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

WFS1和谷氨酸脱氢酶对胰腺β细胞功能和胰岛素分泌调节作用的研究

基本信息

批准号：
14370338
负责人：
TANIZAWA Yukio
金额：
$ 8.7万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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

WFSI基因编码ar. WFSI基因是内质网（ER）膜驻留蛋白，WFSI基因的纯合性功能丧失突变导致W综合征，其特征在于胰岛素缺乏型糖尿病和视神经萎缩。在患者的胰岛中，β细胞选择性丢失。在当前的研究中，我们证明β细胞是WFSI表达的主要位点。化学损伤诱导ER应激可上调WFSI的表达。用试剂如毒胡萝卜素或衣霉素处理成纤维细胞和MIN 6细胞增加了WFSI mRNA和蛋白水平。在秋田小鼠来源的胰岛素-2 ^<96 Y/Y>胰岛素瘤细胞中，WFSI表达也增加。在这些细胞中，内质网应激本质上是由错误折叠的突变体胰岛素表达诱导的。WFSI基因启动子-荧光素酶报告系统显示，人WFSI启动子在MIN 6细胞中被化学诱导的ER应激激活，并且该启动子在胰岛素诱导的ER应激中更有活性。关于我们在胰岛素瘤细胞中，在胰岛素-2 96 Y/Y>细胞中的胰岛素水平高于在胰岛素-2 <野生型/野生型>细胞中的胰岛素水平。总的来说，我们的数据表明，WFSI表达与ER应激，并建议WFSI和ER应激反应之间的功能联系。WFSI基因突变可导致内质网应激诱导的胰岛β细胞功能丧失。谷氨酸脱氢酶（Glutamate dehydrogenase，GDH）催化L-谷氨酸可逆氧化脱氨为α-酮戊二酸。酶活性受几种变构效应物调节。GDH基因的功能获得性突变引起高胰岛素血症/高氨血症（HI/HA）综合征。GDH 266 C是一种组成型激活的突变酶。通过在MINE小鼠胰岛素瘤细胞中过表达GDH 266 C，我们证明了GDH活性的不受调节的升高使细胞在胰岛素分泌中对谷氨酰胺有反应。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，我们使用过表达GDH 266 C的MIN 6细胞（MIN 6-GDH 266 C）研究了细胞谷氨酸代谢。谷氨酰胺抑制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5 mmol/l葡萄糖无谷氨酰胺时，谷氨酰胺氧化也增加，谷氨酸含量降低，胰岛素分泌增加。葡萄糖氧化没有改变。GDH 266 C过表达的大鼠胰岛细胞的胰岛素分泌曲线与MIN 6-GDH 266 C相似。这些结果表明，GDH激活后，将谷氨酸氧化为α-酮戊二酸，从而通过为TCA循环提供底物刺激胰岛素分泌。