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

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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.