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

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

Abbreviations: 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; C_m, 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; K_ATP 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

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 ß-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 ß-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^+/– ß-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 (N⁶-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.

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