Effects of Testosterone Levels on Mortality and Cardiovascular Risk in Men with Type 2 Diabetes Thesis submitted to The University of Sheffield For the degree of Doctor of Philosophy By Vakkat Muraleedharan Department of Oncology and Metabolism University of Sheffield Medical School September 2018 1 Contents Page List of figures 12 List of tables 17 Summary of findings 20 Acknowledgements 22 Publications and presentations arising from the thesis 24 Schematic representation of studies conducted over the course of PhD and personnel 30 contribution to each section Chapter One 31 General Introduction 1.1 Introduction 31 1.2. Testosterone and mortality 40 1.2.1 Population or community studies 40 1.2.2 Studies in specific disease populations 44 1.3. Relationship of testosterone to cardiovascular mortality and morbidity 46 1.4 Testosterone and mortality in renal disease 50 1.5 Metabolic syndrome, diabetes, cardio-metabolic markers and 51 testosterone 1.5.1. Metabolic syndrome 51 1.5.2. Testosterone in the metabolic syndrome diabetes and insulin 54 resistance 1.5.3. Effect of androgen suppression on metabolic syndrome, type 2 60 diabetes and cardiovascular risk profile 1.5.4. The role of testosterone in dyslipidaemia, hypertension and 62 atherosclerosis 2 1.6. Role of androgen receptor in diabetes, obesity and metabolic 66 syndrome 1.6.1. The androgen receptor and androgen receptor gene 66 1.6.2. The androgen receptor CAG (AR CAG) repeat polymorphism 68 1.6.3. Clinical correlations of the AR CAG polymorphism in men 69 1.7 Testosterone replacement therapy 70 1.7.1. Effect of Testosterone replacement therapy on glycaemic control, 71 insulin resistance and lipid profile 1.7.2. Effect of testosterone replacement therapy on hypertension 75 1.7.3 Long term effects of testosterone replacement therapy in men with 76 hypogonadism 1.8 Role of SHBG in cardio-metabolic risk profile 79 1.9 Potential metabolic mechanisms of testosterone action 83 1.9.1 Testosterone action in muscle tissue 83 1.9.1.1 Testosterone action on carbohydrate metabolism in muscle tissue 84 1.9.1.2 Testosterone action on lipid metabolism in muscle tissue 85 1.9.2 Testosterone action on Liver 86 1.9.2.1 Testosterone action on glucose metabolism in the liver 86 1.9.2.2 Testosterone action on lipid metabolism in liver 87 1.9.3 Testosterone action on adipose tissue 88 1.10 Testicular Feminised (Tfm) mouse 91 1.11 Conclusions and Prelude to Thesis 93 3 Chapter Two 97 Longitudinal Study: Long term effect of testosterone on mortality in men with type 2 diabetes 2.1 Background and Introduction 97 2.2 Hypothesis 100 2.3 Research Design and Methods 100 2.4 Clinical and biochemical assessment 103 2.4.1 Recording of demography, medical history and drug history 103 2.4.2 Measurement of weight and body composition 103 2.4.3 Measurement of waist and hip circumference 104 2.4.4 Measurement of blood pressure 104 2.4.5 Biochemical measurements 105 2.4.5.1 Sampling technique 105 2.4.5.2 Measurement of samples in Barnsley hospital clinical chemistry 105 laboratory 2.4.5.3 Measurement of serum bioavailable testosterone- summary 106 2.4.5.4 Measurement of serum bioavailable testosterone- method 106 2.4.5.5 Calculation of free and bio available testosterone 107 2.5 Statistical analysis 108 2.6 Results 109 2.6.1 All-cause mortality in low and normal testosterone groups 111 2.6.2 Cardiovascular mortality in low and normal testosterone groups 116 2.6.3 Effect of testosterone replacement therapy on survival in men with 118 low testosterone 2.7 Discussion and conclusion 122 4 2.8 Limitations 124 Chapter three 127 Longitudinal study: Long term effect of testosterone levels on cardiovascular risk profile and cardiovascular events 3.1 Background and Introduction 127 3.2 Hypothesis 130 3.3 Research Design and Methods 130 3.4 Clinical and biochemical assessment 132 3.4.1 Recording of demography, medical history and drug history 132 3.4.2 Measurement of weight and body composition 132 3.4.3 Measurement of waist and hip circumference 133 3.4.4 Measurement of blood pressure 133 3.5 Biochemical measurements 134 3.5.1 Sampling technique 134 3.5.2 Measurement of samples in Barnsley hospital clinical chemistry 134 laboratory 3.5.3 Measurement of serum bioavailable testosterone- summary 135 3.5.4 Measurement of serum bioavailable testosterone- method 135 3.5.5 Calculation of free and bio available testosterone 136 3.6 Statistical analysis 137 3.7 Results 138 3.7.1 Cardiovascular Medications 140 3.7.2 Hospital admission and cardiovascular events 141 3.7.3 Effect of testosterone levels on cardiovascular risk profile during 142 follow up 5 3.7.3.1 Glycaemic control 144 3.7.3.2 Body composition 146 3.7.3.3 Lipid profile and blood pressure 146 3.7.4 Effect of baseline SHBG on the cardiovascular risk profile during 149 follow up 3.7.4.1 Cardiovascular events 149 3.7.4.2 Cardiovascular risk profile in relation to SHBG levels 149 3.8 Discussion 153 3.9 Limitations 157 Chapter four 159 Cross-sectional Study: Effect of Testosterone on cardiovascular risk profile in men with type 2 diabetes 4.1 Introduction 159 4.2 Hypothesis 161 4.3 Research Design and Methods 161 4.4 Clinical assessment 163 4.4.1 Recording of demography, medical history and drug history 163 4.4.2 Measurement of weight and body composition 164 4.4.3 Measurement of waist and hip circumference 164 4.4.4 Measurement of blood pressure 165 4.5 Biochemical measurements 165 4.5.1 Sampling technique 165 4.5.2 Measurement of samples in Barnsley hospital clinical chemistry 165 laboratory 6 4.5.3 Measurement of serum bioavailable testosterone- summary 166 4.5.4 Measurement of serum bioavailable testosterone- method 167 4.5.5 Calculation of free and bio available testosterone 168 4.6 Assessment of arterial wall parameters-carotid artery ultrasound 168 4.6.1 Carotid ultrasound Measurement for carotid Intima media thickness 170 4.6.2 Calculation of Carotid Stiffness index β 171 4.7 Statistical analysis 171 4.8 Results 172 4.8.1 Baseline characteristics 172 4.8.2 Cardiovascular risk profile 174 4.8.2.1 Effect of Testosterone on Cardiovascular risk profile 174 4.8.2.2 Effect of SHBG on cardiovascular risk profile 178 4.8.3 CIMT and Carotid Stiffness index β 184 4.5 Discussion and conclusion 185 4.6 Limitations 187 Chapter five 189 Cross- sectional study: The relationship between the androgen receptor CAG polymorphism, ratio of testosterone to AR CAG and cardiovascular risk profile in men with type 2 diabetes 5.1 Introduction 189 5.2 Hypothesis 192 5.3 Research Design and Methods 192 5.4 Clinical assessment 193 5.4.1 Recording of demography, medical history and drug history 193 7 5.4.2 Measurement of weight and body composition 193 5.4.3 Measurement of waist and hip circumference 194 5.4.4 Measurement of blood pressure 194 5.5 Biochemical measurements 194 5.5.1 Sampling technique 194 5.5.2 Measurement of samples in Barnsley hospital clinical chemistry 195 laboratory 5.5.3 Measurement of serum bioavailable testosterone- summary 196 5.5.4 Measurement of serum bioavailable testosterone- method 196 5.5.5 Calculation of free and bio available testosterone 197 5.6 Measurement of AR CAG – summary 198 5.6.1 Extraction of DNA from human lymphocytes 198 5.6.2 Polymerase chain reaction (PCR) to amplify section of androgen 199 receptor gene containing AR CAG 5.6.3 Agarose gel electrophoresis to confirm PCR product 200 5.6.4 Magnetic separation and use of automated sequencer 201 5.7 Assessment of arterial wall parameters-carotid artery ultrasound 202 5.7.1 Carotid ultrasound Measurement for carotid Intima media thickness 203 5.7.2 Calculation of Carotid Stiffness index β 203 5.8 Statistical analysis 204 5.9 Results 205 5.9.1 AR CAG and cardiovascular risk profile 205 5.9.2 Total testosterone AR CAG ratio and cardiovascular risk profile 208 5.9.3 Bioavailable Testosterone AR CAG ratio ( cBioT/AR CAG) and 216 Cardiovascular risk profile 8 5.10 Haematocrit, Haemoglobin and cardiovascular risk profile in relation 226 to testosterone 5.11 Discussion and conclusions 226 5.12 Limitations 228 Chapter six 230 Animal Study: The effect of testosterone on the glucose and lipid metabolism in liver, muscle and adipose tissue of Tfm mice 6.1 Introduction 230 6.2 Hypothesis 234 6.3 Materials and methods 234 6.3.1 The Testicular Feminised Mouse 234 6.3.2 Animal Husbandry 235 6.3.3 Experimental treatments 237 6.3.3.1 Promotion of Metabolic Syndrome State 238 6.3.3.2 Testosterone treatment 238 6.3.4 Sry gender determination of animals 239 6.3.5 Collection of animal tissues 239 6.3.5.1 Serum Collection 240 6.3.5.2 Tissue collection 240 6.3.6 RNA extraction procedure from the liver, subcutaneous tissues and 241 muscle 6.3.7 cDNA preparation using QuantiTect Reverse Transcription Procedure 243 6.3.7.1 Reverse transcription protocol 244 6.3.7.2 Protocol for Quantitative, Real-Time PCR using SYBR Green I 247 9 6.4 Statistical analysis 251 6.5 Results 251 6.5.1 Target enzymes of carbohydrate metabolism 253 6.5.1.1 Effect of high fat diet on Tfm mice as compared to XY littermates 253 6.5.1.2 Effect of testosterone administration Tfm mice as compared to Tfm 254 littermates without testosterone 6.5.2 Target enzymes of lipid metabolism 260 6.5.2.1 Target enzymes of fatty acid metabolism 260 6.5.2.1.1 Effect of high fat diet on Tfm mice compared to wild type XY 261 littermates 6.5.2.1.2 Effect of testosterone administration Tfm mouse as compared to Tfm 26 1 littermates without testosterone 6.5.2.2 Target enzymes of cholesterol metabolism 266 6.5.2.2.1 Effect of high fat diet on Tfm mice compared to wild type