Regulation of Digestive Vesicular Glutamate Transporter

说明（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起重要作用，包括组胺诱导的胃酸分泌以及胃和肠的收缩。 作为细胞内信使，谷氨酸参与葡萄糖诱导的胰腺β细胞中的胰岛素胞吐和胰腺β细胞中的胰高血糖素胞吐。 功能性代谢能系统在包括胃、肠和胰腺的消化器官中具有特征。 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。 我们的实验室最近克隆和功能特点的神经元囊泡谷氨酸转运蛋白（VGLUT 2），在胰腺α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控。 本研究的主要目的是探讨葡萄糖对胰腺囊泡谷氨酸转运体基因表达的调控机制。 本提案中待检验的假设是，分别通过高浓度和低浓度葡萄糖对β细胞和α细胞中VGLUT 2的慢性调节是通过转录机制进行的。 这一假设得到了强有力的初步数据的支持，包括（i）高葡萄糖浓度（12.8 mM）增加囊泡谷氨酸转运，（细胞和低葡萄糖浓度（2.8 mM）增加β细胞中的囊泡谷氨酸转运，（ii）β细胞中的高葡萄糖浓度和α细胞中的低葡萄糖浓度增加VGLUT2 mRNA表达，放线菌素D可阻断VGLUT2mRNA表达的变化。（3）在非胰岛素依赖型糖尿病遗传小鼠模型中，VGLUT2mRNA表达增加。 我们建议通过三个具体目标来研究VGLUT 2的调节。 首先，研究VGLUT2对细胞外葡萄糖浓度变化的反应机制。 第二，鉴定VGLUT2启动子并鉴定葡萄糖应答顺式作用调控元件。 第三，鉴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VGLUT2调节中的功能作用。 这项研究的结果应该提供葡萄糖对VGLUT2的调节，以及其在葡萄糖诱导的胰岛素和胰高血糖素胞吐中的功能作用的基本信息。

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.