Medicine (Austin & Northern Health) - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemIdentification and characterisation of genes associated with hyperglycaemia susceptibility and reduced insulin secretion( 2017)The pathogenesis of type 2 diabetes (T2D) involves a complex interplay of genetic and environmental factors which result in impaired insulin secretion in the presence of insulin resistance. A permissive genetic susceptibility is considered a predominant determinant to the onset of hyperglycaemia, however, it has a complex aetiology that is yet to be fully defined. The aim of this thesis was to identify and characterise genes associated with hyperglycaemia. A genetically diverse mouse reference population, the Collaborative Cross (CC), was utilised to identify and characterise genetic loci influencing blood glucose concentrations. Therefore, a large-scale phenotypic screening of random blood glucose, insulin sensitivity and body weight across 652 males from 53 inbred CC strains (n=3-15) and 467 females from 48 CC strains (n=3-10) was conducted. The CC mouse population exhibits an abundant diversity in blood glucose levels as well as in insulin sensitivity and body weight. Interesting to note that strains which have blood glucose levels within the top range are rather lean and sensitive to insulin-mediated glucose lowering, indicative of impaired insulin secretion. To identify the genetic loci contributing to this variation in blood glucose concentrations, an unbiased genome-wide association study (GWAS) and quantitative trait loci (QTL) mapping were performed using the random blood glucose data. Two independent loci on chromosome 7 were identified to be significantly (p <5x10e-8) associated with high blood glucose, moreover, the contributory founder haplotype was demonstrated to be the NZO-derived alleles at both loci according to a haplotype analysis. These implicated loci were then named by the genes nearby peak SNPs as the E2F8 locus (Chr7: 52.6-56.7 Mb) and Dlg2 locus (Chr7: 98.5-101.55 Mb). Potential gene candidates in both loci were determined by sequence analyses throughout all genes in the linkage disequilibrium (LD) blocks, moreover, the property of genes and variants were also taken into account during the selection process. Finally, we narrowed the number of gene candidates down to seven for each locus: Gfy, Hsd17b14, Sphk2, E2F8, Ntn5, Abcc8 and Kcnj11 were identified at the E2F8 locus; Dlg2, Ccdc90b, Andkrd42, Pcf11, Ddias, Rab30 and Prcp for the Dlg2 locus. It was important to find that, to some extent, both E2F8 and Dlg2 loci have been implicated in diabetes-related abnormalities, but the functional defects underlying these deleterious alleles have yet to be fully defined. In order to study the impact of the NZO-derived hyperglycaemia susceptibility alleles on glucose homeostasis, we conducted in vivo and in vitro examinations on the hyperglycaemic CC strains, PIPING and PUB mice, which bear the deleterious alleles from NZO mice at susceptibility loci. Compared with the C57BL/6 mice as a reference strain, the hyperglycaemia susceptible strains are severely glucose intolerant in an oral glucose tolerance test (2 g/kg glucose), due to substantial reduction in insulin secretion in response to glucose and arginine stimulation in vivo. This secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta-cell mass and pancreatic insulin content, but rather as the result of inherent pancreatic beta-cell dysfunction. On the other hand, the genetic predisposition to hyperglycaemia appears to drive the susceptible mice to become more sensitive to dietary fat-induced weight gain and beta-cell decompensation. As evidenced by the findings that PIPING and PUB mice were susceptible to high fat diet-induced weight gain, which in partly due to increased fat intake but seems independent of higher caloric intake. Furthermore, PIPING and PUB mice developed advanced glucose intolerance on high-fat diet as a result of failure to raise insulin secretion for the increased demand. These results provide evidence that the hyperglycaemia susceptibility loci, E2F8 and Dlg2 loci, have substantial influences on pancreatic beta-cell function. Further investigation on the relationship of candidate gene expression with diabetes susceptibility was determined by identifying differential gene expression in primary islets between C57BL/6 (diabetes-resistant) and NZO (diabetes-susceptible) mice. On the basis of results from genetic mapping and expression analysis, E2F8 and Dlg2 genes were prioritised as promising candidates for reduced pancreatic insulin secretion. To determine whether E2F8 and Dlg2 are involved in pancreatic insulin secretion, E2F8 and Dlg2 were knocked down using siRNA and lentiviral transduction of shRNA, respectively, in a mouse pancreatic beta-cell line, MIN6 cells. The results of insulin secretion in E2F8 knockdown cells showed that down-regulation of E2F8 resulted in reduced insulin secretion in response to glucose as well as arginine and tolbutamide but not to GLP-1 and KCl, suggesting E2F8 mediates pancreatic insulin secretion in a K+ATP -channel dependent manner. Furthermore, knockdown of Dlg2 expression in MIN6 cells led to a generalised attenuation of insulin secretion regardless of glucose or other non-glucose secretagogues. These data showed the first evidence that the expression of E2F8 and Dlg2 affect insulin secretory function in pancreatic beta-cells, which can therefore influence individual’s susceptibility to diabetes. Taken together, this thesis provides evidence of two important loci for hyperglycaemia susceptibility which are linked to impaired insulin secretory function in the pancreatic beta-cells. Importantly, our results identified a number of novel candidate genes that are likely to alter insulin secretion in the pancreatic islets. Among these, E2F8 and Dlg2 were validated and recognised as novel genes to be involved in the pathogenesis of hyperglycaemia through altering pancreatic insulin secretion. This thesis delineates the use of the CC to identify novel genes for complex traits and understand the molecular control of insulin secretion.