Interaction Between Munc13-1 and RIM Is Critical for Glucagon-Like Peptide-1–Mediated Rescue of Exocytotic Defects in Munc13-1–Deficient Pancreatic β-Cells

• Published in: Diabetes (New York, N.Y.) Vol. 56; no. 10; pp. 2579 - 2588
• Main Authors: Kwan, Edwin P, Xie, Li, Sheu, Laura, Ohtsuka, Toshihisa, Gaisano, Herbert Y
• Format: Journal Article
• Language: English
• Published: United States American Diabetes Association 01.10.2007
• Subjects: Animals; ATP-Binding Cassette Transporters - physiology; Chemical properties; Cyclic AMP - metabolism; Exocytosis - drug effects; Exocytosis - physiology; Glucagon-Like Peptide 1 - pharmacology; Insulin - deficiency; Insulin - metabolism; Insulin Secretion; Insulin-Secreting Cells - drug effects; Insulin-Secreting Cells - physiology; Mice; Mice, Knockout; Nerve Tissue Proteins - deficiency; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - physiology; Pancreatic beta cells; Patch-Clamp Techniques; Protein research; Protein-protein interactions; Canada
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/db06-1207

Interaction Between Munc13-1 and RIM Is Critical for Glucagon-Like Peptide-1–Mediated Rescue of Exocytotic Defects in Munc13-1–Deficient Pancreatic β-Cells Edwin P. Kwan 1 , Li Xie 1 , Laura Sheu 1 , Toshihisa Ohtsuka 2 and Herbert Y. Gaisano 1 1 Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada 2 Department of Clinical and Molecular Pathology, University of Toyama, Toyama, Japan Address correspondence and reprint requests to Dr. Herbert Y. Gaisano, Medical Sciences Bldg., Rm. 7226, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. E-mail: herbert.gaisano{at}utoronto.ca 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 β-cells exhibited reduced insulin secretion, causing glucose intolerance. Munc13-1 binds Epac2 and Rim2, but their functional interactions remain unclear. RESEARCH DESIGN AND METHODS— We used Munc13-1 +/− islet β-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 +/− β-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. 8-pCPT-2′-O-Me-cAMP, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate AUC, area under the curve BBDC, Banting and Best Diabetes Center CIHR, Canadian Institutes of Health Research Cm, membrane capacitance DAG, diacylglycerol Epac, exchange protein directly activated by cAMP F-PIS, first-phase insulin secretion GLP-1, glucagon-like peptide-1 GSIS, glucose-stimulated insulin secretion GST, glutathione S-transferase IBMX, isobutylmethylxanthine KATP channel, ATP-sensitive K+ channel KRBH, Krebs-Ringer bicarbonate HEPES buffer N6-Bnz-cAMP, N6-benzoyladenosine-cAMP PKA, protein kinase A RIA, radioimmunoassay Rim, Rab3A-interacting molecule RRP, readily releasable pool SNAP, soluble N-ethylmaleimide–sensitive factor attachment protein SNARE, SNAP receptor S-PIS, second-phase insulin secretion SUR1, sulfonylurea receptor 1 Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 16 July 2007. DOI: 10.2337/db06-1207. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1207 . E.P.K. and L.X. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted July 10, 2007. Received August 30, 2006. DIABETES