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

融合膜的能力是所有释放激素进入循环的细胞的基本特性。我的实验室广泛研究了肠道内分泌L细胞分泌胰岛素营养激素，胰高血糖素样肽-1 (GLP-1)，以响应钙依赖性（如葡萄糖）或钙非依赖性（如油酸，通过PKCzeta和胰岛素，通过RhoGTPase， Cdc42）。尽管已经研究了钙依赖的胞外“SNARE”机制的组成部分，但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，本研究的目的是确定GLP-1作为一种原型肠道激素的钙不依赖胞吐的分子介质。（目的1）为了确定参与油酸和胰岛素诱导的GLP-1释放的SNARE蛋白，我们将使用经过验证的GLUTag L细胞系分析SNARE蛋白敲除后的分泌（通过RIA）和胞吐（通过TIRF显微镜）。这些结果将随后使用L细胞的生理模型进行验证（目标2）。因此，syntaxin1A无效小鼠（或对照）将被给予鲁米纳油酸或胰岛素加葡萄糖，并将确定GLP-1释放的变化（与野生型对照相比）。对于胚胎/新生儿致死的VAMP2、syntaxin1B和SNAP25缺失小鼠，将将胎儿肠置于培养中，以确定分泌剂诱导的GLP-1释放的变化（与对照组相比）。这些方法也将允许未来分析其他肠道激素的分泌。（目的3）最后，为了确定PKCzeta、Cdc42和SNARE机制之间的相互作用，GLUTag细胞将对所有这些蛋白进行共免疫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）钙非依赖性胞吐作用的分子机制，并将有助于鉴定操纵其他在营养稳态中起重要作用的肠道激素分泌的靶分子。

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.