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起重要作用，包括组胺诱导的胃分泌酸，胃和肠的收缩。谷氨酸作为细胞内信使，参与葡萄糖诱导的胰腺（-）细胞胰岛素分泌和胰高血糖素分泌。在消化器官中，包括胃、肠和胰腺，都有功能性的谷氨酸系统。谷氨酸通过囊泡转运体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并功能表征了在胰腺α细胞和β细胞中表达的神经元囊泡谷氨酸转运蛋白（VGLUT2）。本实验室的长期目标是研究消化系统中泡状谷氨酸转运蛋白的调控。本提案的主要目的是确定葡萄糖诱导的胰腺泡状谷氨酸转运蛋白基因表达调控机制。本研究要验证的假设是，在β细胞和α细胞中，分别通过高浓度和低浓度葡萄糖对VGLUT2的慢性调节是通过转录机制进行的。这一假设得到了大量初步数据的支持，包括：(1)高葡萄糖浓度（12.8 mM）增加细胞中谷氨酸囊泡转运，低葡萄糖浓度（2.8 mM）增加β细胞中谷氨酸囊泡转运，(2)β细胞中高葡萄糖浓度和α细胞中低葡萄糖浓度增加VGLUT2 mRNA表达，放线菌素D可以阻断mRNA表达的变化。（iii）非胰岛素依赖型糖尿病小鼠遗传模型中VGLUT2 mRNA表达升高。我们提出从三个方面研究VGLUT2的调控。首先，表征VGLUT2响应胞外葡萄糖浓度变化的转录机制。其次，表征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调控的反式作用蛋白因子，确定其在VGLUT2调控中的功能作用。本研究的结果将为葡萄糖调控VGLUT2及其在葡萄糖诱导的胰腺胰岛素和胰高血糖素分泌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.