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

WFSI基因编码ar。内质网(ER)膜驻留蛋白、WFSI基因纯合子功能缺失突变导致Wolfram综合征，以胰岛素缺乏性糖尿病和视神经萎缩为特征。在患者的胰岛中，β-细胞被选择性地丢失，在本研究中，我们证明了β-细胞是WFSI表达的主要部位。化学胁迫诱导的内质网应激可上调WFSI的转录水平。用thapsigargin或衣霉素等试剂处理成纤维细胞和MIN6细胞可增加WFSI的mRNA和蛋白水平。WFSI在秋田小鼠来源的胰岛素瘤细胞中的表达也增加。在这些细胞中，内质网应激是由错误折叠的突变胰岛素表达内在地诱导的。Wfsi基因启动子-荧光素酶报告系统显示，在化学诱导的内质网应激中，人wfsi启动子在MIN6细胞中被激活，并且在胰岛素…中活性更高。胰岛素瘤细胞比野生型/野生型细胞多。总之，我们的数据表明WFSI的表达与内质网应激有关，并提示WFSI与内质网应激反应之间的功能联系。谷氨酸脱氢酶催化L-谷氨酸可逆氧化脱氨为β-酮戊二酸，WFSI基因功能突变的缺失可能导致内质网应激诱导的细胞凋亡。酶的活性受几个变构效应器的调节。GDH基因功能获得突变导致高胰岛素血症/高氨血症(HI/HA)综合征。GDH266C是一种结构性激活的突变酶。通过在小鼠胰岛素瘤细胞中过表达GDH266C，我们证明了GDH活性的异常升高使细胞对胰岛素分泌中的谷氨酰胺有反应。有趣的是，在低糖浓度下，这些细胞的基础胰岛素分泌被夸大了。为了阐明GDH在胰岛素分泌调节中的作用，我们利用高表达GDH266C(MIN6-GDH266C)的MIN6细胞研究了细胞内的谷氨酸代谢。谷氨酰胺估计的胰岛素分泌与谷氨酰胺氧化增加和细胞内谷氨酸含量降低有关。同样，在不含谷氨酰胺的5 mmol/L葡萄糖浓度下，谷氨酰胺氧化增加，谷氨酸含量降低，胰岛素分泌增加。葡萄糖氧化未见改变。高表达GDH266C的大鼠胰岛的胰岛素分泌谱与MIN6-GDH266C相似。这些结果表明，激活后，GDH氧化谷氨酸为α-酮戊二酸，从而通过为TCA循环提供更少的底物来刺激胰岛素的分泌

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