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

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 P-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 P-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(+/-) P-cells, 2) Epac-selective agonist [8-(4-chlorophenylthio)-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.