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

Abstract

Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca2+ -induced Ca 2+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 Ca 2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca 2+ 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. 2+ /calmodulin-regulated protein kinase II; cAMP-GEF, cAMP-regulated guanine nucleotide exchange factor; CICR, Ca 2+ -induced Ca 2+ release; Epac, exchange protein directly activated by cAMP; ER, endoplasmic reticulum; ESCA-AM, Epac-selective cAMP analogue acetoxymethyl ester; EYFP, enhanced yellow fluorescent protein; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; GPCR, G protein-coupled receptor; KO, knockout; NAADP, nicotinic acid adenine dinucleotide phosphate; NP-EGTA, o-nitrophenyl ethylene glycol tetraacetic acid; Phosphate AM3, phosphate tris (acetoxymethyl) ester; PI, phosphoinositide; PKA, protein kinase A; PKC, protein kinase C; PLC-ε, phospholipase C-ε; Sp-8-pCPT-2 -O-Me-cAMPS, 8-(4-chlorophenylthio)-2 -O-methyladenosine-3 ,5 -cyclic monophosphorothioate; SES, standard extracellular saline; VDCC, voltage-dependent Ca 2+ channels; WT, wild-type.

Role of Epac2A/Rap1 Signaling in Interplay Between Incretin and Sulfonylurea in Insulin Secretion

“…In pancreatic b-cells, Rap1 mediates Epac2A-dependent amplification of insulin secretion (9). Rap1 has recently been shown to mediate the potentiation of insulin secretion through activation of phospholipase C-e. Phospholipase C-e activated by Rap1 possibly potentiates insulin secretion by promoting Ca 2+ -induced Ca 2+ release through the production of inositol triphosphate (32,33). Costimulation by sulfonylurea and cAMP has been found to induce a larger change in intracellular Ca 2+ level than stimulation by sulfonylurea alone (34)(35)(36).…”

Activity of PLCε contributes to chemotaxis of fibroblasts towards PDGF

“…We further extended our analysis using a panel of different extracellular agonists, including growth factors for tyrosine kinase receptors (EGF, FGF and VEGF) and agonists for G-protein coupled receptors (LPA, thrombin, ephinephrine, carbachol, bombesin and ATP) some of which have been previously linked to activation of PLCe (Bunney and Katan, 2006;Smrcka et al, 2012). We also analysed several compounds that could act on potential intracellular mediators of PLCe activation such as 8-pMeOPT (Dzhura et al, 2010). As shown in Fig.…”

Endocrinology of the Single Cell

“…The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) by Rap1-activated PLC-ε is suggested to increase K ATP -channel responsiveness to ATP and also to have a role in Ca 21 -induced Ca 21 release thereby contributing to Ca 21 -dependent insulin granule exocytosis. 36,38 In addition to GLP-1-induced increase in β-cell cAMP, earlier studies established that elevated glucose can result in a modest increase in total cAMP content of β-cells but that cAMP by itself does not lead to insulin secretion (as reviewed in refs 8,31 ). Questions still linger as to the mechanism for this glucose effect on increasing cAMP with most attention directed to a role for increased ATP and possibly Ca 21 -dependent calmodulin on a soluble adenylyl cyclase, which is structurally distinct from the transmembrane forms.…”