G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells

Abstract

ObjectiveThe mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells.MethodsUsing epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia.ResultsAt 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect.ConclusionGW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

<p>Agonism of Gpr40 Protects the Capacities of Epidermal Stem Cells (ESCs) Against Ultraviolet-B (UV-B)</p>

“…GW9508 is a Gpr40 agonist that has been recently used in studies on atherosclerosis and insulin secretion. 18 , 19 In the present study, we demonstrate the ability of Gpr40 agonism by GW9508 to rescue the protective functions of ESCs exposed to UV-B radiation. These findings demonstrate a novel use of GW9508 as a treatment for UV-B-induced skin damage.…”

<p>Activation of GPR40 Suppresses AGE-Induced Reduction of Type II Collagen and Aggrecan in Human SW1353 Chondrocytes</p>

“…In current researches, GW9508 has been demonstrated to potentiate glucose-stimulated insulin secretion. 18 Also, activation of GPR40 by GW9508 also alleviated epileptic activity in model in vitro. 19 The purpose of this study is to investigate whether the agonism of GPR40 with GW9508 has a beneficial effect against AGE-induced damages in human SW1353 chondrocytes.…”

The Pancreatic β-Cell: The Perfect Redox System

“…PKCs isoenzymes, which belong to the family of serine/threonine kinases, are activated by DAG, which is their main effector [ 221 , 222 , 223 , 224 ]. PKCs are important for a broad range of cellular processes [ 225 ].…”

“…The results revealed that insulin secretagogues induce transient DAG microdomains, which rapidly recruit both cPKCs (specifically isoforms PKCα, PKCβI and PKCβII) and nPKCs (isoforms PKCδ, PKCε and PKCη), emphasizing the role of DAG in the rapid kinetic control of PKC-mediated phosphorylation. The pharmacological stimulation of GPR40 also activated PKCε at a substimulatory (glucose), while in addition to PKCε, PKCα was also activated at high insulin-stimulating (glucose) [ 224 ]. nPKCs were found to affect cytoskeleton dynamics in a way that eases IGV exocytosis [ 227 ].…”