Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Abstract

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.

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).…”

cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells

“…We therefore tested whether changes in cAMP might be involved in the STIM1 translocation responses of the two cell types. These experiments were done at a basal glucose concentration to prevent cAMP from promoting IP 3 receptor-mediated Ca 2ϩ release, which only occurs in ␤-cells exposed to higher concen- trations of the sugar (36,37). Because [Ca 2ϩ ] i in ␤-cells is low and stable under these conditions, the reported effects of [Ca 2ϩ ] i elevation on STIM1 translocation (38) should not influence the results.…”

Exchange Protein Directly Activated by cAMP (epac): A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions

“…In transgenic mice that lack the Epac effector PLC-«, the Epac activator 8-pCPT-29-O-Me-cAMP loses the ability to potentiate glucose-induced insulin secretion. Importantly, however, these mice also lose their ability to increase calcium in pancreatic b-cells (Dzhura et al, 2011), indicating that a Rap1-regulated PLC-« also links Epac2 to Ca 2+ -dependent insulin granule excocytosis. Since a recent study in cardiomyocytes reported on scaffolding of PLC-« to mAKAP-b , it is tempting to speculate that cellular PLC-« scaffolding contributes to its ability to potentiate glucoseinduced insulin secretion as well.…”

“…Importantly, arginine 447 of Epac2 determines the isoform-selective activation by sulfonylureas (Herbst et al, 2011), strongly suggesting that a direct interaction underlies the molecular link between sulfonylureas and Epac2. Indeed, several early and recent studies demonstrate that Epac2 participates in glucose-, glucagon-, and incretin-enhanced insulin secretion from pancreatic b-cells through multiple mechanisms including inhibition of ATP-sensitive K + -channels, activation of the ryanodine-sensitive Ca 2+ -channel Ryr2 and subsequently Ca 2+ -induced Ca 2+ -release from the endoplasmic reticulum, recruitment of insulin granules to the plasma membrane, and Rap1-regulated activation of PLC-« (Ozaki et al, 2000;Kang et al, 2001Kang et al, , 2003Holz et al, 2006;Shibasaki et al, 2007;Kelley et al, 2009;Dzhura et al, 2011;Leech et al, 2010). Despite this evidence, experts in the field judge the relevance of this phenomenon controversial Rehmann, 2012).…”

“…In addition, the hydrolysis of PIP 2 through Epac2 is expected to increase ATP-sensitive K + -channels responsiveness to ATP and subsequently to promote their closure (Holz et al, 2006), leading to insulin secretion. Indeed, recent studies show that PLC-« links Epac2 to the potentiation of insulin secretion in mouse islets of Langerhans (Dzhura et al, 2011;Fig. 6).…”