Traditional plant treatments have provided the source of many important drugs. Obesity and Diabetes mellitus are debilitating and often life-threatening diseases with increasing incidence in both industrialised and rural populations throughout the world. New therapies are needed because of the inability of current therapies to control all of the pathological aspects of diabetes and obesity, and plant-based medicines are required as a result of the high cost and poor availability of current therapies for many rural populations, particularly in developing countries. A scientific investigation of traditional herbal remedies for obesity and diabetes may also provide valuable leads for the development of novel drug treatments. Studies carried out in db/db mice treated with Artemisia judaica extract (Chapter 3) support the anecdotal claims of its anti-diabetic activity. Though the Artemisia extract treatment did not result in normoglycaemia, any reduction in the blood glucose level was not due to an increased plasma insulin level. Further findings in the current study are that the anti-diabetic~ efficacy is more apparent during the first few days of treatment than later days. This may be due to the diabetogenic principle or some receptor or post-receptor down regulation. The lower concentrations of Artemisia extracts were active but the higher doses were ineffective. This may indicate the presence of multiple components, both pro and anti-diabetic. In isolated hepatocytes (Chapter 4) Artemisia extract reduced glucose production from lactate/pyruvate. Enzyme crossover studies showed that Artemisia extract might inhibit the PEPCK enzyme (phosphoenolpyruvate carboxykinase) activity, but there was no direct inhibition of the enzyme in vitro. Such inhibition could be potentially useful in a clinical setting in reducing hepatic glucose output and further work is required to fractionate the Artemisia extract to identify the active component.

The oxidation of low-density lipoproteins (LDL) is considered a key step in the development and progression of atherosclerosis. Single bouts of aerobic exercise cause transient increases in free radical production that may enhance the susceptibility of LDL to oxidation and create a more atherogenic LDL particle. In contrast, chronic exercise has often been considered an effective tool in improving metabolic profile through changes in aerobic capacity, lipid profile, fuel utilization and oxidative stress in both healthy and disease populations. Despite this, less is known about how it may benefit the prevention of LDL oxidation and the mechanisms by which this may occur, particularly in aged and patients with type II diabetes who have oxidative stress. The primary aim of the work contained in this thesis, is to examine the effects of aerobic exercise on the susceptibility of LDL to oxidation in both young, aged and type II diabetic subjects. The findings of study 1 demonstrate that an acute bout of moderate intensity exercise can increase the susceptibility of LDL III in both young and aged subjects regardless of any change in LDL lipid composition. Study 2 demonstrates that chronic aerobic exercise of moderate intensity is effective at improving the resistance of the LDL I sub fraction against oxidation, as shown by an increase in T1I2max, despite no change in LDL lipid composition. This intervention was also beneficial in altering maximal aerobic capacity in both young and aged subjects. Study 3 demonstrates that chronic low and moderate intensity aerobic exercise has no effect on LDL oxidative susceptibility. However, chronic moderate intensity exercise increased catalase activity and decreased protein oxidation. The collective findings of this work provide evidence that acute exercise may increase LDL oxidation while chronic exercise may prevent the oxidation of LDL particularly in aged subjects. Further research with greater subject numbers is required to determine the precise mechanism by which exercise influences the susceptibility of LDL oxidation.

Type 2 diabetes is a growing health problem worldwide, resulting from the body's inefficiency at utilising insulin or reduced insulin production. For those diagnosed with the chronic health condition careful self-management is required, including following complex medication regimes, exercise, diet and ongoing monitoring by clinicians. Type 2 diabetes is however characterised by poor adherence to self-care behaviours, therefore increasing risk of hypertension, weight gain, renal and nerve damage. A systematic literature review was conducted to scrutinise literature examining psychosocial factors affecting adherence to self-care behaviours. Diverse psychosocial factors were found to affect and facilitate adherence to self-care in those with Type 2 diabetes. Strongest predictors related to social support, depression, self-efficacy and availability of financial resources. Relationship status, employment status, diabetes knowledge, health beliefs, motivation and level of education were also suggested to correlate with adherence. Coping and religion appeared equivocally related. Little research was elicited examining alcohol or smoking, however findings suggested a correlation with reduced adherence to self-care. A quantitative study expanded upon available literature, examining the prevalence of alcohol consumption in the UK Type 2 diabetic population and whether alcohol use correlated with adherence to self-care. A small pilot study examined the efficacy of an intervention to reduce alcohol consumption, and whether this would correlate with improved self-care. Results revealed 9% of the Type 2 diabetic population were consuming alcohol at levels placing them at risk of alcohol-related health problems, with males consuming more than females. A relationship was revealed between increased alcohol consumption and decreased adherence to self-care. Findings have clinical implications regarding the inclusion of screening for alcohol use in routine diabetes care. Due to feasibility issues a small sample were recruited to the pilot study which reduced the ability to confidently infer clinical implications from findings.

Genome-wide association (GWA) studies have provided significant insight into the underlying genetic components of common human diseases such as type 2 diabetes (T2D). However, the translation of such genetic findings into biological and clinical insight remains a major challenge. One of the genes implicated in T2D pathogenesis and effects on related glycaemic and lipidaemic traits by the GWA approach is GCKR, encoding glucokinase regulatory protein (GKRP). GKRP inhibits the glycolytic enzyme glucokinase (GCK) in the liver, sequestering it in an inactive form in the nucleus. Together, GCK and GKRP exert a significant proportion of control of hepatic glycolytic flux, facilitating glucose uptake and disposal in the fed state and inhibiting glycolysis in the fasting state. A common nonsynonymous variant in GCKR, p.P446L, was identified by a combination of genetic and functional approaches as the likely causative variant underlying the GWA findings. The P446L variant protein has been shown to have diminished capacity to inhibit GCK relative to wild-type (WT) GKRP, resulting in enhanced hepatic glycolytic flux and activation of liver synthetic pathways, including those generating triglycerides. Genes such as GCKR harbouring common variants of discernible functional effect may also contain rare variants of large effect. Accordingly, I aimed to comprehensively identify and functionally characterise nonsynonymous variants across the allelic frequency spectrum in GCKR, both by applying and adapting existing kinetic techniques and by developing methodologies to assess the effects of variants on the subcellular localisation of GKRP and GCK. Additionally, I aimed to relate these findings back to clinically important phenotypes, and to further characterise the binding of GCK and GKRP by searching for novel small- molecule modulators of this interaction. Using fluorescent fusion proteins transiently transfected into HeLa cells, I showed that human GKRP localises to the nucleus and sequesters human GCK. I also demonstrated that the common P446L variant significantly reduces the ability of G KRP to localise to the nucleus and sequester GCK. I then investigated the cellular and kinetic characteristics of 18 additional nonsynonymous GCKR variants identified in the National Institutes of Health's ClinSeqTM cohort, determining that the majority of variants affected protein function, and that these effects could be divided into distinct sub-classes. Variants causing a significant loss of function were associated with increased lipid levels in the cohort. Finally, I developed robust assays capable of measuring the interaction of re comb in ant GKRP and GCK in a format suitable for quantitative high-throughput screening of up to 400,000 small-molecule compounds. While no compounds that specifically affected this interaction have been identified to date, the assays developed could be useful in future studies of GCK and GKRP. These data provide further insight into the critical regulatory role of GCKR in metabolism, the structure and function of the GKRP protein, and the potential pathogenic consequences of human variants within GCKR.

In diabetics, impaired ADAMTS13 proteolysis of highly-thrombogenic VWF multimers may accelerate renal and cardiovascular complications, that are major causes of illness and death in these patients. Restoring physiological VWF handling might contribute to ACE inhibitors (ACEi) reno- and cardioprotective effects. In this thesis is presented a study on the 1,163 normoalbuminuric type 2 diabetics from the BENEDICT phase A trial assessed to evaluate the interaction of the common nonsynonymous ADAMTS13 polymorphisms associated with impaired protease activity levels with ACEi therapy in predicting renal and cardiovascular complications. In this population the p.Pr0618Ala ADAMTS13 polymorphism was a major independent predictor of renal and cardiovascular events as well as of reno- and cardioprotective effects of ACEi therapy. Ala carriers showed the highest risk of events and, at the same time, had the largest benefit from ACEi therapy. Excess risk of events appeared to be associated with defective ADAMTS 13 activity. Indeed, in a sub-study on the same population, the 618Ala variant associated with less proteolytic activity, higher risk of chronic complications and better response to ACEi therapy. Thus, p.Pr0618Ala screening may help identifying diabetics at highest risk who may benefit the most from early reno- and cardioprotective therapy. Furthermore, among all ACEi treated microalbuminuric diabetics of the BENEDICT phase B, Ala carriers had lower risk of progression to macroalbuminuria and also higher risk of regression to normoalbuminuria. Thus, ACEi appears to be renoprotective both in the context of prevention, and as a treatment to ameliorate renal function. As a final evaluation, a genetic screening study on a third COhOli of diabetics with different extents of renal damage, revealed a higher prevalence of ADAMTS13 mutations in patients with severe nephropathy in respect to those without, sustaining the role of ADAMTS13 defects in diabetic nephropathy.

The aetiology of type 2 diabetes is complex with both genetic and environmental factors playing a role. Largely through genome-wide association studies, some proportion of the genetic component of type 2 diabetes and its related traits has been identified. However, there are some limitations in those study designs including possible overestimation of disease risk in the general population and a large proportion of unexplained heritability in the disease or trait of concern. There are several approaches that can be taken, to better understand the aetiology of type 2 diabetes. These include studying closely related quantitative traits such as fasting glucose levels and expanding the current efforts in discovering susceptibility variants to other classes of variation beyond common variants. Although, observational associations between type 2 diabetes and related quantitative traits may provide further clues to the aetiology of type 2 diabetes and its related traits causal nature of such associations are not clear. This thesis presents a series of studies that address the above concerns, and explore approaches of how known common variants associated with metabolic traits can be used to understand their role in the aetiology of the associated disease or trait. Chapter 1 is an introduction to the molecular genetics of type 2 diabetes, fasting glucose and birth weight and discusses the biological implications of these genetic findings. Chapter 2 describes a study that shows how genetic factors are important regardless of how patients are recruited using a common variant robustly 2 ociated with type 2 diabetes in the TCF7L2 locus. This was the first study to empirically show that the risk estimates from genetic studies involving genetically enriched cases are likely to be overestimated. Chapter 3 describes a study that was designed to identify rare or low frequency variants associated with fasting glucose levels in the G6PC2 gene where common variation in the same gene is robustly associated with fasting glucose levels. We found no evidence that the identified rare variants were associated with normal variation in fasting glucose levels. Chapter 4 describes a study that was designed to test the hypothesis that common genetic variants associated with fasting glucose and triglyceride levels would alter the same traits in pregnancy and influence fetal growth and development. We found evidence that the common variants alerting fasting glucose and triglyceride levels outside pregnancy largely alters these traits in pregnancy to the same extent. We also found preliminary evidence that the relationship between raised mothers glucose levels and increased birth weight is causal. Chapter 5 describes a study that utilises the Mendelian randomisation approach to test whether the observed correlations between raised circulating triglyceride levels and type 2 diabetes, fasting glucose and fasting insulin levels are causal. We found no evidence that raised circulating triglycerides are causal for type 2 diabetes, fasting glucose or fasting insulin levels. The observed correlations are therefore confounded or likely to be secondary to the diabetes disease process.

Introduction Diets rich in green leafy vegetables have been shown to lower blood pressure and lower the risk of cardiovascular disease. Green leafy vegetables are particularly rich in inorganic nitrate. Dietary nitrate supplementation, via sequential reduction to nitrite and nitric oxide, has previously been shown to lower blood pressure and improve endothelial function in healthy humans. Aims To develop a nitrate-depleted beetroot juice placebo in order to conduct a randomised double blind control trial. To determine if supplementing dietary nitrate with beetroot juice, a rich source of nitrate, will lower BP, improve endothelial function and insulin sensitivity in individuals with type 2 diabetes (T2DM). To determine if supplementing dietary nitrate will lower BP in healthy older adults. Methods The beetroot juice nitrate concentration was lowered using an ion exchange resin. The nitrate content of 250 ml of active juice was 7.5 mmol, whilst nitrate- depleted juice had 0.002 mmol of nitrate in 250ml. Twenty-seven patients, age 67.2 +/-4.9 years, (18 male) were recruited for a double blind, randomised, placebo-controlled crossover trial. Participants were randomised to begin in either order a 2 week period of supplementation with 250 ml beetroot juice daily (active, 7.5 mmol of nitrate) or 250 ml nitrate-depleted beetroot juice (placebo 0.002 mmol of nitrate) followed by a 4 week washout period before entering the second arm of the study. At the conclusion of each intervention period 24 hour ambulatory blood pressure monitoring, vascular tests and a hyperinsulinaemic euglycaemic clamp were performed. Seventeen healthy controls, age 60.5 ± 3.6 (11 male) were recruited to a more limited protocol examining the effect of dietary nitrate supplementation on blood pressure. Results The mean systolic BP was unchanged: 134.6 ± 8.4 mm Hg vs 135.1 ± 7.8 mm Hg (mean±SD) placebo vs active - mean difference of -0.5 mm Hg (placebo- active) p=0.737 (95% Cl -3.9 to 2.8) in the T2DM group. The plasma nitrate concentration was 31IJM (median, IOR; 19.8-41.6) vs 150IJM (122.7, 200), p<0.001. The plasma nitrite concentration was 232 nM (200, 265) vs 390nM (312, 537), p<0.001, for placebo vs active. There were no differences in endothelial function or insulin sensitivity between supplementation arms. The healthy control group mean systolic BP was unchanged at 129.2 ± 9.8 mm Hg vs 131.0 ± 13.1 mm Hg, with a mean difference of -1.8 mm Hg, p=0.320 (95% CI-5.4 to 1.9). The plasma nitrate concentration was 19.81JM (10.6, 37.7) vs 116.6 IJM (91.9, 158.9), p<0.001, and the plasma nitrite concentration 285 nM (245,348) vs 335 nM (296, 357), p=0.127. Conclusion Supplementation of the diet with 7.5 mmol of nitrate per day caused an increase in the plasma nitrite concentration, but did not lower blood pressure, improve endothelial function or improve insulin sensitivity in individuals with T2DM in this study. Dietary nitrate supplementation did not lower BP in healthy older adults in this study.

Type 2 diabetes is caused by defective insulin secretion in the presence of insulin resistance. It usually occurs in late adulthood and is associated with high body mass index (BM I). Genome-wide association studies (GWASs) have identified more than 200 genetic variants associated with metabolic traits, including more than 100 associated with type 2 diabetes, obesity and insulin resistance. This number is increasing as larger meta-analyses of GWASs are performed. However, there has been a bottleneck in translating GWAS findings into improved knowledge of biology and disease mechanisms. This thesis presents a series of post-GWAS strategies to find the genetic background between type 2 diabetes and metabolic traits, to use known genetic variants to understand some of the mechanisms that lead to insulin resistance and its relationship with type 2 diabetes and cardio vascular disease and to find the precise genes involved for most of the variants using gene expression data. Chapter 1 is an introduction to the discovery of genes associated with type 2 diabetes risk and metabolic traits from genome-wide association studies and some of the recent mechanistic insights derived from the genetic association studies about type 2 diabetes and its link with metabolic traits and diseases. Chapter 2 presents the largest genome-wide association study of birth weight. Five novel common genetic variations were identified to influence birth weight, in addition to an interesting finding about the genetic links between fetal growth and postnatal growth and metabolism. Chapter 3 describes a study designed to identify the genetic link between birth weight and type 2 diabetes. This study used a novel approach to account for opposing effect of maternal and fetal genotypes and found sets of type 2 diabetes genes that influence birth weight either directly through the fetal genotype or indirectly through the maternal genotype. Chapter 4 describes a study that uses Mendelian randomisation approaches to test the causal association between adiponectin and risk of insulin resistance , I and type 2 diabetes. This largest study of adiponectin did not provide any consistent evidence that interventions aimed at increasing adiponectin levels will improve insulin sensitivity or risk of type 2 diabetes. Chapter 5 presents a study designed to utilise metabolic features of monogenic forms of insulin resistance in a novel approach to characterise less penetrant variants associated with common form of insulin resistance. This study identified an interesting cluster of 11 common variants consistent with a "polygenic lipodystrophy" phenotype characterised by an increased risk of type 2 diabetes, cardio-vascular disease and hypertension but with reduced BMI. Chapter 6 describes the largest study designed to identify common genetic variants influencing the expression levels of their neighbour genes ('cis' hits) and genes far from them (,trans' hits). These 'trans' hits provided insights into the downstream effects of complex trait associated SNPs, such as altered TAGAP and LDLR expression in celiac disease and cholesterol metabolism, respectively, and altered C1 QB and type 1 interferon (IFN-a) gene expression in systemic lupus erythematosus. Chapter 7 discusses the research findings and limitations from previous chapters, describes future research plans in the genetics of insulin resistance and type 2 diabetes and presents conclusions.

Summary As the field of molecular endocrinology advances, more genes, enzymes and receptors are being defined as possible therapeutic targets for therapy in type 2 diabetes, obesity, and the metabolic syndrome as a whole. These novel therapeutics are hoped to rival and surpass current pharmacological compounds in efficacy and duration of action; but also minimise the contraindications and the side-effects involved. Given the wide range of drug targets in the literature, it was decided that this thesis would focus on two active research areas - glucocorticoids and K-cell derived therapies. High levels of l lb-hydroxysteroid dehydrogenase type 1 in adipose and liver are associated with type2 diabetes and the metabolic syndrome. Non-specific inhibition of 11~-HSDl utilising carbenoxolone, a naturally occurring 11~-HSDl inhibitor in mice with high-fat diet induced insulin resistance and obesity, had no effect on food intake, but significantly lowered body weight and non -fasting plasma glucose levels without reduction in circulating plasma corticosterone levels. Insulin sensitivity and glucose tolerance in these mice were also improved, while 11~-HSDl gene expression was significantly down-regulated in liver. Glucocorticoid receptor gene expression was increased in both liver and adipose tissue following carbenoxolone treatment. These results support current findings for the potential value of specific 11~-HSDl inhibition as a new target for the development of selective drugs for the treatment of type 2 diabetes, obesity and related metabolic disorders. As such, we employed a compound designed by Biovitrum (named BVT.2733) to test the effects of specific inhibition of l1~-HSDl in a chronic genetic model of obesity and diabetes. Twice daily treatment in ob/ob mice with BVT.2733 had no effect on food intake or bodyweight while non-fasting blood glucose levels were attenuated from day 1 of the study; however these results were independent of improvement in insulin sensitivity or glucose tolerance. PEPCK gene expression was attenuated in liver, whereas glucocorticoid receptor and 11~-HSDl gene expression was not affected in either liver of adipose tissue following treatment. Taken together, these studies highlight the potential value of selective 11 ~-HSD 1 inhibition as a new target for the treatment of type 2 diabetes and obesity. XXll The glucocorticoid receptor is another attractive therapeutic target, and as such we employed the glucocorticoid receptor antagonist RU486 to block glucocorticoid action. Results in BRIN-BDll cells showed that incubation with RU486 protected from the inhibitory effects of dexamethasone on insulin secretion, which may be in part due to a reduction in glucokinase expression. Long-term results in obese diabetic mice receiving RU486 daily over 28 days showed a significant reduction in blood glucose, improved glucose tolerance and insulin sensitivity, independent of changes in food intake and body weight. Gene expression analysis revealed a significant reduction in hepatic mRNA PEPCK, G6Pase and l1~-HSDl, while in adipose tissue 11 ~- HSD 1 and LPL were reduced. These data demonstrate wide ranging effects of glucocorticoid receptor antagonism on gene expression and metabolism, illustrating the therapeutic potential of specific glucocorticoid receptor in obesity-diabetes. Glucose-dependent insulinotropic polypeptide (GIP) is a major physiological incretin with important insulin releasing and glucose-lowering properties both within and outside the pancreatic islets. Antagonism of GIP in this situation through the use of (Pr03)GIP has proven beneficial, attenuating hyperinsulinemia, hyperglycaemia, and body weight. Therefore a study was designed to characterise the actions of novel truncated (Pro3) based GIP antagonist peptides. At the cellular level (Pro3)GIP(2- 42), (Pr03)GIP(3-42) and (P2•3)GIP(2-42) had no significant effects, however (p1•2•3)GIP was resistant to the actions of DPP-IV, attenuated the insulin-stimulatory effects of native GIP and inhibited GIP stimulated cAMP production. In vivo, (p1•2•3)GIP acutely elevated overall glucose concentration over 60-minutes when compared to GIP control long-term dosing of (p1•2•3)GIP did not however significantly affect the 'diabesity' syndrome observed in leptin deficient ob/ob mice. These data demonstrate that although (p1,2,3)GIP was established as a novel antagonist of the GIP receptor in vitro, and acutely in vivo, it did not improve upon the biological effectiveness of the established GIP receptor antagonist (Pr03)GIP in a genetic model of diabetes and obesity. Xenin-25 is eo-secreted from the GIP producing K-cell but its effects on glucose homeostasis have been poorly examined. Results in this thesis demonstrated that in BRIN-BDll cells, xenin-25 has a stimulatory effect on insulin secretion at IIlM at nonno and hyper-glycaemic glucose levels, as well as an additive effect to GIP, GLP-l and neurotensin mediated insulin secretion. Xenin-25 did not have a stimulatory effect on cAMP, which is contrary to the effects of GIP. Ex vivo and in vivo studies showed xenin-25 to be degraded rapidly over 6 hours in murine plasma, to have short acting satiety effects at high concentrations, as well as having acute glucose homeostatic properties in normal mice in fed and fasted states. These effects were however found to be short acting as a similar response to glucose bolus was not observed dosed l h previously with xenin-25. Collectively these results will hopefully prompt further investigation into longer-acting and more potent mimetics and degradation products ofxenin in models of diabetes and obesity. As a whole this thesis demonstrates the attractive qualities of glucocorticoid blockade and the use ofK-cell derived factors in the treatment of type 2 diabetes.

hisulin resistance is a characteristic halhnark of type 2 diabetes and contributes to impaked glucose disposal and utilization in insulin-sensitive tissues (liver, skeletal muscle and fat) and inadequate suppression of postprandial hyperglycaemia. Elevated hepatic and intramuscular triglyceride (TG) content in type 2 diabetes has been postulated to underpin the insulin resistance seen in each of these issues. The reasons for this increased tissue TG content is poorly understood. Reduction in liver TG content with weight loss normalizes basal rates of EGP. Likewise peroxisome proliferator activated receptor-7 agonists (PPAR-y) and peroxisome proliferator activated receptor-a agonists (PPAR-a) also decrease liver TG content, but their effects on EGP is unclear. A mutation in PPP1R3A, the gene encoding muscle-specific glycogen-regulating subunit, a key regulator of muscle glycogen metabolism has recently been reported. The physiological implication of this genetic variant on human muscle glycogen metabolism is unknown.