title: Role of vesicle-associated membrane protein 2 in exocytosis of glucagon-like peptide-1 from the murine intestinal L cell.
abstract: OBJECTIVE
  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 attachment 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 interactions, 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 expression rescued GLP-1 secretion (p < 0.01-0.001).
  
  
  CONCLUSIONS
  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 secretagogues. 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.
authors.affiliation: Department of Physiology, Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, ON, M5S 1A8, Canada.
authors.forename: Samantha K
authors.initials: SK
authors.lastname: Li
authors.forename: Dan
authors.initials: D
authors.lastname: Zhu
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Brubaker
mesh_terms: D000818:Animals; D002460:Cell Line; D019858:Enteroendocrine Cells / Q000378:metabolism*; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D052216:Glucagon-Like Peptide 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D050988:Vesicle-Associated Membrane Protein 2; D052216:Glucagon-Like Peptide 1