ARTICLE Role of vesicle-associated membrane protein 2 in exocytosis of glucagon-like peptide-1 from the murine intestinal L cell Samantha K. Li & Dan Zhu & Herbert Y. Gaisano & Patricia L. Brubaker Received: 22 August 2013 /Accepted: 22 November 2013 /Published online: 20 December 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Aims/hypothesis Glucagon-like peptide-1 (GLP-1), secreted by the enteroendocrine L cell, is an incretin hormone that potently stimulates insulin secretion. Although signalling pathways promoting GLP-1 release are well characterised, the mechanisms by which GLP-1-containing granules fuse to the L cell membrane are unknown. As soluble NSF attach- ment proteins (SNAREs) are known to mediate granule– membrane fusion, the role of vesicle-associated membrane proteins (VAMPs) in GLP-1 exocytosis was examined. Methods SNARE expression was determined in murine GLUTag L cells by RT-PCR and immunoblot and in primary murine L cells by immunofluorescence. Co- immunoprecipitation was used to examine SNARE interac- tions, while tetanus toxin (TetX)-mediated cleavage of VAMP was used with a GLP-1 secretion assay and total internal reflection fluorescence microscopy to determine the role of VAMP2 in exocytosis. Results VAMP2 was expressed in murine L cells and localised to secretory granules in GLUTag cells. VAMP1/3 and the core membrane proteins syntaxin1a and synaptosomal- associated protein 25 kDa (SNAP25) were also detected. TetX cleaved VAMPs in GLUTag cells. However, only VAMP2 interacted with syntaxin1a, as did SNAP25 and Munc18-1. TetX treatment of GLUTag cells prevented glucose-dependent insulinotrophic peptide- and oleic-acid-stimulated GLP-1 secretion ( p <0.05–0.01), as well as K + -stimulated single-cell exocytosis ( p <0.05–0.001), while TetX-resistant VAMP2 ex- pression rescued GLP-1 secretion ( p <0.01–0.001). Conclusions/interpretation Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag L cell in response to a variety of established secre- tagogues. An improved understanding of the mechanisms governing the release of GLP-1 may lead to new therapeutic approaches to enhance the levels of this incretin hormone in patients with type 2 diabetes. Keywords Exocytosis . GLP-1 . Incretin . L cell . Secretion . SNARE . Tetanus toxin . TIRF . VAMP Abbreviations GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFP Green fluorescent protein GLP-1 Glucagon-like peptide-1 GIP Glucose-dependent insulinotrophic peptide NPY Neuropeptide Y pCMV Porcine cytomegalovirus R Resistant SNARE Soluble NSF attachment protein receptor SNAP25 Synaptosomal-associated protein 25 kDa TetX Tetanus toxin TIRF Total internal reflection fluorescence microscopy VAMP Vesicle-associated membrane protein WT Wild type Introduction It is estimated that 347 million people worldwide have diabetes [1], the majority of whom have type 2 diabetes. Current Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-3143-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users. S. K. Li : H. Y. Gaisano : P. L. Brubaker (*) Department of Physiology, Medical Sciences Building, 1 King’ s College Circle, University of Toronto, Toronto, ON M5S 1A8, Canada e-mail: p.brubaker@utoronto.ca D. Zhu : H. Y. Gaisano : P. L. Brubaker Department of Medicine, University of Toronto, Toronto, ON, Canada Diabetologia (2014) 57:809–818 DOI 10.1007/s00125-013-3143-2