UBC Theses and Dissertations
The role of Npas4 and Rgs2 in the regulation of pancreatic β-cell function Speckmann, Thilo
Pancreatic β-cells regulate systemic glycemia by releasing the glucose-lowering hormone insulin. Diabetes, a chronic metabolic disease characterized by insulin insufficiency, is linked to β-cell dysfunction with perturbed calcium homeostasis. The activity-induced, calcium-dependent transcription factor, NPAS4, reduced insulin secretion and promoted β-cell health, in part through its target gene, the GTPase-activating protein RGS2. Because our mechanistic understanding of this process remains incomplete, studying the normal physiology of calcium-dependent β-cell function may uncover new avenues for the treatment or prevention of diabetes. The overall goal of my thesis was to establish whether activity-induced NPAS4 and RGS2 expression could optimize β-cell function. Initially, I uncovered a role for CaMKII, calcineurin, and PKB in membrane depolarization-induced Npas4 mRNA and protein expression in MIN6 cells and mouse islets. Calcineurin inhibition and concurrent loss of NPAS4 showed cytotoxic increases in cleaved caspase 3 expression, which was reversed by adenovirally reinstating NPAS4 in MIN6 cells. Co-immunoprecipitation studies in MIN6 cells then uncovered competition between NPAS4 and a related transcription factor, HIF1α, for the shared heterodimerization partner, ARNT. Accordingly, HIF1α target gene expression was lower in human and mouse islets overexpressing Npas4, and higher in β-cell-specific Npas4 knockout mouse islets (N4KO). Because excessive HIF1α signalling compromises β-cell function by switching energy production from oxidative phosphorylation to anaerobic glycolysis, I examined whether N4KO mice developed functional defects. Indeed, N4KO islets showed lower oxygen consumption rate, and HFD-fed N4KO mice developed mild glucose intolerance. To understand how NPAS4 may counteract these defects, I identified shared DNA binding sites of NPAS4 and ARNT in MIN6 cells using ChIP-Seq. Among the shared sites, I observed NPAS4 and ARNT binding near Rgs2, corroborating an earlier study. I then demonstrated that RGS2 is a negative regulator of glucose-stimulated insulin secretion (GSIS), because Rgs2-overexpressing MIN6 cells and mouse islets showed reduced GSIS, due to lower calcium influx and oxygen consumption, whereas Rgs2 knockout cells exhibited increased GSIS. In sum, I demonstrated that NPAS4 and its target gene, RGS2, are important regulators of β-cell function. This suggests that these two factors could be promising therapeutic targets to promote β-cell health and optimize insulin secretion in diabetes.
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