Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation
Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation
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DOI：
10.1152/ajpendo.00380.2003
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发表时间：
2004-02-01
影响因子：
5.1
通讯作者：
Tanizawa, Y
中科院分区：
文献类型：
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作者：
Anno, T;Uehara, S;Tanizawa, Y
谷氨酸脱氢酶（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。高胰岛素血症/高氨血症（HI/HA）综合征是由GDH功能获得性突变引起的一种新的高胰岛素血症性低血糖症，它的发现突出了GDH在葡萄糖稳态中的重要性。GDH 266 C是我们在HI/HA综合征患者中鉴定的组成型激活突变酶。通过在MIN 6小鼠胰岛素瘤细胞中过表达GDH 266 C，我们先前证明了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。在此，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。谷氨酰胺刺激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。来自GDH 266 C过表达的分离的大鼠胰岛的胰岛素分泌谱与来自MIN 6-GDH 266 C的胰岛素分泌谱相似，表明MIN 6细胞中的观察结果与天然β细胞相关。这些结果表明，在活化时，GDH将谷氨酸氧化为α-酮戊二酸，从而通过为TCA循环提供底物来刺激胰岛素分泌。没有证据支持激活的GDH通过逆反应产生谷氨酸（最近提出的胰岛素分泌的第二信使）以刺激胰岛素分泌的假设。
Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of L-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously 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. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated 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, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.