Medicine (Austin & Northern Health) - Theses
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ItemInvestigating the relative contribution of obesity and glucose in the development of β-cell dysfunction( 2016)Insulin resistance and impaired insulin secretion are hallmark features that contribute to the development of hyperglycaemia in type 2 diabetes (T2D) and other complications such as kidney failure, stroke and cardiovascular disease. Obesity is considered to be one of the main drivers in expediting hyperglycaemia by inducing insulin resistance in the liver, muscle and fat. These defects place additional stress on the β-cell to increase insulin output to compensate for the prevailing glucose and over time, can result in declined β-cell function and T2D. Pre-clinical and clinical studies investigating interventions that reduce obesity in pre-diabetes have shown that the incidence of T2D can be attenuated by preserving β-cell function through enhanced insulin sensitivity. However, as obesity clearly induces hyperglycaemia, it has become inherently difficult to dissociate the relative contribution of each in the progression of impaired glucose tolerance (IGT) to T2D. Accordingly, the scope of this thesis was to use dietary and pharmacological interventions to determine the contribution of obesity and glucose in the development of metabolic defects associated with T2D, namely glucose intolerance, insulin resistance and β-cell dysfunction. The overall hypothesis was that both obesity and excess glucose contribute to these defects. In order to address the overall aim of this thesis, it was necessary to firstly characterise a pre-clinical model that does not rely on the presence of obesity to drive hyperglycaemia and subsequently, insulin resistance and β-cell dysfunction. We therefore utilised the phosphoenolpyruvate carboxykinase (PEPCK) transgenic rat, which is characterised by a 2-3 fold induction of PEPCK in the liver and kidney that leads to the impaired suppression of endogenous glucose production. Through the use of in vivo and in vitro experimental techniques, we show that the PEPCK transgenic rat develops defective glucose-stimulated insulin secretion in parallel with the worsening of glucose tolerance at 14 weeks of age, and that this is primarily due to the significant reduction in β-cell Glut2 gene expression and the inability of the constituents that make up the K+ATP channel, Sur1 and Kir6.2, to function properly. This defect in insulin secretion progressively worsens by 20 weeks due to the combination of β-cell dysregulation and reduced β-cell mass. We next investigated the potential of the selective glucose-lowering SGLT2 inhibitor, dapagliflozin, in preventing the progression of insulin resistance and β-cell dysfunction in the PEPCK transgenic rat. We show that in older animals with established insulin resistance and β-cell dysfunction, dapagliflozin treatment for 6 weeks resulted in lower body weight gain despite the compensatory increase in food intake due to energy loss from the urine, reduced plasma glucose and insulin levels, and improvements in glucose tolerance which was associated with enhanced insulin sensitivity and glucose uptake in muscle and fat. In addition, dapagliflozin treatment in PEPCK transgenic rats significantly improved GLUT4 protein content in fat while adipocyte number was increased and the size reduced. A subset of PEPCK transgenic rats were also calorie-restricted in order to prevent further weight gain so that they could be used to account for any potential weight-induced insulin-sensitising benefits seen with dapagliflozin treatment. The prevention of weight gain in these PEPCK transgenic rats greatly enhanced peripheral insulin sensitivity to levels comparable with dapagliflozin treatment. Interestingly, dapagliflozin treatment did not preserve β-cell mass or improve the insulin secretory response to glucose. These data suggest that dapagliflozin elicits its effects on the β-cell in an indirect manner by increasing insulin sensitivity and providing an islet β-cell sparring effect. To dissociate the relative contribution of obesity and glucose per se in IGT and β-cell dysfunction, obesity and glucose excess were prevented by commencing calorie-restriction and dapagliflozin treatment prior to any metabolic defects in 5 week-old PEPCK transgenic rats. Our findings show that preventing either obesity or glucose improves glucose tolerance but does not directly increase the insulin secretory capacity when assessed by a hyperglycaemic clamp or static incubation of isolated islets. These results support the hypothesis that both obesity and glucose per se contribute to the development of glucose intolerance, insulin resistance and β-cell dysfunction in the PEPCK transgenic rat and that enhancing insulin sensitivity with either intervention could effectively prevent the decline in β-cell function with age.