Epac2-dependent mobilization of intracellular Ca2+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in [beta]-cells of phospholipase C- knockout mice

Calcium can be mobilized in pancreatic [beta]-cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in [beta]-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the [beta]-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse [beta]-cells were loaded with the photolabile Ca2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca2+ generated CICR in only 9% of the [beta]-cells tested, whereas CICR was generated in 82% of the [beta]-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in [beta]-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO [beta]-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT [beta]-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in [beta]-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type. The glucagon-like peptide-1 receptor (GLP-1R) expressed on pancreatic [beta]-cells is the molecular target of a new class of blood glucose-lowering agents designated as the 'incretin mimetics'. Exendin-4 is an incretin mimetic, and it acts via the GLP-1R to stimulate [beta]-cell insulin secretion, thereby explaining its usefulness as a novel treatment for type 2 diabetes mellitus. Here we report that exendin-4 mobilizes calcium in [beta]-cells, and that this action of exendin-4 is mediated, at least in part, by a phospholipase C- that is regulated by Epac2 and Rap1,