Regulation of Digestive Vesicular Glutamate Transporter

描述(申请人提供)：谷氨酸是一种信号分子，在胃肠道的正常生理功能中发挥重要作用，包括组胺诱导的胃酸分泌，以及胃肠的收缩。谷氨酸作为一种细胞内信使，参与了葡萄糖诱导的胰岛细胞的胰岛素分泌和胰岛β细胞的胰升糖素分泌。功能性谷氨酸系统主要存在于胃、肠和胰腺等消化器官中。囊泡谷氨酸转运体摄取谷氨酸进入分泌颗粒是谷氨酸释放的限速步骤。我们实验室最近克隆了一个神经元囊泡谷氨酸转运蛋白(VGLUT2)，并对其进行了功能鉴定，该转运蛋白在胰腺的α细胞和β细胞中表达。本实验室的长期目标是研究囊泡谷氨酸转运体在消化系统中的调节。本研究的主要目的是确定葡萄糖对胰腺囊泡型谷氨酸转运体基因表达的调节机制。这项提议中要检验的假设是，在β细胞和α细胞中，VGLUT2分别受到高和低浓度葡萄糖的长期调节，是通过转录机制实现的。这一假说得到了强有力的初步数据的支持，包括(I)高糖浓度(12.8 mM)增加了囊泡谷氨酸在细胞内的转运，而低糖(2.8 mM)增加了囊泡谷氨酸的转运；(Ii)高糖浓度(12.8 mM)和低糖浓度(2.8 mM)分别增加了β细胞和α细胞中VGLUT2 mRNA的表达，并且这种变化可被放线菌素D阻断；(Iii)在遗传性非胰岛素依赖型糖尿病小鼠模型中，VGLUT2 mRNA的表达增加。我们建议通过三个具体目标来研究VGLUT2的调控。首先，研究VGLUT2在胞外葡萄糖浓度变化时的转录机制。其次，鉴定VGLUT2启动子，并鉴定葡萄糖反应顺式调控元件。第三，确定参与葡萄糖诱导的VGLUT2调节的反式作用蛋白因子，并确定它们在调节VGLUT2中的功能。这项研究的结果将为葡萄糖对VGLUT2的调节以及它在葡萄糖诱导的胰腺胰岛素和胰升糖素胞吐中的功能作用提供基础信息。

DESCRIPTION (provided by applicant): Glutamate is a signaling molecule that plays an important role in the normal physiological function of gastrointestinal tract including histamine-induced acid secretion in stomach, and contractility of the stomach and intestine. As an intracellular messenger, glutamate is involved in glucose-induced insulin exocytosis in pancreatic (-cells and glucagon exocytosis in pancreatic beta cells. The functional glutamaterigic systems have been characterized in digestive organs including stomach, intestine, and pancreas. Glutamate uptake into the secretory granules by vesicular glutamate transporter is a rate-limiting step for glutamate release. Our laboratory has recently cloned and functionally characterized a neuronal vesicular glutamate transporter (VGLUT2) that is expressed in pancreatic alpha and beta cells. The long-term goal of our laboratory is to study the regulation of vesicular glutamate transporter in digestive system. The primary purpose of this proposal is to determine the mechanisms of glucose-induced regulation of vesicular glutamate transporter gene expression in pancreas. The hypothesis to be tested in this proposal is that chronic regulation of VGLUT2 in beta and alpha cells, by high and low concentrations of glucose, respectively, is via transcriptional mechanisms. This hypothesis is supported by strong preliminary data including (i) high glucose concentration (12.8 mM) increases vesicular glutamate transport in ( cells and low glucose concentration (2.8 mM) increases vesicular glutamate transport in beta cells, (ii) VGLUT2 mRNA expression is increased by high glucose concentration in beta cells and by low glucose concentration in alpha cells, and the changes of mRNA expression can be blocked by actinomycin D, (iii) VGLUT2 mRNA is increased in genetic mouse model of non-insulin-dependent diabetes. We propose to study the regulation of VGLUT2 by three specific aims. First, characterize the transcriptional mechanism of VGLUT2 in response to changes of extracellular glucose concentration. Second, characterize VGLUT2 promoter and identify glucose-response cis-acting regulatory elements. Third, identify trans-acting protein factors involved in glucose-induced regulation of VGLUT2 and determine their functional role in the regulation of VGLUT2. The results of this study should provide fundamental information on the regulation of VGLUT2 by glucose, and on its functional roles in glucose--induced insulin and glucagon exocytosis in the pancreas.