EFFECTS OF GLUCOSE ON ENDOTHELIAL FUNCTION IN PREGNANCY AND THE INFLUENCE OF DIABETES A thesis submitted to The University of Manchester for the degree of PhD in the Faculty of Medical and Human Sciences 2006 Haiju Henry Chirayath School of Medicine LIST OF CONTENTS ABSTRACT 17 CHAPTER 1: INTRODUCTION 26 1.1 Diabetes Mellitus 27 1.1.1 Background 27 1.1.2 Rationale of study 28 1.1.3 Definition of diabetes 28 1.1.4 Classification of diabetes 29 1.1.5 Diagnosis of diabetes 29 1.1.6 Type 1 diabetes 30 1.1.7 Type 1 diabetes in pregnancy 31 1.1.8 Type 2 diabetes 31 1.1.9 Type 2 diabetes in pregnancy 32 1.1.10 Gestational diabetes 32 1.1.10.1 Definition and diagnosis 32 1.1.10.2 Features of gestational diabetes 35 1.1.11 Other specific types of diabetes 36 1.2 Animal models of diabetes 36 1.2.1 Animal models of type 1 diabetes 37 1.2.2 Animal models of type 2 diabetes 37 1.2.3 Animal models of diabetes in pregnancy 37 1.2.4 Advantages of animal models of diabetes 38 1.2.5 Limitations of animal models of diabetes 38 1.3 Pregnancy 39 1.3.1 Carbohydrate metabolism in pregnancy 39 1.3.2 Cardiovascular changes in pregnancy 40 1.4 Arteries 41 1.4.1 Blood flow to the fetus 41 1.4.2 Resistance arteries 41 2 1.5 The Endothelium 42 1.5.1 Endothelial function 42 1.5.2 Endothelium-dependent mediators of relaxation 43 1.5.2.1 Nitric Oxide 44 1.5.2.2 Prostacyclin 45 1.5.2.3 Endothelium Derived Hyperpolarizing Factor 46 1.5.3 Endothelium-derived vasoconstrictors 47 1.5.4 Other factors affecting endothelial function 48 1.5.4.1 Age 48 1.5.4.2 Smoking 48 1.5.4.3 Ethnicity 48 1.5.4.4 Cholesterol 48 1.5.5 Endothelial function in pregnancy 48 1.5.6 Methods of measuring endothelial function 49 1.5.6.1 Venous occlusion plethysmography 50 1.5.6.2 Brachial artery flow-mediated vasodilation 50 1.5.6.3 Limitations of brachial artery FMD 51 1.6 Endothelial dysfunction in diabetes 51 1.6.1 Animal studies of endothelial dysfunction in diabetes 52 1.6.2 Human studies of endothelial dysfunction in diabetes 52 1.6.3 Mechanisms of endothelial dysfunction in diabetes 53 1.6.4 Endothelial dysfunction and regional variations 55 1.6.5 Other factors influencing endothelial dysfunction in diabetes 56 1.6.5.1 Control of diabetes 56 1.6.5.2 Obesity 56 1.6.5.3 Lipids 57 1.6.5.4 Insulin resistance 58 1.6.5.5 Gender differences in diabetes 58 1.7 Endothelial dysfunction of diabetes in pregnancy 58 1.7.1 Type 1 diabetes 58 1.7.2 Gestational diabetes 59 1.7.3 Previous gestational diabetes 59 1.8 Glucose levels 61 1.8.1 HbA1C 62 3 1.9 Hyperglycaemia 62 1.9.1 Definition 62 1.9.2 Estimating hyperglycaemia 62 1.9.3 Degree of hyperglycaemia 63 1.9.4 Duration of hyperglycaemia 63 1.9.5 Pathophysiology of hyperglycaemia 64 1.9.5.1 Oxidative stress 64 1.9.5.2 Hyperglycaemia and endothelial mediators 66 1.9.6 Acute Hyperglycaemia 66 1.9.7 Hyperglycaemia and endothelium-dependent relaxation 67 1.10 Hypoglycaemia 69 1.10.1 Hypoglycaemia and pregnancy 69 1.10.2 Hypoglycaemia and endothelial function 70 1.11 Blood glucose in pregnancy 70 1.12 Summary of Introduction 72 1.13 Hypotheses 73 1.14 Aims 73 CHAPTER 2 MATERIALS AND METHODS 74 2.1 Ethics 75 2.2 Human subjects 75 2.2.1 Healthy women 75 2.2.1.1 Non-pregnant women 75 2.2.1.2 Pregnant women 75 2.2.2 Diabetic patients 75 2.2.2.1 Type 1 and type 2 diabetes in pregnancy 75 2.2.2.2 Gestational diabetes 76 2.3 Mice 76 2.4 Tissue Collection 76 2.4.1 Non-pregnant women 76 2.4.2 Pregnant women 77 2.4.3 Mice 77 4 2.5 Stereomicroscopic dissection 77 2.6 Resistance arteries 77 2.7 Choice of methodology 77 2.8 Wire myography 78 2.8.1 Multi Myograph System 610M 79 2.8.2 Advantages of wire myography 80 2.8.3 Limitations of wire myography 81 2.9 Normalisation of vessel lumen 81 2.10 Drugs 82 2.10.1 Potassium chloride 82 2.10.2 Bradykinin 82 2.10.3 Phenylephrine 83 2.10.4 Acetylcholine 83 2.10.5 U46619 83 2.10.6 L-NNA 83 2.10.7 Indomethacin 83 2.10.8 Streptozotocin 83 2.10.9 Choice of drugs 84 2.10.9.1 Vasoconstriction 84 2.10.9.2 Endothelium-dependent relaxation 84 2.11 Solutions 85 2.11.1 Glucose physiological saline solutions 85 2.11.2 60 mmol/L KPSS 85 2.11.3 Hyper-osmolar solution 86 2.11.4 Modified Krebs’ buffer 86 2.12 Experimental protocols 86 2.12.1 Protocol 1: The effect of glucose levels on maximum constriction 86 2.12.2 Protocol 2: The effect of glucose levels on endothelium-dependent relaxation 88 2.12.3 Protocol 3: The effect of hyper-osmolarity 89 2.12.4 Protocol 4: The effect of blockers to endothelium-dependent relaxation 90 5 2.13 Optimising experiments 92 2.13.1 General measures 92 2.13.2 Optimising mice experiments 92 2.14 Creating an animal model of diabetes 93 2.14.1 Restrictions in human research 93 2.14.2 Rationale for creating an animal model of diabetes 94 2.14.3 Obtaining a Home Office Licence 94 2.14.4 Species and strain 94 2.14.5 Duration of diabetes 95 2.14.6 Diabetogenic drug 95 2.14.7 Procedure 95 2.15.8 Impediments in creating an animal model of diabetes 96 2.15 Data analysis 97 2.15.1 Constriction 97 2.15.2 Endothelium-dependent relaxation 97 2.16 Statistical analysis 97 2.16.1 General Principles 97 2.16.2 Statistical analysis of data from patients 98 2.16.3 Statistical analysis of myography data 98 2.16.3.1 Constriction 98 2.16.3.1 Endothelium-dependent relaxation 98 CHAPTER 3 THE GLUCOSE LEVELS OF WOMEN WITH DIABETES IN PREGNANCY 99 3.1 Introduction 100 3.2 Aims 101 3.3 Methodology 101 3.3.1 Inclusion criteria 102 3.3.2 Exclusion criteria 102 3.3.3 Data collection 103 3.3.4 Statistical analysis 103 6 3.4 Results 103 3.4.1 Clinical characteristics 103 3.4.2 Glucose levels 106 3.4.3 Glucose levels of type 1 diabetes in pregnancy 107 3.4.4 Glucose levels of type 2 diabetes in pregnancy 108 3.4.5 Glucose levels of gestational diabetes 108 3.4.6 HbA1C levels: comparison across types 112 3.4.7 HbA1C levels in each group 112 3.4.7.1 Type 1 diabetes 112 3.4.7.2 Type 2 diabetes 112 3.4.7.3 Gestational diabetes. 112 3.4.8 Glucose levels in GDM: effect of insulin 114 3.4.9 Glucose levels and outcomes in type 1 diabetes 116 3.4.10 HbA1C and outcomes in type 1 diabetes 116 3.5 Discussion 119 3.5.1 Glucose levels of women with diabetes in pregnancy 119 3.5.2 Glucose levels in type 1 diabetes 120 3.5.3 Glucose levels in type 2 diabetes 120 3.5.4 Glucose levels in gestational diabetes 120 3.5.5 Adverse outcomes 120 3.5.5.1 Type 1 and 2 diabetes 121 3.5.5.2 Gestational diabetes 122 3.5.6 HbA1C and glucose levels 122 3.5.7 Limitations of study 123 3.5.8 Summary 123 CHAPTER 4 THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN HEALTHY NON-PREGNANT AND PREGNANT WOMEN 125 4.1 Introduction 126 4.2 Aims 127 4.3 Materials and Methods 127 4.4 Results 128 7 4.4.1 Clinical characteristics 128 4.4.2 Arterial diameters 129 4.4.3 Constriction: effect of glucose (non-pregnant group - KCl) 131 4.4.4 Endothelium-dependent relaxation: effect of glucose (non-pregnant group – KCl) 131 4.4.5 Constriction: effect of glucose (non-pregnant group - U46619) 131 4.4.6 Endothelium-dependent relaxation: effect of glucose (non-pregnant group – U46619) 131 4.4.7 Constriction: effect of glucose (pregnant group - KCl) 136 4.4.8 Endothelium-dependent relaxation: effect of glucose (pregnant group – KCl) 136 4.4.9 Constriction: effect of glucose (pregnant group–U46619) 139 4.4.10 Endothelium-dependent relaxation: effect of glucose (pregnant group – U46619) 139 4.4.11 Comparison of endothelium-dependent relaxation in non-pregnant and pregnant groups 144 4.4.12 Effect of prolonged exposure (2 hours) of 2, 5, 8 and 12 mmol/L glucose 146 4.5 Discussion 148 4.5.1 Summary of results 148 4.5.2 Low glucose concentration and impaired relaxation 148 4.5.2.1 The effect of vasoconstrictors 149 4.5.2.2 The effect of pregnancy 150 4.5.3 Other findings 150 4.5.3.1 Constriction 150 4.5.3.2 Effect of pregnancy on endothelium-dependent relaxation 150 4.5.3.3 Effect of prolonged incubation 151 4.5.3.4 High glucose concentrations 152 4.5.4 Explanations for the lack of effect of high glucose levels 152 4.5.4.1 Inadequate duration of high glucose levels 152 4.5.4.2 Inadequate degree of high glucose levels 152 4.5.4.3 Systemic factors 153 8 4.5.4.4 Endothelial vasodilators 154 4.5.4.5 Bradykinin 154 4.5.4.6 Branch-order 155 4.5.4.7 Age 155 4.5.4.8 Nature of vascular bed 156 4.5.4.9 Gender 156 4.5.4 Summary 157 CHAPTER 5 THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN DIABETES COMPLICATING PREGNANCY 158 5.1 Introduction 159 5.2 Aims 160 5.3 Materials and methods 161 5.4 Results 161 5.4.1 Clinical characteristics – gestational diabetes 161 5.4.2 Diameter of arteries – gestational diabetes 163 5.4.3 Constriction: effect of glucose (GDM-KCl) 163 5.4.4 Endothelium-dependent relaxation: effect of glucose (GDM-KCl) 166 5.4.5 Constriction: effect of glucose (GDM-U46619) 166 5.4.6 Endothelium-dependent relaxation: effect of glucose (GDM-U46619) 166 5.4.7 GDM: effect of vasoconstrictor on endothelium-dependent relaxation 170 5.4.8 Constriction: effect of glucose (type 1 and 2 diabetes) 171 5.4.9 Endothelium-dependent relaxation: effect of glucose (type 1 and 2 diabetes) 171 5.4.10 Comparison between GDM and healthy pregnant women constriction 175 5.4.10.1 Using KCl 175 5.4.10.2 Using U46619 175 9 5.4.11 Comparison between GDM and healthy pregnant women: endothelium-dependent relaxation 175 5.4.11.1 Using KCl 175 5.4.11.2 Using U46619 175 5.5 Discussion 179 5.5.1 Summary of results 179 5.5.2 Endothelial dysfunction in GDM 180 5.5.2.1 Explanations for impaired relaxation 180 5.5.2.2 Implications of findings 182 5.5.2.3 Limitations of study 183 5.5.3 Patient characteristics 183 5.5.4 The effect of