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Abstract
Many studies investigating mechanisms of diet-induced insulin resistance impose controlled experimental conditions (constant insulin stimulation, constant glucose and/or fatty acid availability) designed to make interpretation of results simpler. These models may alter important parameters such as insulin or glucose to levels that are not ever experienced by the animal during normal feeding or fasting. The aim of this thesis was to investigate mechanisms of insulin resistance in the muscle of rodents fed a high- fat, high-sucrose diet (HFHS) under physiologically relevant conditions. We examined glucose uptake and markers of insulin action in the muscle of chow and HFHS rats at four time-points during the normal diurnal cycle. Chow and four week HFHS-fed rats were implanted with chronic venous catheters and were infused with a bolus of glucose tracer in the conscious state to determine glucose dynamics. HFHS animals displayed hyperinsulinemia but had reduced systemic glucose disposal and impaired muscle glucose uptake at the time-points studied across the feeding period. There were no differences in the phosphorylation of the canonical insulin signalling protein Akt or its downstream targets, AS160 and GSK3B in the muscle of chow and HFHS rats. Transfection of a constitutively active Akt into the tibialis muscle of HFHS rats did not rescue diet-induced reductions in insulin-stimulated glucose uptake. Phosphoproteomic analysis of the red quadriceps muscle comparing chow and HFHS identified differentially regulated phosphopeptides that tended to be from proteins involved directly in energy metabolism rather than protein kinases. Proteins involved in the regulation of pyruvate dehydrogenase (PDH) were altered in both the phospho, and total, proteomic analysis. In order to investigate the role of PDH in insulin-stimulated glucose uptake, we utilised the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA). Both acute and chronic administration of DCA in rodents did not affect insulin sensitivity systemically or in muscle, measured by hyperinsulinemic-euglycemic clamp. Overall, these studies suggest that insulin resistance in muscle induced by a relatively short-term of HFHS-feeding is not associated with defective canonical insulin signalling. Additionally, although PDH activity in muscle is reduced by HFHS-feeding, activation of PDH does not rescue lipid-induced insulin resistance. The work in this thesis highlights the advantage of conducting investigations under a variety of experimental conditions (in vivo, ex vivo, free-living) to determine the relevance of molecular processes to whole body metabolism particularly in the context of insulin sensitivity.