title: Interaction between Munc13-1 and RIM is critical for glucagon-like peptide-1 mediated rescue of exocytotic defects in Munc13-1 deficient pancreatic beta-cells.
abstract: OBJECTIVE
  Glucagon-like peptide-1 (GLP-1) rescues insulin secretory deficiency in type 2 diabetes partly via cAMP actions on exchange protein directly activated by cAMP (Epac2) and protein kinase A (PKA)-activated Rab3A-interacting molecule 2 (Rim2). We had reported that haplodeficient Munc13-1(+/-) mouse islet beta-cells exhibited reduced insulin secretion, causing glucose intolerance. Munc13-1 binds Epac2 and Rim2, but their functional interactions remain unclear.
  
  
  METHODS
  We used Munc13-1(+/-) islet beta-cells to examine the functional interactions between Munc13-1 and Epac2 and PKA. GLP-1 stimulation of Munc13-1(+/-) islets normalized the reduced biphasic insulin secretion by its actions on intact islet cAMP production and normal Epac2 and Rim2 levels.
  
  
  RESULTS
  To determine which exocytotic steps caused by Munc13-1 deficiency are rescued by Epac2 and PKA, we used patch-clamp capacitance measurements, showing that 1) cAMP restored the reduced readily releasable pool (RRP) and partially restored refilling of a releasable pool of vesicles in Munc13-1(+/-) beta-cells, 2) Epac-selective agonist [8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate] partially restored the reduced RRP and refilling of a releasable pool of vesicles, and 3) PKA blockade by H89 (leaving Epac intact) impaired cAMP ability to restore the RRP and refilling of a releasable pool of vesicles. Conversely, PKA-selective agonist (N(6)-benzoyladenosine-cAMP) completely restored RRP and partially restored refilling of a releasable pool of vesicles. To determine specific contributions within Epac-Rim2-Munc13-1 interaction sites accounting for cAMP rescue of exocytosis caused by Munc13-1 deficiency, we found that blockade of Rim2-Munc13-1 interaction with Rim-Munc13-1-binding domain peptide abolished cAMP rescue, whereas blockade of Epac-Rim2 interaction with Rim2-PDZ peptide only moderately reduced refilling with little effect on RRP.
  
  
  CONCLUSIONS
  cAMP rescue of priming defects caused by Munc13-1 deficiency via Epac and PKA signaling pathways requires downstream Munc13-1-Rim2 interaction.
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Edwin P
authors.initials: EP
authors.lastname: Kwan
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Laura
authors.initials: L
authors.lastname: Sheu
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohtsuka
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D052216:Glucagon-Like Peptide 1 / Q000494:pharmacology*; D007328:Insulin / Q000172:deficiency / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D018408:Patch-Clamp Techniques
chemical_list: D018528:ATP-Binding Cassette Transporters; C512841:Abca4 protein, mouse; D007328:Insulin; D009419:Nerve Tissue Proteins; C503321:Unc13a protein, mouse; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP