Poster Session 2 - E8
1Jhenielle Campbell, 1,2Patricia Brubaker
1 Dept. of Physiology, University of Toronto; 2 Dept. of Medicine, University of Toronto
Background: The incretin hormone, glucagon-like peptide-1 (GLP-1) is an integral player in the maintenance of glucose homeostasis. GLP-1 is secreted by the intestinal epithelial L cell; it acts to enhance glucose-stimulated insulin secretion from pancreatic beta cells following nutrient intake. As a result, GLP-1 receptor agonists are currently used as a therapy for Type II Diabetes (TIID). A new paradigm in GLP-1 based TIID therapeutics is focused on the prospect of increasing the endogenous secretion of GLP-1 from the L cell. The targets of such therapies require an understanding of the regulation of GLP-1 exocytosis, which remains elusive in the field. There are multiple signalling pathways leading to the exocytosis of GLP-1 containing secretory vesicles from the L cell, depending on the acting secretagogue. However, all of these pathways converge on the activation of Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. Syntaxin, vesicle associated membrane protein (VAMP), and SNAP25 are the three core SNARE proteins which mediate membrane fusion. Previous studies on VAMP2 and syntaxin-1a in the L cell show that they play an essential role in GLP-1 secretion. Rationale: Syntaxin-1a is regulated by Munc18-1, a SNARE regulatory protein. Munc18-1 binds to syntaxin-1a to induce syntaxin-1a’s open conformation, allowing it to associate with the other core SNAREs. It has been previously shown that Munc18-1 co-immunoprecipitates with syntaxin-1a, and that Munc18-1 levels are positively correlated to peak GLP-1 secretion in mGLUTag cells – a murine intestinal L cell line. Hypothesis: In accordance with these findings, we hypothesize that Munc18-1 is an integral regulator of GLP-1 exocytosis from the L cell, and that the mGLUTag cells are an appropriate in vitro model in which to evaluate this hypothesis. Methods: A 2-hour GLP-1 secretion assay was conducted on the mGLUTag cells. The cells were treated with either 50 µM forskolin with 50 µM IBMX (3-isobutyl-1-methylxanthine) or 10-7 M glucose-dependent insulinotropic polypeptide (GIP), both known GLP-1 secretagogues. Peptides were collected from the cells and media; GLP-1 was quantified by radioimmunoassay in both compartments to calculate percent secretion. Normal mRNA and protein levels of Munc18-1 in the mGLUTag cells were also assessed with real time – quantitative PCR (RT-qPCR) and western blot, respectively. Munc18-1 knockdown (KD) studies were also conducted in the cells using an siRNA mediated reverse transfection technique. The results of the transfection were quantified by RT-qPCR. Results: The mGLUTag cells exhibited characteristic GLP-1 secretion levels in response to the treatments. Both forskolin/IBMX and GIP significantly increased GLP-1 secretion (n=4) in comparison to basal levels (p<0.01 and, p>0.05, respectively). In addition, the presence of Munc18-1 mRNA and protein in the cells was confirmed. mGLUTag cells transfected with siRNA (n=2) demonstrated a greater than 50% reduction in Munc18-1 mRNA compared to cells transfected with scrambled RNA (scRNA; control). Conclusion: The mGLUTag cells are an appropriate model with which to assess the role of Munc18-1 in GLP-1 secretion from the L cell. The cells exhibit a suitable response to GLP-1 secretagogues and can be successfully transfected to create Munc18-1 KD L cells for use in future GLP-1 secretion assays.