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

Abstract

Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in β-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-ε (PLC-ε) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-ε gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the β-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse β-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 β-cells tested, whereas CICR was generated in 82% of the β-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 β-cells of PLC-ε 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-ε KO β-cells with recombinant PLC-ε rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-ε with a mutated Ras association (RA) domain, or a H1640L PLC-ε that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT β-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-ε exists in β-cells, and that the activities of Epac2 and PLC-ε are key determinants of CICR in this cell type.The glucagon-like peptide-1 receptor (GLP-1R) expressed on pancreatic β-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 β-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 β-cells, and that this action of exendin-4 is mediated, at least in part, by a phospholipase C-ε that is regulated by Epac2 and Rap1, and that was not previously known to exist in β-cells. Our new findings concerning phospholipase C-ε» provide an unexpected explanation for how GLP-1R agonists act independently of protein kinase A to stimulate calcium-dependent exocytosis of insulin from the β-cells. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.