprotocol.Table39.Plasmaglucose,%glycosylatedhemoglobinandlipidlevelsfromcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed)orcontrolconditions.- -ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=7)(n=5)Glucose12.4±1.610.7±0.4335.8±2.8*34.4±2.7*(mmol/L)GlycosylatedHb2.87±0.112.91±0.165.68±0.19*6.11±0.14*(%)Triglycerides0.96±0.110.58±0.144.76±0.59*2.09±0.41*(mmolIL)Cholesterol1.96±0.181.39±0.112.3±0.151.91±0.21(mmolIL)Valuesaremean±S.E.M.*p.<001significantlydifferentfromcontrol.Table40.Pre-ischemicfunctionalmeasurementsinheartsfromcontrolanddiabetic(twelveweeks’duration)maleWistar ratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.r%)ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=7)(n=5)Developedpressure117.5±5.1126.0±5.095.1±5.1*93.1±7.1*(mmHg)+dP/dTm4,879±2255,423±1694,117±232*3,981±341*(mmHglsec)dP/dTmax3,822±1464,270±1072,741±190*2,788±242*(mmHg/sec)Enddiastolicpressure10.2±2.36.7±1.78.8±1.28.4±1.3(mmHg)Valuesaremean±S.E.M.*p<0.05.Significantdifferencerelativetocontrol.Table41.Comparisonofbaselineandpost-reperfusionfunctionalindicesinheartsfromcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressoraexposed)orcontrolconditions.ControlStressor-DiabeticDiabetic!stressorexposedexposed(n=6)(n=6)(n=7)(n=5)DevelopedBaseline117±5126±595±593±7pressure(mmHg)Reperfusion64±7*85±10*67±5*69±7*+dPIdtmaxBaseline4,900±2005,400±1504,100±2504,000±350(mmHg/see)Reperfusion2,650±200*3,500±400*2,900±200*2,850±350*dP/dtmaxBaseline3,800±1504,250±1002,750±2002,800±250(mmHg/see)Reperfusion2,200±200*2,750±350*1,850±200*1,950±200*EnddiastolicBaseline10±27±29±18±Ipressure(mmHg)Reperfusion24±7*227*164*11±2*Valuesaremean±S.E.M.Pairedcomparisonsmadeofstress,diabeticanddiabetic/stressor-exposedatbaseline(20mmstabilization)withthesamegroupfollowing20mmreperfusion.Significantdifferencerelativetobaseline*p<0.01.L3 C)Table42.Post-reperfusionantioxidantstatusinheartsfromcontrolanddiabetic(twelveweeks’duration)maleWistarratsexposedtothestressprotocol(stressor-exposed) orcontrolconditions.Valuesaremean±S.E.M.GSSG-RD=glutathionereductase;GSH=glutathione.*p<OO1significantlydifferentfromcontrol.1\)ControlStressor-DiabeticDiabeticlstressorexposedexposed(n=6)(n=6)(n=7)(n=5)GSH0.73±0.070.75±0.060.99±0.120.94±0.06(nmol/mgwetwt)VGSSG-RD0.312±0.0220.297±0.020.408±0.02*0.41.±0.03*(nmolNADPHmin1mg1wetwt)Ascorbate53.5±6.745.4±4.953.1±9.441.3±5.8(ig/gtissue)-215-Fig. 43. Post-lschemic percent developed pressure in hearts from control anddiabetic (twelve weeks’ duration) Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions.Values are mean ± S.E.M. n = 6 for Control, Stress, n = 7 for Diabetic, n = 5 forDiabetic/Stress. Comparisons made of Stress, Diabetic and Diabetic/Stress withcontrol and Diabetic/Stress with Diabetic. Significant difference relative to control,p<0.01.CON-STR——--DIAB——-DIAB/STR100*90TZ,,r—--.--_————=3“---“Post-IschemicTime(mm)-217-Fig. 44. Post-ischemic percent +dP!dt, in hearts from control and diabetic(twelve weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions.Values are mean ± S.E.M. n = 6 for Control, Stress, n = 7 for Diabetic, n = 5 forDiabetic/Stress. Comparisons made of Stress, Diabetic and Diabetic/Stress withcontrol and Diabetic/Stress with Diabetic. Significant difference relative to control,<0.01.CON--STRDIAB-DIAB/STR10090f——I/.——.—.—.—.L80//_//÷/II.LLLPOstIschemicTime(mm)-219-Fig. 45. Post-lschemic Percent -dP!dt in hearts from control and diabetic(twelve weeks’ duration) male Wistar rats exposed to the stress protocol(stressor-exposed) or control conditions.Values are mean ± S.E.M. n = 6 for Control, Stress, n = 7 for Diabetic, n = 5 forDiabetic/Stress. Comparisons made of Stress, Diabetic and Diabetic/Stress withcontrol and Diabetic/Stress with Diabetic. Significant difference relative to control,p<0.01.CONSTR——-DIAB——-DIAB/STR10090**80IIPost-IschemicTime(mm)-221 -Fig. 46. Post-reperfusion peroxide-induced TBARS formation in hearts fromcontrol and diabetic (twelve weeks’ duration) male Wistar rats exposed to thestress protocol (stressor-exposed) or control conditions.Hydrogen peroxide concentration 1.0 mmol. Values are mean ± S.E.M. n = 6 forControl, Stress, n = 7 for Diabetic, n = 5 for Diabetic! Stress. Comparisons made ofStress, Diabetic. and Diabetic/Stress with control and Diabetic/Stress with Diabetic.Significant difference relative to control, < 0.01, or diabetic tp < 0.01.Absorbanceat532nmppppp000010010C) 0 -I 0 Cr, CD Cl) 0 CD 0CD®0 0.L3-223-4. DISCUSSION4.1. Effects of Sympathectomy on Plasma Catecholamine Levels andAntioxidant Status4.1.1. Effects of 6-OH and AdrenalectomyThe finding that adrenalectomy and 6-OH treatment resulted in reduced plasmalevels of adrenaline and noradrenaline, respectively, while a combination of treatmentsled to a reduction in the levels of both catecholamines, made it possible to compare thedifferential effects of reduced levels of adrenaline and noradrenaline on antioxidantstatus individually, and in combination. Treatment with 6-OH is known to induce amarked and long-lasting depletion of noradrenaline in various sympatheticallyinnervated organs,142’217 including heart,28°kidney305 and lung,29°through destruction ofperipheral adrenergic nerve endings.28° On the other hand, the adrenal gland appearsto be resistant to its direct actions.1 Tranzer and Thoenen28° have demonstrated thatseveral days following treatment with 6-OH, although adrenergic nerve endings ofsympathetically innervated organs were in the process of degeneration, thesurrounding Schwann’s cells, the smooth muscle cells and the cholinergic nerveendings of various tissues adjacent to the degenerating adrenergic nerve terminals allappeared ultrastructurally normal. Therefore, the effects of 6-OH on antioxidant statusare probably directly attributable to a reduction in the release of noradrenaline at the-224-tissue level or to a reduction in the catecholamine content of tissues rather than toalterations in adrenal catecholamines or a non-specific toxic effect on tissues unrelatedto the effect on adrenergic nerve terminals.The antioxidant changes found in adrenalectomized groups may, in part, be aresult of reduced levels of corticosterone. This is suggested by the finding thatmetyrapone-induced suppression of corticosterone production delays lung antioxidantenzyme maturation in neonatal rats.2 However, a direct effect of alteredcorticosterone levels on tissue antioxidant status in adult animals has, to ourknowledge, not been demonstrated. Furthermore, a study of the potential antioxidantproperties of steroids using the free radical initiator 2,2’-azobis(2-amindino-propane)and a phycoerythrin fluorescence-based assay for peroxy radicals concluded thatcortisol and corticosterone showed no antioxidant effects and in fact had very mild(probably insignificant) pro-oxidant properties.182 However, the well known effects ofstress-induced release of glucocorticoids in stimulating catecholamine synthesis in theadrenal medulla21’15°’304 may indirectly act to alter antioxidant capacity. On the otherhand, catecholamines can undergo enzyme or metal-catalysed oxidation with resultingproduction of reactive-oxygen derived substance121’218 which have been implicated incatecholamine-induced myocardial necrosis.2 While these findings might suggest amajor role for catecholamine-induced alterations of antioxidant components, furtherexperiments will be necessary to determine the relative contributions of catecholaminesand corticosterone to these changes.The majority of effects on antioxidant status following sympathectomy wereproduced by adrenalectomy rather than 6-OH treatment and thus the changes appear-225-to be primarily due to alterations in levels of adrenaline (and possibly corticosterone)as opposed to noradrenaline. This is exemplified by the increased glutathionereductase activities and reduced peroxide-induced TBARS formation in liver and lungfollowing adrenalectomy alone but not following 6-OH treatment alone. In addition,most of the antioxidant alterations following adrenalectomy were not further changed bysubsequent 6-OH treatment, suggesting that noradrenaline does not potentiate theeffects of adrenaline in this regard. Exceptions to this generalization occurred in lungglutathione peroxidase activity and erythrocyte glutathione levels, both of which wereincreased only following the combination of adrenalectomy and 6-OH treatment.The finding that sympathectomy reduced the susceptibility of several tissues(liver, kidney and lung) to peroxide-induced lipid peroxidation is interesting in light ofthe proposal by Singal et al2 that catecholamine-induced tissue damage mightinvolve an increase in lipid peroxidation. The finding that pretreatment with dietarytocopherol can protect against isoproterenol-induced ultrastructural damage in ratmyocardium2is consistent with the pro-oxidant properties of catecholamines. It hasalso been reported that severe emotional stress induced by pain, associated with anexcessive release of catecholamines, resulted in increased peroxidation of myocardiallipids.170 The mechanism of reduced peroxide-induced TBARS formation in our studiesmay, in part, involve an improvement in glutathione status found in the lung and liver asshown by increases in glutathione reductase activity (lung and liver) and in glutathionelevels (liver); however, since the reduction in kidney lipid peroxide formation was notaccompanied by changes in these glutathione-related antioxidant components,alterations in the levels of other non-enzymatic antioxidants, such as tocopherol, may-226-be involved. In this regard, the relative insensitivity of heart tissue fromsympathectomized rats to changes in peroxide-induced TBARS formation incomparison to lung, liver and kidney, is interesting in light of the effects of high levels ofcatecholamines on the heart discussed previously. We have shown levels oftocopherol to be significantly higher in heart relative to kidney and liver.276 In addition,heart homogenates from control rats in the present study were less susceptible toperoxide-induced lipid peroxidation relative to kidney and liver. It follows, therefore,that the elevated tocopherol levels in the heart might reduce its sensitivity to peroxideinduced lipid peroxidation when catecholamine levels are low and that this protectionmay be lost when catecholamine levels are high, such as in severe emotional stress.17°On the basis of TBARS formation alone, it might have been concluded thatfunctional antioxidant capacity of the myocardium is unaltered by adrenalectomy.However, an independent index of antioxidant status, namely susceptibility to peroxideinduced sulfhydryl group depletion, suggested a decrease following adrenalecomy.This increased susceptibility to peroxide-induced glutathione depletion occurred in theabsence of a reduction in other components associated with glutathione status,including glutathione peroxidase, glutathione reductase activities and glutathionelevels. Our previous studies of erythrocyte alterations in clinical and experimental275diabetes have also revealed a discrepency between changes in susceptibility toperoxide-induced TBARS formation (increased) and gI utathione depletion (decreased).The diabetes-related decrease in glutathione depletion was shown to result fromhyperglycemia-induced stimulation of the hexose monophosphate shunt, therebyelevating intracellular availibility of NADPH which may be limiting to the glutathione-227-reductase system under conditions of in vitro oxidative stress. If catecholamines havean effect on the regulation of NADPH producing enzymes such as glucose-6-phosphatedehydrogenase in the heart, the reduced levels of catecholamines followingsympathectomy might result in decreased levels of available NADPH and thus lead toelevated susceptibility to peroxide-induced glutathione depletion.The elevation of heart glutathione reductase activity following sympathectomysuggests a role for both adrenaline and/or corticosterone and noradrenaline inmodulating the activity of this enzyme. This, in turn, could have implications underconditions associated with increased oxidative stress, including ischemia/reperfusioninjury,80 contributing to an impairment in antioxidant capacity. Chronic reduction incatecholamine levels might, therefore, be expected to alter recovery fromischemia/reperfusion injury; however, recent experiments have shown that treatment ofrabbits with 6-OH prior to coronary artery ligation-induced ischemia was not associatedwith a reduction in infarct size.1 In light of the present study, these results could beattributed, in part, to the persistence of circulating levels of adrenaline, which are notreduced by 6-OH treatment and which could decrease the levels of some endogenousantioxidant components. In a study by Botting et al., it was shown that thecombination of adrenomedullectomy and 6-OH treatment accentuated the adverseeffects of coronary ligation in rats; however, the authors suggested that the deleteriouseffects of sympathectomy might be a result of reduced blood pressure induced by thetreatment.-228-4.1.2. Effects of Reserpine and AdrenalectomyThe reduction of plasma adrenaline and noradrenaline levels inadrenalectomized animals treated with reserpine or 6-OH were comparable. Similar tothe effects of 6-OH alone, reserpine treatment in non-adrenalectomized animalsresulted in an approximately 50% reduction of plasma noradrenaline levels and minimalchanges in adrenaline levels. While the effects of 6-OH and reserpine on plasmalevels of catecholamines were similar, the mechanism of action of these two agentsdiffers, with 6-OH treatment resulting in destruction of adrenergic nerve terminals andreserpine leading to depletion of catecholamines from adrenergic nerve terminals.Similar to 6-OH, reserpine treatment results in profound reduction of catecholaminelevels in various tissues including the heart.37 It has been shown that relatively highdoses of reserpine causing sedation can result in elevated plasma levels ofcorticosterone; however, the dose and treatment schedule used in the present studywere shown not to increase adrenal gland weight or basal corticosterone levels.181Thus, some comparisons can be made regarding the effects of sympathectomy usingeach of these treatments.The suggestion that sympathectomy-induced changes in antioxidant status areprimarily due to adrenaline (and possibly corticosterone) is supported by the findingsparallel to the 6-OH study of reduced peroxide induced TBARS formation in liver andlung following adrenalectomy alone or in combination with reserpine but not withreserpine treatment alone. Furthermore, TBARS formation was similarly unaffectedfollowing adrenalectomy and/or reserpine treatment, while sulfhydryl group depletionwas increased. Thus, in general the observed effects on antioxidant status are-229-probably a result of sympathectomy rather than non-specific effects of the drugs used.Sympathectomy following adrenalectomy and 6-OH treatment resulted in a number oftissue changes in glutathione reductase activity which, although elevated, did notconsistently reach statistical significance when 6-OH was replaced with reserpine. Onthe other hand, sympathectomy using reserpine resulted in mimimal changes in otherantioxidant components, paralleling the findings with 6-OH.In summary, sympathectomy produced by adrenalectomy and/or 6-OH orreserpine treatment altered antioxidant status in a number of tissues in the rat.Measures of antioxidant status which appeared particularly sensitive tosympathectomy-induced changes included glutathione reductase activity (increased)and susceptibility to peroxide-induced lipid peroxidation (reduced) in a number oftissues. Differences between surgical and chemical sympathectomy on antioxidantcomponents are suggestive of differential actions of adrenaline and noradrenaline ontissue antioxidant status which may have important implications under conditionsassociated with elevations in levels of these catecholamines, including chronic stressand myocardial infarction.18’77”24.2. Effects of Acute and Chronic Stress on Plasma Levels ofCorticosterone, Catecholamines and Glucose as Measured UsingIndwelling Catheters in Control and Diabetic RatsThe finding that plasma levels of corticosterone, adrenaline and noradrenaline innonstressed control rats remained stable throughout the sampling periods suggestedthat the blood sampling procedure did not disturb the animals. The sustainedelevations in corticosterone, adrenaline and noradrenaline levels found during the first-230-exposure to restraint stress in non-diabetic rats verified that the stressor caused asubstantive physiological stress response. It has been well established that bloodlevels of adrenaline, noradrenaline and corticosterone provide a biochemicalassessment of the functional activity of the sympathetic-adrenal and adrenocorticalsystems and these can serve as a sensitive indicator of stressor intensity.105’9°’924.2.1. Effects of Acute Stress in Non-Diabetic RatsIt has been suggested that the neuronal and adrenomedullary branches of thesympathoadrenal system show differential responses to physical and psychologicalcomponents of stressor exposure, with elevations in plasma noradrenaline beingrelated more to the physical components of the response (i.e., skeletal muscle activityincluding struggling, shivering and grooming behaviors) and elevations in plasmaadrenaline more related to the psychological components.50’1 Restraint stress hassome physical component, but is primarily considered a psychological stressor. Therestraint stressors used in the present study were designed to minimize movement; thegreater change in adrenaline relative to noradrenaline levels observed during the firstexposure to stress is consistent with the above suggestion. However, the stressinduced elevation in noradrenaline levels was not unexpected, considering that inanimals exposed to acute stress, up to 45% of noradrenaline is derived from theadrenal medulla. Our finding of persistent hyperglycemia during acute stress in nondiabetic rats in association with elevated catecholamine levels is in agreement withprevious reports of elevated plasma glucose levels in acute restraint or foot shock1stress.-231 -4.2.2. Effects of Repeated Stress in Non-Diabetic RatsExposure to stress on days 7 and 14 of the chronic stress protocol continued toresult in a significant stress response as indicated by reduced weight gain, ageneralized indicator of stress,1 and maintenance of elevated plasma corticosteronelevels at all sampling periods. Thus, under non-pathological conditions, the model ofchronic variable stress employed in this study did not lead to adaptation of theadrenocortical response. On the other hand, some degree of catecholamineadaptation was apparent, as shown by fewer statistically significant increases atindividual sampling periods relative to non-stressed controls. However, comparison ofthe stress responses assessed through AUC analysis of the data across sampling daysfailed to show any significant reduction in the overall catecholamine response over thedays of exposure to stress. Thus, results from this study of chronic-intermittent stresssuggest an independence of the various components of the stress response toadaptation. The stress-induced changes in glucose appear to parallel changes incatecholamine levels throughout the study. For example, while the stress-inducedelevation in plasma glucose levels persisted on day 7, by day 14 plasma glucose levelswere elevated only following 60 mm stress and were no longer significantly elevatedrelative to samples from stressor-exposed animals on -days I and 7.Differences in the pattern of the noradrenaline, adrenaline and corticosteronestress responses have been reported previously. For example, following short-termchronic stress (exposure to 20 pulses of white noise each of 4 mm duration delivered atrandom times in a 24 hour period), noradrenaline and corticosterone levels were foundto be comparable to those from rats exposed to the first pulse while adrenaline levels-232-were significantly reduced. While results from other studies of chronic stressemploying a repetitive stress regimen have shown adaptation of both thecorticosterone124’231 and catecholamine stress responses,’1 it has generally beenassumed that unpredictibility in the nature and timing of stress would diminish thetendency towards adaptation.222 However, in a study comparing 26 days of 30 mmexposures to chronic predictable versus unpredictable stress, Konarska et al.1 foundadaptation of the sympatho-adrenal medullary responses occurred with both stressregimens, although weight gain was reduced to a greater extent following the variablestress protocol. The factors involved in adaptation or sensitization to the stressresponse are highly complex and depend on stressor intensity, duration, frequency andthe time between exposures and appear to be influenced by numerous other factorsincluding prior exposure to stress, age, gender and species.5°’215’312 In addition, thepatterns of adaptation seem to differ depending on the particular biochemical marker ofstress examined.4.2.3. Effects of Diabetes on Levels of Stress HormonesSeveral lines of evidence suggest that diabetes is a sustained stimulus to theadrenocortical and sympathetic axes in rats, although this has been difficult todocument consistently under basal (resting) conditions. In consideration of thepossibility that stress may have different consequences in insulin-dependent (IDDM)versus insulin independent (NIDDM) diabetes, it should be mentioned thatstreptozotocin-induced diabetes, the type used in these studies, is classified as a typeof IDDM diabetes in which animals are under relatively poor insulin control. It hasbeen previously shown that while insulin levels in streptozotocin-diabetic rats are-233-severely reduced, they are not entirely absent and the animals can survive withoutinsulin therapy.While some authors have reported increased basal levels of corticosterone instreptozotocin-induced (rats) and genetic (ob/ob mice) diabetes,2 others havereported decreased thymus weights, increased urinary excretion of corticosterone andincreased corticosterone levels when averaged over the light hours of the day in theabsence of elevated basal levels.2” In addition, abnormal corticoid circadian rhythmshave been observed in human diabetics147 and in rats, as shown by elevated plasmalevels observed during the light hours of the day.2°4 Further, Bellush et al. havereported elevated basal urinary noradrenaline levels in streptozotocin-diabetic rats.16In the present study, although there were no significant elevations in resting levels ofthe three stress hormones in non-stressed diabetic animals following 14, 21 and 28days of diabetes, the values of corticosterone reported in all groups were elevatedabove usually reported basal levels due to the presence of the catheter and thereforeare not representative of absolute basal levels.4.2.4. Effects of Acute Stress in Diabetic RatsIn diabetic rats the levels of corticosterone, adrenaline and noradrenalinefollowing the first exposure to stress were elevated relative to non-stressed diabeticrats but the response differed from that of the non-diabetic stressor-exposed group.For example, the levels of corticosterone in diabetic stressor-exposed rats weresignificantly elevated above those of non-diabetic rats exposed to stress. Conversely,the levels of adrenaline were increased to a lesser degree in the diabetic relative to thenon-diabetic group exposed to stress, as shown using AUC analysis of the data,-234-although the levels of noradrenaline were equivalent in both groups of stressedanimals. The finding of an exaggerated corticosterone response in the absenca ofelevated resting levels concurrent with a blunted adrenaline response has, to ourknowledge, not been previously reported and indicates that the response to acutestress is altered by diabetes. Other investigators have also noted exaggeratedelevations in plasma corticosterone levels in diabetic relative to non-diabetic ratsfollowing an acute episode of intraperitoneal cold water injections or restraint stress.7Further, in diabetic rats subjected to 24 hrs cold (4°C) stress, total urinary excretion ofboth noradrenaline and adrenaline was elevated relative to non-stressed diabeticcontrols, whereas noradrenaline but not adrenaline was elevated relative to non-diabetic rats exposed to the same treatment.16 The relative increase in noradrenalinelevels in the diabetic stressor-exposed group may be a specific thermoregulatoryresponse to the physical stress of extreme cold and thus may not be directlycomparable to hormone responses elicited in our model of stress. In a study of acutefootshock stress in diabetic rats, Lee et al. divided diabetic rats into reactive and nonreactive responders based on basal adrenaline levels which were either elevated fromor similar to those of control animals.1 While all groups exposed to stress showedsimilar elevations in post-stress levels of noradrenaline, reactive responders showedlevels of adrenaline which exceeded those of non-diabetic animals exposed to stress.While the existence of a subgroup of diabetic rats with elevated sensitivity to stresscould in theory explain some differences among studies of diabetes and stress, thediabetic animals in our study exhibited a very narrow range of resting plasma levels of-235-adrenaline which would seem to preclude the division of animals into reactive and nonreactive responders.Results from the present study failed to show any evidence of a stress-inducedchange in the extent of hyperglycemia found in diabetic animals. It has beensuggested that blood glucose levels in subjects with diabetes exposed to acute stresswould be elevated since little or no endogenous insulin is secreted to offset thecatecholamine-induced increases in blood glucose.4° The plasma glucose status maybe a factor in the response of diabetic animals to stress. For example, while our studyemployed uncontrolled diabetic animals in which marked hyperglycemia was presentprior to stress exposure, other studies demonstrating stress-induced elevations inplasma glucose have used animals under some degree of glycemic control with insulininjections.148’293 The stress-induced elevations in glucose levels under such conditionsmay be due to the anti-insulin effects exhibited by corticoids.6”’314.2.5. Effects of Repeated Stress in Diabetic RatsA temporal change in the stress response of diabetic rats was observed on days7 and 14 of the stress protocol but this was not apparent in the non-diabetic groupexposed to stress. Thus, exposure of diabetic rats to the stressor on day 7 resulted inlevels of both catecholamines and corticosterone that were not significantly differentfrom values found in the non-stressed diabetic group, indicating a reduced stressresponse. Following 14 days, however, the stress response appeared more similar tothat found on the first exposure to stress, with levels of corticosterone elevated at allsampling periods and levels of adrenaline and noradrenaline elevated at severalsampling points. Interestingly, these alterations in stress hormone levels, as in acute-236-stress, did not result in changes in diabetes-associated hyperglycemia on days 7 or 14.Thus, while non-diabetic rats showed a persistent elevation in corticosterone valuesand a diminishing catecholamine response with an increasing number of exposures tostress, diabetic rats showed a biphasic stress response over the 14 day stress period.It is currently not known if these alterations in hormonal responses of the diabeticanimals to chronic stress are characteristic of the particular model of stress used in thisstudy or if this response can be generalized to other types of chronic stress. To ourknowledge, there are no other studies in which stress hormones have been measuredthroughout a chronic stress regime in diabetic animals. Further study of the hormonalresponses of diabetic animals to different chronic stressor regimes would certainlyseem warranted. In addition, our results show an increasing corticosterone andcatecholamine stress response between 7 and 14 days of stress exposure, raisingquestions as to the consequences of longer-term stress in diabetic animals.4.2.6. Effects of Repeated Stress on Levels of the Non-EnzymaticAntioxidants Tocopherol and AscorbateAs will be discussed in a later section, we found a significant number ofalterations in diabetes-associated antioxidant status following exposure to the stressprotocol used in this study. This work has been extended to include measurement ofthe non-enzymatic antioxidants tocopherol and ascorbate. Unlike the foregoingchanges, which involve alterations in enzymatic antioxidant components and in vitrosusceptibility of tissues to peroxide-induced glutathione depletion and lipidperoxidation, there were no corresponding changes in the levels of plasma and tissueascorbate and tocopherol in animals exposed to chronic stress despite diabetes--237-associated alterations in tissue and plasma levels of these antioxidants in non-stressedanimals.In summary, we have have examined the effects of acute and chronicintermittent variable restraint stress on hormonal, glycemic and antioxidant indices innormal and diabetic rats. Acutely, the restraint stress protocol produced a substantialstress response in both diabetic and non-diabetic rats which showed differentialadrenal and glycemic responses. Continued exposure to stress in normal rats led tosome adaptation of the sympathetic and adrenomedullary, but not adrenocorticalresponses. In contrast, diabetic rats exhibited a biphasic stress response in whichsome adaptation was observed after 7 days but responses more similar to the first dayof stress were seen on day 14 of the stress protocol. These results suggested thatdiabetic rats show differential reactivity to both acute and chronic stress which couldhave consequences in the development of long-term complications associated withdiabetes.4.3. Antioxidant Status in Diabetes of Short- (4 week) and Long-(12 week) Duration: Effects of Chronic-Intermittent StressThe aim of this work was to investigate the independent and synergistic effectsof short-term (4 week) and long-term (12 week) diabetes and chronic intermittent stresson antioxidant status in a number of tissues in rats. Tissues were chosen for analysisbecause of their association with complications arising from diabetes (heart, kidrey),exposure to sustained high oxygen tensions (lung) or as the major site of oxidativemetabolism (liver). Erythrocytes. were analysed to assess their potential clinical use asearly predictors of antioxidant changes in tissues.-238-4.3.1. Effects of Short-Term (4 Week) Diabetes on Antioxidant StatusThe diabetic state was found to be associated with complex alterations in organand blood antioxidant status. For example, basal glutathione levels were unaltered inerythrocytes and kidney, increased in heart and decreased in liver. Glutathionereductase activity was unaltered in the lung and kidney, and increased in heart andliver. The direction of some alterations was consistent with other published reports,notably the elevated heart glutathione reductase activity299 and reductions in livercatalase299 and glutathione peroxidase2activity, as well as basal glutathione,155’299andcatalase activity of kidney;2 however, there were also inconsistencies in otherantioxidant alterations, including liver Cu,Zn-superoxide dismutase activity,4211’299which may relate to differences in duration of diabetes, rat strain or gender.The functional consequences of diabetes-associated antioxidant changes interms of peroxide-induced glutathione depletion and TBARS formation have notpreviously been examined. In the present study, erythrocytes and tissue homogenatesboth showed alterations in susceptibility to in vitro peroxide challenge. In general, mosttissues from diabetic rats, including erythrocytes, heart and liver but not kidney,exhibited a reduced susceptibility to hydrogen peroxide-induced sulfhydryl groupdepletion. Previous work in our laboratory has demonstrated reduced susceptibility oferythrocytes from twelve week alloxan or streptozotocin diabetic rats to hydrogenperoxide-induced sulthydryl group depletion.2 As mentioned previously, we haveshown that this reduced susceptibility is likely related to stimulation of the hexosemonophosphate shunt by hyperglycemia, thereby elevating intracellular availability ofNADPH which may be limiting to the glutathione reductase system under conditions of-239-in vitro oxidative stress. The same mechanism may also be applicable to the tissuesshowing decreased susceptibility to peroxide-induced glutathione depletion. However,the reason for the unaltered susceptibility of kidney to sulthydryl group depletion isunknown. Conversely, most tissues showed an increased susceptibility to hydrogenperoxide-induced lipid peroxidation including erythrocytes, liver, kidney and lung. Instriking contrast, TBARS formation was reduced in myocardial homogenates. Thisfinding is in agreement with a previously published finding, in six week alloxan andstreptozotocin diabetic rats, of decreased myocardial susceptibility to lipid peroxidationinduced by an in vitroFe2/ascorbate free radical generating system.197 The decreasein myocardial susceptibility to lipid peroxidation may be related to the increases(possibly compensatory) in glutathione levels and glutathione reductase activity, toalterations in membrane lipid fatty acid composition’ known to occur in diabeticrats207 or to alterations in non-enzymatic antioxidants. Diabetes-related alterations inlevels of tocopherol14’”8and dihydroascorbic acid1’252 have previously beenreported. Consistent with the finding of reduced susceptibility of the diabetic heart toperoxide-induced lipid peroxidation is our observation of increased tocopherol levels inthe heart.2 The increased tocopherol levels are probably not caused byhyperglycemia as control of hyperglycemia with insulin did not reduce tocopherol levelsin diabetic rats.’18 Based on these findings, it has been suggested that the elevation ofaipha-tocopherol may be a defence mechanism rather than a result of uncontrolledlipolysis associated with the insulin-deficient state which could result in mobilization oftocopherol from adipose tissue.”8-240-In some instances, increased susceptibility to lipid peroxidation, as found inother tissues, can be rationalized in terms of impaired enzyme activities. For example,the increased hepatic lipid peroxidation in diabetic rats could be related to decreases inlevels of glutathione as well as in the activities of catalase and glutathione peroxidase.In the kidney, reduced catalase and in the lung reduced glutathione peroxidaseactivities (in the absence of increases in other enzyme activities) may have contributedto the increased susceptibility of these tissues to lipid peroxidation. However, it is notknown to what extent antioxidant enzymes are active in the in vitro peroxidizingsystems.4.3.2. Effects of Long-Term (12 Week) Diabetes on Antioxidant StatusOur finding that long-term diabetes is associated with a complex pattern ofalterations in tissue antioxidant components is consistent with previous reports from ourlaboratory.275’°° Some diabetes-associated alterations do not appear to beinfluenced by the duration of diabetes. In the heart, for example, glutathione reductaseactivity and basal glutathione levels were elevated and in the liver basal glutathionewas reduced following diabetes of both 4275 and 12 weeks duration. Antioxidantenzyme activities that were not altered following diabetes of 4 or 12 weeks durationinclude& Cu,Zn-superoxide dismutase (erythrocytes, heart, liver, kidney), catalase(erythrocytes, heart), gI utathione reductase (kidney) and g lutathione peroxidase(erythrocytes, heart, kidney).Alterations in enzyme activities that were found to be influenced by the durationof uncontrolled diabetes occurred in the liver and kidney. Although the activities of liverglutathione peroxidase and catalase were decreased and glutathione reductase-241 -increased following diabetes of 4 weeks duration,275 no changes in the activities ofthese enzymes were apparent following 12 weeks of diabetes. Similarly, althoughkidney catalase activity was reduced at 4 weeks, it was unchanged following 12 weeksof diabetes. This apparent “normalization” of certain early antioxidant enzymealterations later in the course of the disease may reflect some form of compensation,possibly involving alterations in levels of non-enzymatic antioxidants such astocopherol.27°In terms of the functional consequences of diabetes-associated antioxidantchanges, erythrocytes, heart, liver and kidney all exhibited a reduced susceptibility tohydrogen peroxide-induced sulfhydryl group depletion. The results for erythrocytes,heart and liver are in agreement with our findings in 4 week diabetic animals. Inaddition, previous work in our laboratory has also demonstrated reduced susceptibilityof erythrocytes from 12 week alloxan or streptozotocin diabetic female rats, to hydrogenperoxide-induced sulfhydryl group depletion.2 The mechanism of reducedsusceptibility to sulfhydryl group depletion discussed in the previous section, involvingstimulation of the hexose monophosphate shunt by hyperglycemia resulting in anelevation in intracellular availability of NADPH would be expected to occur independentof the duration of diabetes, given the persistence of hyperglycemia.In contrast to the generalized reduction in tissue sensitivity to peroxide-inducedsulfhydryl group depletion, susceptibility to hydrogen peroxide-induced lipidperoxidation varied among tissues following 12 weeks of diabetes. The diabetesinduced reduction in TBARS formation in the heart is in agreement with our previousstudy in 4 week diabetic rats. This decrease in myocardial susceptibility to lipid-242-peroxidation may be related to the increases in glutathione status as shown byelevated glutathione levels and glutathione reductase activity found following both 4and 12 weeks diabetes. In this regard, liver and kidney tissue did not show increasesin glutathione reductase activity or glutathione levels, and did not exhibit reductions inperoxide-induced TBARS formation.The diabetes-induced elevation in plasma tocopherol found following 12 weeksof diabetes supports previous findings14111 although the increase following 4 weeksdiabetes did not reach statistical significance in this regard. It has been suggested thatthe elevation in tocopherol levels in diabetes may, in part, be attributable to theassociated hyperlipidemia, given that lipoproteins are involved in the transport oftocopherol; however, after adjustment for non-HDL cholesterol, triglycerides were notfound to be correlated with tocopherol concentrations in diabetic patients.23 In ourstudy of diabetes of 12 weeks duration, plasma levels of triglycerides but notcholesterol were elevated. Thus, our finding of elevated plasma tocopherol levels indiabetes is probably not attributable to the associated hyperlipidemia alone. Contraryto the findings in heart and plasma, erythrocyte tocopherol levels have been shown tobe reduced in diabetes.119 This latter observation would be consistent with our findingof increased susceptibility of erythrocytes to hydrogen peroxide-induced lipidperoxidation in diabetes of both 4 and 12 weeks duration.We have shown that hearts from rats with uncontrolled diabetes have reducedcontractile function following 12 but not 4 weeks diabetes. This diabetes-associatedtime-dependent reduction in contractile function is in agreement with previous studiesin rats262 corresponding to the long-term development of cardiovascular complications-243-in humans, including diabetic cardiomyopathy which can occur independently ofatherosclerotic and hypertensive heart disease. 22310 Interestingly, however, wedid not find a generalized deterioration in antioxidant status in hearts from rats withincreased duration of diabetes. In fact, diabetes-associated alterations involvingglutathione reductase activity, glutathione levels and susceptibility to in vitro peroxide-induced sulfhydryl group depletion and lipid peroxidation remained remarkablyconsistent with increased duration of the disease. It should be noted, however, that themechanisms involved in failure of the diabetic heart are multifactorial and not fullyunderstood.24.3.3. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (4 Weeks)In non-diabetic stress-exposed rats, the absence of changes in resting plasmaglucose, triglyceride and cholesterol levels, is consistent with the results of previouslypublished studies.°’72 In addition, in the experiments involving collection of bloodsamples by remote sampling, we did not find changes in resting glucose levels prior tostress on days 1, 7 and 14 of the stress protocol. In contrast, Tsopanakis et. aL282reported a reduced level of serum cholesterol in animals subjected to swimming stress,but the confounding metabolic effects due to exercise cannot be readily dissociatedfrom those of non-exercise related stress factors. For example, while an increase ininsulin sensitivity is associated with exercise,171179 an increase in insulin resistance isassociated with stress.6’216 Paralleling the findings of the present study, humansexposed to significant psychological stress were found to exhibit no correspondingchanges in lipid and lipoprotein levels.197-244-Diabetic rats exposed to the stress protocol had elevated resting plasma glucoselevels relative to the non-stressed diabetic animals in samples collected the morningfollowing the last stress. This elevation might occur through the combined effects ofinsulin resistance and elevations in corticosterone and adrenaline, promotinggI uconeogenesis and liver glycogenolysis, respectively. As mentioned previously,plasma glucose levels were not increased in diabetic rats during exposure to stress. Ithas been suggested that stress-induced elevations in glucose levels may occur hoursafter exposure to stress and may not be observed during stress. The observation thatthe elevations in plasma triglycerides and cholesterol in diabetic animals were loweredto control levels following exposure to chronic stress was unexpected, given theaforementioned absence of changes in plasma levels of these lipids in non-diabeticanimals subjected to the same stress protocol. These changes are not a consequenceof altered food intake since the weights of animals in the diabetic-stressed group didnot differ from their non-stressed diabetic counterparts. Elevations in triglyceride levelsin diabetic rats have, in part, been attributed to a reduction in triglyceride lipase activityassociated with an increase in liver lipid peroxides.2°° Normalization of this enzymeactivity following treatment with tocopherol has been shown to be associated withcorresponding decreases in liver peroxide levels and triglycerides.219 It might,therefore, be postulated that stress-induced normalization of triglycerides could be theresult of an increase in lipoprotein lipase activity. A later section of this discussionfocuses on findings regarding the effects of our chronic intermittent stress protocol onplasma lipid levels in hyperlipidemia of dietary rather than diabetic origin.279 Briefly,stress did not reduce lipid levels in hyperlipidemic animals, suggesting that the-245-normalizing effects of stress on diabetes-induced elevations in plasma lipids is aparticular feature of the diabetic ètate and cannot be solely ascribed to the associatedhyperlipidemia per Se.4.3.4. Effects of Chronic-Intermittent Stress on Resting Levels of PlasmaGlucose and Lipids in Control and Diabetic Rats (12 Weeks)In non-diabetic, stress-exposed rats, the absence of changes in resting plasmaglucose and cholesterol levels is consistent with the results of previously publishedstudies5°’282 including our parallel study of 4 weeks duration. The finding of reducedtriglyceride levels following the completion of the chronic stress protocol may be aresult of prolonged catecholamine-induced de-esterification of triglycerides, withsubsequent release of free fatty acids into the circulation,28 or may be related toreduced food intake in stress-exposed animals, as suggested by a significantlydecreased percent weight gain associated with exposure to stress. It is interesting thatin the parallel study of 4 weeks duration, non-diabetic rats exposed to the stressprotocol did not show reduction in resting triglyceride levels despite the finding thatanimals showed a significantly decreased rate of weight gain.Diabetic rats exposed to the stress protocol did not exhibit elevated restingplasma glucose or cholesterol levels relative to their non-stressed counterparts. In ourparallel study of 4 weeks duration, stress exacerbated the diabetes-associatedelevation in glucose levels and ameliorated the diabetes associated elevation incholesterol levels. The discrepancy between these two studies suggests that chronicstress has different metabolic consequences in diabetes of 12 as opposed to 4 weeksduration. The observation that the elevation in plasma triglycerides in diabetic animals-246-was abolished following exposure to chronic stress is, however, in agreement withsimilar findings in the parallel study of 4 weeks duration. This stress-induced reductionmay, in part, be explained by prolonged catecholamine-induced de-esterification oftriglycerides28as described above, or due to changes in the levels of triglyceride lipaseactivity;2°°19 however, it is not a consequence of altered food intake, since the weightsof animals in the diabetic-stressed group did not differ from their non-stressedcounterparts.4.3.5. Stress-Related Alterations in Antioxidant Status4.3.5.1. Effects of Chronic-Intermittent Stress on AntioxidantStatus in Non-Diabetic AnimalsA major focus of this thesis was to determine the extent to which chronic-intermittent stress influences tissue antioxidant status in control and diabetic animals.Our findings regarding the effects of stress on antioxidant status reflect a complexseries of positive and negative alterations, the number of which was greater in diabeticas opposed to non-diabetic rats relative to their respective controls. In the 4 weekstudy, the only change in non-diabetic rats exposed to chronic intermittent stress was asignificant elevation in erythrocyte susceptibility to H20-induced sulfhydryl groupoxidation. This increase in sensitivity was not accompanied by changes in erythrocyteantioxidant enzyme activities or peroxide-induced lipid peroxidation. Oxygen radicalshave been shown to induce protein degradation in intact red cells in the absence oflipid peroxidation or detectable membrane damage. Thus, increased sulfhydryl groupsusceptibility to in vitro peroxidation may be an early indicator of stress-inducedalterations in free radical-mediated processes. In the 12 week study, the only change-247-in non-diabetic rats exposed to chronic intermittent stress was a small, but significantreduction in liver glutathione reductase activity. This was not accompanied by changesin functional antioxidant capacity measured by susceptibility toH20-induced sulfhydrylgroup oxidation or TBARS formation.The finding that the chronic-intermittent stress protocol did not appreciably alterantioxidant status in the non-diabetic groups was not unexpected. While otherS171’2have found alterations in antioxidant components in non-diseased animals followingacute stress exposure, as discussed previously, the nature of the stress required toproduce these changes was severe, involving long bouts of hypothermia orunpredictable shock. We question the relevance of an acute stress model whichinitiates numerous antioxidant alterations in an otherwise normal animal, given thatendogenous antioxidant mechanisms have presumably evolved to cope with elevatedlevels of oxidant stress. The chronic intermittent stress protocol in the present studywas designed to determine whether subtle alterations in antioxidant status would occurin a disease state with pre-existing oxidative stress. Our finding that the stress protocolused in this study resulted in minimal changes in antioxidant status in normal animalsbut did produce numerous changes in diabetic animals strengthens the generalizationthat pre-existing pathology may increase the susceptibility of antioxidant systems toperturbation by moderate stress.4.3.5.2. Effects of Chronic-Intermittent Stress on AntioxidantStatus in Diabetic Rats (Short-Term Diabetes)In all tissues studied, some diabetes-associated changes in antioxidant statusreverted to control values in animals exposed to chronic intermittent stress. Whereas-248-antioxidant status was generally improved in erythrocytes and lung, stress reduceddiabetes-associated compensatory change in the heart. The effects of stress onantioxidant enzyme activities for the most part were limited to those enzymes in whichdiabetes-associated changes were present. Furthermore, the direction of changes inenzyme activities seemed more related to a stress-induced normalization rather thanan overall increase or decrease in enzyme activities. For example, the activity ofglutathione peroxidase in diabetic rats was decreased in liver and lung. Followingstress, the activity was increased relative to the diabetic group, but no longersignificantly different from non-diabetic controls. Similarly, liver glutathione reductaseactivity, increased in diabetes, was reduced following stress, but was no longersignificantly different from non-diabetic controls: It should be noted, however, that notall antioxidant components altered by diabetes were affected by stress, including theactivities of heart glutathione reductase and kidney catalase. The normalizing patternnoted above also appeared to be reflected in some functional measures of antioxidantstatus including peroxide-induced sulfhydryl group depletion in heart and TBARSformation in kidney and lung. Some changes in functional antioxidant status occurredcoincident with changes in antioxidant enzyme activity such as a reduction in lungsusceptibility to peroxide-induced TBARS formation in association with an increase inglutathione peroxidase activity. Other changes, however, such as reduced kidneysusceptibility to lipid peroxidation and increased myocardial sensitivity to sulfhydrylgroup oxidation in diabetic stress-exposed rats occurred in the absence of anydetectable antioxidant enzyme changes. The relative increase in liver sensitivity toH20-induced sulthydryl group depletion, which was no longer significantly reduced-249-from controls following stress, occurred without a concurrent change in basalglutathione level, which remained reduced from normal, but was accompanied by areturn of glutathione reductase activity to control from elevated values; glutathioneperoxidase activity, which was reduced in diabetes alone, returned to control valuesfollowing stress. Assuming that the concentrations of H20 used did not cause enzymeinactivation, the decreased ability to regenerate reduced glutathione may be a factor inthe increased susceptibility of the liver to glutathione depletion. It is interesting to notethat sensitivity to peroxide-induced lipid peroxidation in the heart (which was reduced indiabetes) was unaffected by stress. One possibility for this lack of change could haveto do with our previous finding of elevated tocopherol levels in diabetic rat hearts,which appeared to be unaltered by stress.276 In several other tissues studied, includingliver and kidney, in which stress abolished the diabetes-associated increase inperoxide-induced TBARS formation, we did not find corresponding evidence of stress-induced alterations in tocopherol levels in diabetic animals which could explain thisphenomenon.276The finding that chronic-intermittent stress normalized a number of alterations indiabetes of short-term duration, including elevated plasma lipid levels and a number ofantioxidant components and functional measures of antioxidant status, suggests thepossibility that moderate stress may be of some benefit in diabetes. While somechanges, including lowered lipid levels, have obvious implications in long-termdiabetes, the potential benefits of the positive changes in antioxidant status, includingnormalized lung and liver glutathione peroxidase activity and improved peroxideinduced suceptibility to TBARS formation in lung and kidney, remain to be studied. In-250-addition, some stress-induced alterations in the antioxidant status of diabetic animalsoccurred in a negative direction, including liver glutathione reductase activity and heartbasal glutathione, as well as liver and heart peroxide-induced susceptibility tosulThydryl group depletion. Further, following the chronic stress protocol, the diabeticgroup exhibited elevated resting plasma glucose levels. The situation is thus highlycomplex and a detailed understanding of the relative benefits or adverse consequencesof stress-induced antioxidant changes will require more research on organ functionfollowing chronic stress in longer-term diabetes during which the probability ofdevelopment of secondary complications increases.4.3.5.3. Effects of Chronic-Intermittent Stress on AntioxidantStatus in Diabetic Rats (LonQ-Term Diabetes)Stress produced alterations in antioxidant components in all tissues studied in12 week diabetic animals. In the heart, liver and plasma, some diabetes-associatedchanges in antioxidant components reverted to control values and in the kidney anderythrocytes some antioxidant components were altered in the diabetic stressorexposed group that were not changed by stress or diabetes alone. In general, whilethe relative changes in the liver and erythrocytes were increased, changes in the heartand plasma were decreased. In the kidney, a shift in glutathione status occurred with areduction in glutathione reductase activity and an increase in glutathione peroxidaseactivity. We have previously shown chronic stress to modify antioxidant components indiabetes of 4 (as opposed to 12) weeks duration. While chronic-intermittent stressappears to reduce antioxidant status in the diabetic heart regardless of the duration ofdiabetes, the nature of the changes seems to be influenced by the duration of the-251 -diabetic state. For example, in the 4 week study, the diabetes-associated elevation inheart basal glutathione level was no longer significant following stress and in vitrosensitivity to sulfhydryl group oxidation was significantly increased relative to thediabetic state alone; however, the diabetes-associated elevation in glutathionereductase activity was unchanged with stress. In 12 week diabetic animals, a stress-induced loss in the diabetes-associated increase of glutathione reductase activityoccurred in the absence of other changes. A major influence of stress in diabetesseems to be the normalization of changes in antioxidant status. For example, in theliver, following 4 weeks of diabetes, stress reversed the diabetes-associated reductionin glutathione peroxidase activity and the elevation in glutathione reductase activity,while in the 12 week study there were no diabetes and/or stress-related changes in theactivity of these enzymes. It is not known whether normalization of antioxidantcomponents is a common effect of chronic stress in other diseases with associatedalterations in antioxidant status.4.3.5.4. Generalizations Reciardinci Effects of Chronic IntermittentStress on Antioxidant Status in Short- and Long-TermDiabetesWe have confirmed the presence of diabetes-associated changes in antioxidantcomponents, which for the most part appear to be increased in the myocardium andreduced in the other organs studied in short-term diabetes. In long-term diabetes,while changes in antioxidant status did not reflect a general deterioration incomponents in various tissues in comparison with short-term diabetes, there appear tobe somewhat different consequences on antioxidant status as the disease progresses-252-in some tissues, although antioxidant status does appear to remain increased in theheart. Exposure of rats to chronic-intermittent stress in the absence of an underlyingdisease state was found to have minimal effects on antioxidant status in both the 4 and12 week studies. Our results have shown that chronic stress can modify diabetes-related alterations in carbohydrate and lipid metabolism; however, some changesappear to depend on the duration of diabetes. Some changes in lipid metabolism may,in part, be related to the return of several diabetes-associated functional antioxidantindices to control levels. Following both 4 and 12 weeks diabetes and chronic stress,some diabetes-associated changes were abolished; however, unlike the former study,some antioxidant components that had been unaltered in long-term diabetes showedsignificant changes, suggesting that the interaction of stress and antioxidant status indiabetes varies as the disease progresses. In addition, while some antioxidant enzymeactivity changes paralleled the functional changes, others did not, implicating apossible role for non-enzymatic antioxidants in this regard. In general, the effects ofstress on antioxidant status in the diabetic rat depended on the organ studied and,therefore, generalizations about the ability of stress to enhance or decrease antioxidantcapacity must be made at the level of the organ rather than the whole organism.4.4. Effects of Chronic-Intermittent Stress on Plasma Lipids andAntioxidant Status in Rats with Diet. induced as Opposed toDiabetes-Induced HyperlipidemiaIn the previous studies of antioxidant status and stress in diabetes of short- andlong-term duration, some diabetes-associated changes in antioxidant status returned tocontrol values following stress, coincident with some measures of plasma lipids,-253-suggesting that the changes may be related. It was, therefore, of interest to explore theinfluence of stress on plasma lipid profiles and tissue antioxidant status in non-diabeticrats with diet induced hyperlipidemia.The cholesterol-supplemented diet used in this experiment produced elevationsof plasma cholesterol comparable to those found in our previous studies in diabeticrats.275’899 However, although plasma triglycerides were significantly elevated in ratson the cholesterol-supplemented diet for a period of four weeks, they did not reach thelevels found in rats diabetic for the same duration, but were similar to those in ratsdiabetic for a duration of twelve weeks. Thus, the diet-induced hyperlipidemia in thepresent study appears to mimic some aspects of diabetes-associated hyperlipidemia.Studies of chronic-intermittent stress have used a decreased weight gain as ageneral indicator of the presence of a significant stress effect.2°5 In the present study,animals subjected to the chronic-intermittent stress protocol showed an average weightgain which was 50% less than that in unstressed controls. The finding thathyperlipidemic rats gained weight at the same rate as control animals and that stressaffected the rate of weight gain in both groups to the same degree eliminates thepotential confounding factor of weight differences in the interpretation of our results.One aspect of this study was to investigate the effect of stress on hyperlipidemiaer se in the absence of diabetes. In a previous study dealing with the influence ofchronic-intermittent stress in animals with .diabetes of 4 weeks duration, wedemonstrated that diabetes-induced increases in cholesterol and triglycerides wereattenuated following exposure to stress. A stress-induced reduction in lipid levels inanimals with diabetes-related hyperlipidemia might occur through increased beta--254-oxidation of fatty acids, which is known to occur in both stress and diabetes.27’13°Alternatively, a stress-induced reduction in food intake might play a role. The stress-induced reduction in plasma lipid levels in diabetic rats was probably not a result ofaltered food intake, since weight gain in diabetic rats was found to be unaffected bystress. Further, although stress reduced the rate of weight gain in hyperlipidemic rats,there were no associated changes in plasma lipid levels. The lack of stress-inducedchanges in hyperlipidemic animals when combined with the fact that plasma cholesterollevels actually increased in the normolipidemiclstressed rats suggests a minimal role ofbeta-oxidation in mediating the stress-induced reduction in lipid levels in diabetic rats.Hence, it may be suggested that an as yet unidentified feature of the diabetic state isresponsible for stress-induced changes in lipid levels under these conditions.A second aspect of the present study was to examine the influence of stress andhyperlipidemia on antioxidant status in the absence of complicating effects related todiabetes. Numerous studies have shown elevations in glutathione reductase activity inerythrocytes and certain tissues in rats with diabetes of 4 or 12 weeks duration. Giventhe absence of detectable changes in the activity of this enzyme in hyperlipidemiclnondiabetic rats in the present study, it can be concluded that the elevation in glutathionereductase activity in diabetes cannot be simply attributable to hyperlipidemia.Although oxidative processes have been implicated in the development ofatherosclerosis,1°4little is known about the influence of the associated hyperlipidemiaper se on antioxidant enzyme activities. Antioxidant enzyme changes inatherosclerosis might be attributable to a direct influence of the hyperlipidemia, acompensatory response to oxidative stress, or to secondary pathological changes in-255-affected tissues. Based on the present study of diet-induced hyperlipidemia in rats,which are relatively resistant to the development of atherosclerosis,2the first of thesehypotheses seems unlikely.However, the results from our study do not preclude an influence ofhyperlipidemia on antioxidant status as it relates to the development of atherosclerosisin more susceptible species. Species-related differences in antioxidant enzymes andsusceptibility to oxidative damage, particularly between rat (insensitive toatherosclerosis), rabbit and quail (susceptible to diet-induced atherosclerosis) suggestthat studies be conducted relating the development of diet-induced atherosclerosis tochanges in antioxidant Status.81’2 In this regard, susceptible species, including theatherosclerotic rabbit and congenitally obese mouse,’ have shown changes inantioxidant enzyme activities3°’ and lipid peroxide levels3°in a number of tissues. Ourlaboratory is currently studying the time-dependence of changes in endogenousantioxidant components during the course of atherogenesis in a strain ofatherosclerosis-prone Japanese quail.The increased susceptibility of heart and liver to peroxide-induced TBARSformation following chronic intermittent stress in normolipidemic rats raises thepossibility that some deleterious effects of stress on antioxidant systems are related tothe associated elevations in corticosterone and catecholamines. In our other studiesusing the same model of stress, while there was a tendency towards increasedsusceptibility of various tissues to peroxide-induced TBARS formation following stress,the levels did not reach statistical significance. As mentioned previously, evidence ofincreased oxidative activity has recently been found in myocardial tissue of rats-256-exposed to the severe “emotional” stress of intermittent inescapable shock.171’2 Ourdemonstration that susceptibility of some tissues to in vitro peroxide-induced TBARSformation is reduced following chemical sympathectomy and adrenomedul lectomytmalso lends support to this suggestion. As these changes were not found in all tissuesstudied, it supports our previous findings that tissues vary considerably in theirsusceptibility to stress-induced alterations in antioxidant capacity. The elevation inmyocardial TBARS formation in the cholesterol-fed and stress-exposed groups, whichoccured independently but showed no evidence of synergistic effects, is noteworthy.However, it would be of more pathological relevance to re-examine these effects in anatherosclerosis-sensitive species. Elucidation of the biochemical mechanismsdetermining stress-mediated impairment in myocardial antioxidant systems might clarifythe putative role of stress in the exacerbation of cardiovascular disease.4.5. Effects of Chronic-Intermittent Stress on Myocardial FunctionalRecovery Following IschemialReperfusion Injury in Short and Long-Term DiabetesThe work in this thesis has provided additional support for the presence ofdiabetes-associated changes in antioxidant status which appear to be altered bychronic stress. While antioxidant alterations in some tissues including liver and kidney,seem to change with the progression of the disease, interestingly, the changes inantioxidant status in the diabetic heart remained relatively consistent following 4 and 12weeks diabetes, with elevations in glutathione status, including total glutathione levelsand glutathione reductase activity, and elevation of tocopherol (only measured at 4weeks). Furthermore, the in vitro measures of functional antioxidant status including-257-susceptibility to peroxide-induced TBARS formation, and suiThydryl group depletionwere relatively improved. Thus, there appears to be some diabetes-associatedcompensation in antioxidant capacity in the heart, which would be consistent with thehypothesis of increased oxidative stress. Unlike the situation with diabetes alone theadditional effects of stress on the heart seemed to be qualitatively different at the twotime periods studied. Following 4 weeks diabetes and stress, some diabetes-associated compensatory changes in the heart were normalized, including totalglutathione levels and peroxide-induced sulfhydryl group depletion, which wasincreased relative to the diabetic state alone. On the other hand, following 12 weeksdiabetes and stress, normalization of glutathione reductase activity occurred in theabsence of other changes.Diabetes is a dynamic disease associated with the progressive development ofcardiovascular and other complications. Failure of the cardiovascular system isconsidered to be the leading cause of death in diabetic patients.87 Time-dependentreduction in contractile function has been demonstrated in diabetic rats whichcorresponds to the long-term development of cardiovascular complications in humansincluding diabetic cardiomyopathy which can occur independent of atherosclerotic andhypertensive heart disease. 2,2,310The aim of the present work was to examine the functional effects of chronicstress assessed in terms of myocardial susceptiblity to a short period ofischemialreperfusion injury in control and diabetic rats with short (4 weeks) or long-term(12 weeks) diabetes. Effects were assessed in terms of differences in recovery of-258-myocardial function following ischemia and antioxidant capacity following ischemia andreperfusion.4.5.1. Diabetes Associated Alterations in Myocardial Functional Recoveryand Antioxidant Status Following Ischemia/Reperfusion Injury4.5.1.1. Baseline Function Before lschemia/ReperfusionWe have shown that hearts from rats with uncontrolled diabetes have reducedcontractile function as shown by a decrease in developed pressure and ±dP/dt maxfollowing 12274 but not 4273 weeks diabetes. Measurements of cardiac function werecarried out on isolated Langendorff perfused hearts where end-diastolic pressure, heartrate, perfusion temperature and aortic pressure were kept constant. Thus, themeasurements of myocardial function probably reflect the contractile state of the heart.The two periods of diabetes used in these experiments were thought to have somesimilarities to the conditions of short and long-term diabetes with more severecardiovascular complications occurring in long-term diabetes. As mentioned earlier,the rat was chosen as an experimental animal in these studies because it is highlyresistant to the development of atherosclerosis1 and, thus interpretation of thefindings would occur with minimal interference from coronary atheroscleroticcomplications. Therefore, the reduced function in long-term diabetes found in thisstudy is probably not a result of atherosclerosis. In experimental diabetes, using avariety of heart preparations including isolated heart, working heart and the in situheart, reduction in myocardial contractile performance has been noted by others asmeasured by stroke volume, stroke work, cardiac output, peak contractile force andpeak systolic pressure.176212228’2The force generation velocity relationship is also-259-altered as shown by a reduction in ±dP/dt max•76’2 Furthermore, it has been shownthat hearts from diabetic dogs have reduced ventricular compliance as indicated byincreased left ventricular end-diastolic pressure and decreased end diastolic volume inassociation with increased formation of connective tissue as shown by the presence ofincreased glycoprotein and collagen in the interstitium.228 This appears not to be thecase, however, in diabetic rats. While some capillary basement thickening has beenobserved, myocardial collagen was not found to be increased.71’178The mechanism of reduced function in the diabetic heart is clearly multifactorialand not completely understood, but appears to involve, in part, alterations in cardiacmembranes which ultimately affect calcium handling. Numerous reports of changes incalcium handling have been compiled in a recent review in which it was suggested thatdiabetes itself is a disease of calcium metabolism.1 For example, increasedmyocardial Ca2 content has been found in alloxan diabetic mice.187 In streptozotocininduced diabetic rat hearts among other changes, reduction in Ca2 transport byisolated sarcoplasmic reticulum has been observed, including, decreased Ca2 -ATPase activity, ATP-dependent Ca2 uptake, and Na dependent Ca2uptake.76’’1572 In addition, reduction in myosin ATPase activity1 has beenobserved which is closely associated with the shortening speed of the heart.67 It hasbeen suggested that insulin plays a critical role in maintaining the integrity of the cellmembrane, and that changes in insulin availability or action as well as toxic effects ofthe oxidation products of catecholamines can result in membrane defects associatedwith calcium handling, resulting in Ca2+ overload and leading to diabeticcardiomyopathy. Furthermore, in a recent study of 2 month streptozotocin-diabetic-260-rats, heart ventricles were found to have elevated vitamin E-quinone and lipidperoxidation products suggesting the occurrence of oxidative damage.118 It washypothesized that the elevated levels of vitamin E-quinone could be due to increasedutilization of tocopherol in scavenging oxygen radicals generated by thehyperglycemia.251 Quinones are known to undergo reduction by enzymatic ornonenzymatic mechanisms to the semiquinone and can be converted to free radicalmetabolites, which in turn can interact with dioxygen to produce highly toxic activespecies.25’ Thus, accumulation of small amounts of quinones can generate largequantities of active oxygen species which can deplete intracellular glutathione andinhibit key enzymes such as those involved in maintaining intracellular calcium andvitamin E-quinone conversion to vitamin E.25’ The finding of increased accumulation oflipid peroxidation products in the heart when coupled with our findings of a decreasedsusceptibility to peroxide-induced lipid peroxidation in heart and increased glutathionestatus is suggestive of the possibility that this increased myocardial antioxidantcapacity in diabetic rats may not be sufficient to cope with the elevated oxidative stresshypothesized to occur in diabetes.4.5.1.2. Myocardial Function Followinc lschemia/ReperfusionThe ischemia produced in this model, although of short duration (2 mm),involved a complete cessation of aortic flow. In addition, hearts were maintained undernormothermic conditions and cardiac pacing was continued throughout the ischemicperiod. These conditions led to a moderate statistically significant reduction in cardiacfunction when measured during the reperfusion period in all groups relative to theirrespective controls. The ischemic period was probably of short enough duration to-261 -eliminate the complicating factor of cell necrosis normally associated with ischemia of30 mm or longer.214Early into the reperfusion period there was evidence of significantly improvedfunctional recovery in hearts from rats with diabetes of 4 and 12 weeks duration whencompared to similarly treated non-diabetic controls. In the 12 week diabetic group, thisrelative improvement occurred despite the baseline reduction in contractility. Althoughmyocardial function was not significantly different among groups by the end of thereperfusion period, antioxidant status was significantly elevated in hearts from rats withdiabetes of 4 and 12 weeks duration following ischemia/reperfusion as shown byincreased levels of basal glutathione and glutathione reductase activity. Since thelevels of these components are also elevated in freshly harvested hearts from diabeticrats, it would be reasonable to assume that they remain elevated through the ischemicand early reperfusion periods and thus would be available under conditions ofincreased oxidative stress. Impaired glutathione status has been demonstrated to be afeature of myocardial ischemia/reperfusion injury especially during the post-ischemicreperfusion phase.32’ Several studies have shown improved myocardial functionalrecovery following ischemiclreperfusion injury associated with pharmacologicallyinduced elevation of glutathione status. For example, administration of Nacetlycysteine added to the perfusion solution 60 mm prior to ischemia in normal rabbithearts and continuing through the ischemic and reperfusion periods elevated reduced(as opposed to oxidized) glutathione levels prior to ischemia and resulted in increasedpercent developed pressure during reperfusion.42 Furthermore, at the end ofreperfusion, tissue content of glutathione was significantly higher, associated with a-262-decrease in cellular damage as shown by reduction in creatine phosphokinase releaseand impairment of mitochondrial function relative to controls.32 (Incidentally, Nacetlycysteine is a sulfhydryl group donor which is easily transported into the cellwhere it is de-acetylated, increasing the thiol pool, primarily reduced glutathione.69) Inanother series of experiments, chronic pretreatment of rabbits with allopurinol wasfound to elevate myocardial glutathione reductase activity. In rabbits chronicallytreated with allopurinol, following ischemia and reperfusion of the left circumflexcoronary artery, a reduction in the elevation of peroxide-induced susceptibility of theheart to lipid peroxidation and sulfhydryl group depletion was found. It should benoted that allopurinol-induced inhibition of xanthine oxidase would not likely be amechanism in the improved antioxidant capacity as levels of this enzyme are notdetectable in rabbit heart.1 In addition, allopurinol treatment was not associated withchanges in the levels of ATP. Therefore, the increased glutathione status found indiabetic hearts in the present studies could be a contributory factor to the improvedearly functional recovery following ischemia. Furthermorewe have shown levels oftocopherol, a lipophilic antioxidant, to be elevated in diabetic hearts which could beavailable under conditions of increased oxidative stress; however, levels of tocopherolwere not measured following ischemia/reperfusion in the present studies. Others haveshown in non-diabetic rats that pharmacologically elevated levels of myocardialtocopherol (51 %) attenuated lipid pertubations in terms of tissue accumulation ofunesterified fatty acids, and reduced the accumulation of calcium and improvedcontractile function following 25 mm of ischemia and 30 mm reperfusion.1 As ourstudies were not specifically designed to determine the mechanism of improved-263-recovery of diabetic hearts, additional experiments will be necessary before definitiveconclusions can be made.While some authors have noted improved recovery of diabetic hearts followingischemia,1312 others have found diabetic hearts to be more sensitive to ischemiainduced dysfunction.70’209214 Factors relating to the diabetic state including differencesin duration of diabetes and degree of control by insulin and factors related to theischemic insult such as the model of ischemialreperfusion used, the severity andduration of ischemia and in isolated heart preparations, the components in theperfusion solution all may affect post-ischemic recovery of function. In addition,atherosclerosis-sensitive animal models of diabetes in which heart failure isaccompanied by atherosclerosis cannot be used to separate the effects of ischemia onatherosclerosis from the independent development of cardiomyopathy which occursfollowing long-term diabetes. In hearts from rats with diabetes for 48 hrs, improvedfunctional recovery following 30 mm of in vitro global (whole heart) ischemia andreperfusion was associated with a significantly reduced reperfusion Ca2 uptake.2The resistance to ischemia in diabetic hearts was not related to higher tissue levels ofhigh energy phosphates during reperfusion nor to the degree of lactate accumulationduring ischemia. The extent of the recovery of diabetic hearts (100%) was significantlygreater than recovery in hearts from rats with diabetes of 4 and 12 weeks durationshown in the present study, suggesting that the very short duration of diabetes may bea factor in this response. Recently a marked decrease in the activity of the amiloridesensitive Na/H exchanger has been shown in hearts from rats with streptozotocininduced diabetes of 4 weeks duration131 which was associated with decreased rate of-264-recovery of myocardial pH following ischemia. Inhibition of the Na/H exchanger hasbeen demonstrated to reduce accumulation of Na during reperfusion.2 In non-diabetic rats, it has been shown that an increase in intracellular Na causes excessiveCa2 uptake,2 in part mediated by Na - Ca2 exchange resulting in reducedrecovery of cellular function during reperfusion.2 In addition to a protective effect ofreduced Na/H exchange in diabetic rats, it has been suggested that the reducedactivity of the Na - Ca2 exchange also observed in diabetic hearts1 might furthercontribute to protection of the diabetic heart against reperfusion damage. Furthermore,an Na/HCO3 -dependent carrier mechanism, which has previously been shown tocontribute substantially to pH recovery from an ammonium-induced intracellular acidload140 has recently been found to be reduced in diabetes, associated with an improvedpost-ischem ic functional recovery.131 Thus, it seems that diabetes-associatedalterations in membrane functional components may operate conferring an increasedprotection of the diabetic heart to ischemialreperfusion injury in the absence of thecomplicating factor of atherosclerosis. The effects of increased oxidative processes inalteration of membrane components in diabetes, however, is in need of additionalstudy. Furthermore, although study of ischemialreperfusion in isolated hearts as donein these experiments is necessary to assess the direct effects of chronic diabetes onthe heart, additional in vivo experimental studies are necessary to assess the heartunder the biochemical conditions associated with the diabetic state which may exposethe heart to elevated oxidative processes involving for example the presence oftransition metals as discussed previously. For example, in 6 and 12 weekstreptozotocin-diabetic rats, coronary occlusion of the left anterior descending artery in-265-conscious animals was associated with a significant increase in ventricular fibrillation indiabetic rats.194.5.2. Stress Related Alterations in Myocardial Functional Recovery andAntioxidant Status Following Ischemia/Reperfusion Injury4.5.2.1. Non-Diabetic AnimalsThe finding that two weeks of stress did not alter the functional recovery of themyocardium in non-diabetic rats is interesting when viewed in light of our previousstudies of stress in non-diabetic rats. Analysis of stress hormone levels on day 1, 7and 14 of the stress protocol indicated some adaptation in the response of plasmacatecholamines to stress; however, corticosterone levels remained elevated duringeach sampling point through the three days of sampling. Thus, the persistent increasein corticosterone levels found during stress through the stress protocol and theadaptation response of catecholamines did not appear to have a direct influence onmyocardial functional susceptibility to ischemia and reperfusion. In our foregoingstudies of stress on antioxidant status in non-diabetic rats, there were no stress-induced alterations in antioxidant components in the heart. Following Langendorfperfusion and subsequent ischemia and reperfusion, it should be mentioned thatantioxidant components are altered in all groups, as shown by an approximately 40%reduction in glutathione and ascorbate levels and glutathione reductase activity relativeto those values measured in freshly harvested hearts. In the present study of 4 weeksduration, a significant increase in time-dependent peroxide-induced susceptibility tosulfhydryl group depletion occurred in hearts from non-diabetic stressor-exposed ratsfollowing reperfusion in the absence of changes in basal glutathione levels, glutathione-266-reductase activity, or peroxide-induced TBARS formation. Furthermore, this change insusceptibility was not accompanied by a change in myocardial contractility relative tohearts from unstressed rats. Changes in glutathione status have been recorded in theabsence of myocardial functional changes following ischemia and reperfusion in rabbithearts. In general, however, relative to non-stressed rats, there appears to be littleeffect of chronic-intermittent stress of 2 weeks duration on antioxidant status andmyocardial functional recovery following this short period of ischemia and reperfusion.4.5.2.2. Diabetic AnimalsIn diabetic rats exposed to the stress protocol, the early improvement inmyocardial functional recovery associated with diabetes of 4 weeks duration was lost.As with diabetes alone, in animals exposed to stress, myocardial function was notsignificantly different among groups by the end of the reperfusion period. The relativereduction in early myocardial function was associated with a decrease in antioxidantcapacity as shown by significantly reduced levels of glutathione relative to the diabeticgroup. In addition glutathione reductase activity was no longer significantly elevatedfrom control animals. Whether antioxidant capacity was reduced prior to theischemialreperfusion insult following stress is unknown; however, in freshly harvestedhearts from diabetic rats exposed to stress, the diabetes associated elevation inglutathione levels was no longer significant relative to controls, suggesting that thismight be a possibility. On the other hand, antioxidant capacity could have beenreduced as a result of an increased susceptibility to oxidative stress during ischemiaand reperfusion. It is interesting to note that in stress-exposed diabetic animalsanalysis of stress hormone levels on day 1, 7 and 14 of the stress protocol indicated a-267-biphasic stress response which showed some adaptation after 7 days and responsesmore similar to the first day of stress on day 14 of the stress protocol suggesting thatthe diabetic rats have an altered response to chronic stress. Although it would bereasonable to suggest that the differences in stress response could account for somedifferences in susceptibility to ischemia/reperfusion injury, it must be cautioned thatfurther experiments will be necessary to elucidate the mechanism. In a compariscn ofthe effects of 4 and 12 weeks diabetes and stress on recovery fromischemialreperfusion injury, stress did not appear to alter the improved early recoveryfound in 12 week diabetic rats. This lack of effect of stress was also accompanied bythe lack of a significant effect of stress on the diabetes-associated elevation inglutathione reductase activity and glutathione levels found following ischemia andreperfusion. Interestingly, however, peroxide-induced TBARS formation was elevatedin the diabetic group exposed to stress. The qualitative differences between the effectsof stress on functional recovery during early reperfusion and differences in the effectson antioxidant capacity in 4 as opposed to 12 weeks diabetes, are suggestive ofchanges in the diabetic state as the disease progresses, which are in need of furtherstudy.4.6. Summary and ConclusionsSympathectomy produced by adrenalectomy and/or 6-OH treatment alteredantioxidant status in a number of tissues in the rat. Measures of antioxidant statuswhich appeared particularly sensitive to sympathectomy-induced changes includedgI utathione reductase activity (increased) and susceptibility to peroxide-induced lipidperoxidation (reduced) in a number of tissues. Differences between adrenalectomy-268-and 6-OH treatment on antioxidant components are suggestive of differential actions ofadrenaline and noradrenaline on tissue antioxidant status which may have importantimplications under conditions associated with elevations in levels of thesecatecholamines including chronic stress and myocardial infarction. The effects ofreserpine treatment in chemical sympathectomy in general showed similar changes inantioxidant capacity as do those of 6-OH treatment which would suggest that theantioxidant effects in these studies were a result of the resulting changes incatecholamine and corticosterone levels rather than an effect of the chemical used.We have examined the effects of acute and chronic intermittent variable restraintstress on hormonal, glycemic and antioxidant indices in normal and diabetic rats.Acute restraint produced a substantial stress response in both diabetic and non-diabetic rats which showed differential adrenal and glycemic responses. Continuedexposure to stress in normal rats led to some adaptation of the sympathetic andadrenomedullary but not adrenocortical responses. In contrast, diabetic rats exhibiteda biphasic stress response which showed some adaptation after 7 days and responsesmore similar to the first day of stress in diabetic rats on day 14 of the stress protocol.These results suggest that diabetic rats show differential reactivity to both acute andchronic stress which could have consequences in the development of long-termcomplications associated with diabetes.Exposure of rats to chronic-intermittent stress in the absence of an underlyingdisease state was found to have minimal effects on antioxidant status. We haveconfirmed the presence of diabetes-associated changes in antioxidant components in 4week diabetic rats, which for the most part appeared to be increased in the myocardium-269-and reduced in the other organs studied. The finding that stressor exposure in diabeticanimals exacerbated elevated plasma glucose and modulated the increase intriglyceride and cholesterol levels, indicates that chronic stress can modify diabetes-related changes in carbohydrate and lipid metabolism. The latter changes may, in part,be related to the return of several diabetes-associated functional antioxidant indices tocontrol levels. In addition, while some antioxidant enzyme activity changes paralleledthe functional changes, others did not, implicating a possible role for non-enzymaticantioxidants in this regard. Thus, chronic intermittent stressor exposure has multipleeffects in diabetes.Although we have demonstrated altered antioxidant status in long-term (3month) diabetes, the changes did not reflect a general deterioration in components invarious tissues in comparison with short-term diabetes of 4 weeks duration. Chronic-intermittent stress as in the 4 week study appeared to have minimal effects onantioxidant status in non-diabetic rats. Chronic stress exposure in diabetic animalsmodulated the increase in plasma triglycerides, corroborating the similar finding inshort-term diabetes. The effect of stress on antioxidant status in the long-term diabeticrat depended on the organ studied and, therefore, generalizations about the ability ofstress to enhance or decrease antioxidant capacity must be made at the level of theorgan rather than the whole organism. As in the study of 4 weeks diabetes and chronicstress, some diabetes-associated changes were abolished; however, unlike the formerstudy, some antioxidant components that had been unaltered by diabetes alone didshow significant changes, suggesting that the interaction of stress on antioxidant statusin diabetes varies as the disease progresses.-270-The role of the diabetic state per se in determining the effects of stress onhyperlipidemia and antioxidant status explored through study of non-diabetic rats withdiet-induced hyperlipidemia showed a number of findings. In contrast to the situation inthe diabetic state, the severity of dietary hyperlipidemia was not affected by stress. Inaddition, the minor alterations in antioxidant components associated withhyperlipidemia alone were also unaffected by stress. Therefore, the effects of stresson diabetes-induced changes in plasma lipids and antioxidant components cannotsolely be ascribed to the associated hyperlipidemia.In the study of the effects of stress and short-term diabetes on myocardialsusceptibility to ischemialreperfusion injury, following 4 weeks diabetes, baselinemyocardial function in isolated hearts was, not significantly altered and chronic-intermittent stress did not significantly alter myocardial function in normal or diabeticrats. While the brief period of normothermic global ischemia significantly reducedreperfusion function in all groups, initial functional recovery following the ischemicperiod was significantly improved in the diabetic group; however, exposure to chronicstress eliminated this effect. At the end of the reperfusion period, although there wereno differences between groups in percent functional recovery, the diabetic group hadincreased antioxidant status as reflected in elevated basal glutathione levels andglutathione reductase activity. Paralleling the changes in early post-reperfusionfunction, exposure to stress in the diabetic group eliminated the elevation in glutathionereductase activity and glutathione levels. Thus in short-term diabetes, the diabeticmyocardium shows indications of an altered sensitivity to ischemialreperfusion which isreflected in changes in antioxidant status.-271 -In the study of the effects of stress and long-term diabetes on myocardialsusceptibility to ischemia/reperfusion injury, following 12 weeks of diabetes, baseline,myocardial function was significantly reduced. Two weeks of chronic-intermittent stressdid not significantly alter reduced myocardial function in diabetic rats. The brief periodof ischemia significantly reduced functional indices in all groups. Initial functionalrecovery was improved in the diabetic group and stress did not alter this effect. Theimproved functional recovery was only observed in the early stage of reperfusion. Atthe end of the reperfusion period, although there were no differences between groupsin percent functional recovery, both diabetic groups had increased antioxidant capacityas reflected in elevated basal glutathione levels and glutathione reductase activity.However, peroxide-induced lipid peroxidation was less in the non-stressed diabeticgroup and this effect was lost in the diabetic group exposed to stress.In conclusion, evidence has been shown for the modulation of antioxidantcapacity by “stress hormones” including catecholamines and/or glucocorticoids. This issuggested in part by experiments in which a reduction in endogenous stress hormonelevels led to increased antioxidant capacity in various tissues. While the effects ofchronic-intermittent stress on antioxidant status were minimal in otherwise normalanimals, they were intensified with diabetes, a disease associated with increasedoxidative stress. The stress-related changes in antioxidant capacity were tissuedependent and therefore generalizations about the ability of stress to enhance ordecrease antioxidant capacity must be made at the level of the organ rather than wholeorganism. In this regard, stress reduced the early functional recovery of the diabeticheart following ishemia/reperfusion injury, associated with a reduction in antioxidantcapacity. The altered “stress hormone” response of the diabetic rat during the chronic--272-intermittent stress protocol suggests a possible means whereby a differential responseto stress could lead to alterations in antioxidant components. 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