"Role of the SNARE proteins in exocytosis of the intestinal hormone, glucagon-like peptide-1"

“SNARE 蛋白在肠道激素胰高血糖素样肽-1 胞吐作用中的作用”

• 批准号: 418329-2012
• 负责人: Brubaker, Patricia
• 金额: $ 2.91万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=515737
• 关键词: Role; SNARE; proteins; exocytosis; intestinal

The ability to fuse membranes is a fundamental property of all cells that release hormones into the circulation. My laboratory has extensively studied the intestinal endocrine L cells that secrete the insulinotrophic hormone, glucagon-like peptide-1 (GLP-1), in response to secretagogues that are either calcium-dependent (e.g. glucose) or calcium-independent (e.g. oleic acid, via PKCzeta, and insulin, via the RhoGTPase, Cdc42). Although components of the exocytotic 'SNARE' machinery responsible for calcium-dependent have been investigated, nothing is known about their role in oleic acid- or insulin-induced GLP-1 secretion. The goal of the proposed studies is, thus, to identify the molecular mediators of calcium-independent exocytosis of GLP-1, as a prototypic gut hormone. (Aim 1) To determine the SNARE proteins involved in oleic acid- and insulin-induced GLP-1 release, we will analyze secretion (by RIA) and exocytosis (by TIRF microscopy) using the validated GLUTag L cell line following SNARE protein knock-down. These results will then be validated (Aim 2) using physiologic models of the L cell. Hence, syntaxin1A null mice (or controls) will be given luminal oleic acid or insulin plus glucose and changes in GLP-1 release (vs. wild-type controls) will be determined. For embryonic/neonatal lethal VAMP2, syntaxin1B and SNAP25 null mice, fetal intestines will be placed into culture to determine alterations in secretagogue-induced GLP-1 release (vs. controls). These methods will also permit future analysis of the secretion of other gut hormones. (Aim 3) Finally, to determine the interactions between PKCzeta, Cdc42 and the SNARE machinery, GLUTag cells will undergo co-immunoprecipitation and immunoblot analysis for all of these proteins. The results of these studies will elucidate the molecular machinery underlying calcium-independent exocytosis of GLP-1, a prototypic gut hormone, and will lead to identification of target molecules to manipulate the secretion of other intestinal hormones with important roles in nutrient homeostasis.

融合细胞膜的能力是所有将荷尔蒙释放到循环中的细胞的基本特性。我的实验室已经对肠内分泌L细胞进行了广泛的研究，这些细胞分泌胰岛素营养激素-胰升糖素样肽-1(GLP-1)，对钙依赖(例如葡萄糖)或非钙依赖的促分泌剂(例如油酸，通过PKzzeta，以及胰岛素，通过RhoGTPase，CDC42)做出反应。虽然已经研究了导致钙依赖的胞吐“陷阱”机制的成分，但对它们在油酸或胰岛素诱导的GLP-1分泌中的作用尚不清楚。因此，这项研究的目的是确定GLP-1作为一种典型的胃肠激素的钙非依赖性胞吐的分子介质。(1)为了确定参与油酸和胰岛素诱导的GLP-1释放的SNARE蛋白，我们将在SNARE蛋白敲除后，利用验证的GLUTag L细胞系来分析分泌(用放射免疫分析)和胞吐(用放射免疫荧光显微镜)。然后将使用L细胞的生理模型对这些结果进行验证(目标2)。因此，Synaxin1A基因缺失的小鼠(或对照组)将被给予鲁米那油酸或胰岛素加葡萄糖，并将确定GLP-1释放的变化(与野生型对照组相比)。对于胚胎/新生儿致死的VAMP2、Synaxin1B和SNAP25缺失的小鼠，胎儿肠道将被置于培养中，以确定分泌剂诱导的GLP-1释放的变化(与对照组相比)。这些方法也将使未来能够分析其他肠道激素的分泌。(目的3)最后，为了确定PKzzeta、CDC42和圈套机制之间的相互作用，GLUTag细胞将对所有这些蛋白进行免疫共沉淀和免疫印迹分析。这些研究结果将阐明GLP-1--一种典型的肠道激素--钙非依赖性胞吐的分子机制，并将导致识别靶分子来操纵其他在营养动态平衡中具有重要作用的肠道激素的分泌。