Investigation of the roles of WFS1 and glutamate dehydorogenase on the pancreatic β-cell function and regulation of insulin secretion

点击翻译按钮获取中文摘要

The WFSI gene encodes ar. endoplasmic reticulum (ER) membrane-resided protein, Homozygous loss of function mutations of the WFSI gene cause wolfram syndrome, characterized by insulin-defficient diabetes mellitus and optic atrophy. In the patient's islets, β-cells are selectively lost, In the current study, we demonstrated that β-cells were the major site of the WFSI expression. The WFSI expression was transcriptionally up-regulated by chemical insults inducing ER stress. Treatment of fibroblasts and MIN6 cells with reagents such as thapsigargin or tunicamycin increased WFSI mRNA and protein levels. The WFSI expression was also increased in the Akita mouse-derived insulin-2^<96Y/Y> insulinoma cells. In these cells, ER stress was intrinsically induced by the misfolded mutant insulin expression. The WFSI gene promoter-luciferase reporter system revealed that the human WFSI promoter was activated by chemically-induced ER stress in MIN6 cells, and the promoter was more active in the insulin … More -2^<96Y/Y> insulinoma cells than in insulin-2^<wild/wild> cells. Collectively, our data demonstrated that WFSI expression is associated with ER stress, and suggested functional link between WFSI and the ER stress responses. Loss of function mutations of WFSI gene may cause β-cell loss due to ER stress-induced apoptosisGlutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of L-glutamate to α-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Gain-of function mutations of the GDH gene cause hyperinsulinism/hyper-ammonemia (HI/HA) syndrome. GDH266C is a constitutively activated mutant enzyme. By overexpressing GDH266C in MINE mouse insulinoma cells, we demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. To clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutaminestimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C.These results demonstrate that upon activation, GDH oxidizes glutamate to α-ketoglutarate thereby stimulating insulin secretion by providing the TCA cycle with a substrate Less