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

• Published in: PloS one 2019-09, Vol.14 (9), p.e0222179-e0222179
• Main Authors: Hashimoto, Takuya, Mogami, Hideo, Tsuriya, Daisuke, Morita, Hiroshi, Sasaki, Shigekazu, Kumada, Tatsuro, Suzuki, Yuko, Urano, Tetsumei, Oki, Yutaka, Suda, Takafumi
• Format: Article
• Language: English
• Subjects: Activation; Agonists; Alanine; Animals; Biology and Life Sciences; Calcium (intracellular); Calcium - metabolism; Calcium ions; Cell Line, Tumor; Cell Membrane - drug effects; Cell Membrane - metabolism; Cytosol; Cytosol - drug effects; Cytosol - metabolism; Diabetes; Dose-Response Relationship, Drug; Enzyme Activation - drug effects; Fatty acids; Fluorescence; Fluorescence microscopy; G protein-coupled receptors; Glucose; Glucose - pharmacology; Green fluorescent protein; Homeobox; Hypoglycemia; Inhibitors; Insulin; Insulin resistance; Insulin secretion; Insulin Secretion - drug effects; Internal medicine; Isoforms; Isotypes; Kinases; MARCKS protein; Medicine; Medicine and Health Sciences; Methylamines - pharmacology; Microscopy; Myristoylated Alanine-Rich C Kinase Substrate - metabolism; Physical Sciences; Physiology; Plasmids; Propionates - pharmacology; Protein kinase C; Protein Kinase C-alpha - metabolism; Protein Kinase C-epsilon - metabolism; Protein Transport - drug effects; Proteins; Rats; Receptors, G-Protein-Coupled - agonists; Research and Analysis Methods; Secretion; Signal transduction; Signal Transduction - drug effects; Substrates; Translocation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0222179

The 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. Using 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. At 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. GW9508 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.