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

WFSI基因编码一种内质网（ER）膜蛋白，WFSI基因的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-细胞选择性丢失，在目前的研究中，我们证明β-细胞是WFSI表达的主要部位。化学损伤诱导内质网应激可上调WFSI的表达。用thapsigargin或tunicamycin等试剂处理成纤维细胞和MIN6细胞可增加WFSI mRNA和蛋白水平。在秋田小鼠源性胰岛素-2^<96Y/ y>胰岛素瘤细胞中，WFSI的表达也有所增加。在这些细胞中，内质网应激本质上是由错误折叠的突变胰岛素表达诱导的。WFSI基因启动子-荧光素酶报告系统显示，人WFSI启动子在MIN6细胞中可被化学诱导的内质网应激激活，并且该启动子在胰岛素-2^<96Y/Y>胰岛素瘤细胞中比在胰岛素-2^<野生/野生>细胞中更活跃。总的来说，我们的数据表明，WFSI表达与内质网应激有关，并提示WFSI与内质网应激反应之间存在功能联系。谷氨酸脱氢酶（glutamate dehydrogenase， GDH）可催化l -谷氨酸可逆氧化脱胺生成α-酮戊二酸。酶的活性受几种变构效应物的调节。GDH基因获得性功能突变导致高胰岛素血症/高氨血症（HI/HA）综合征。GDH266C是一种组成激活的突变酶。通过在MINE小鼠胰岛素瘤细胞中过表达GDH266C，我们证明了GDH活性不受调节的升高，使细胞对谷氨酰胺的胰岛素分泌有反应。有趣的是，在低葡萄糖浓度下，这些细胞的基础胰岛素分泌被夸大了。为了阐明GDH在调节胰岛素分泌中的作用，我们利用过表达GDH266C的MIN6细胞（MIN6-GDH266C）研究了细胞谷氨酸代谢。谷氨酰胺刺激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5 mmol/l葡萄糖不含谷氨酰胺时，谷氨酰胺氧化也增加，谷氨酸含量随着胰岛素分泌的增加而降低。葡萄糖氧化未发生改变。过表达gdh266c的分离大鼠胰岛胰岛素分泌谱与MIN6-GDH266C相似。这些结果表明，激活后，GDH将谷氨酸氧化为α-酮戊二酸，从而通过为TCA循环提供底物Less来刺激胰岛素分泌

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