G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase C[alpha] and [epsilon] in INS-1 cells

• Published in: PloS one 2019-09, Vol.14 (9), p.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: Cell membranes; Complications and side effects; Diabetes mellitus; Fluorescence microscopy; G proteins; Glucose; Insulin; Medical research; Microscopy; Novels; Pancreatic beta cells; Patient outcomes; Peptides; Protein kinases; Type 2 diabetes
• 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 Ca.sup.2+ ([Ca.sup.2+ ].sub.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 PKC[alpha]-GFP and PKC[epsilon]-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 [alpha]-helix of the homeodomain of Antennapedia. At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca.sup.2+ ].sub.i . At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca.sup.2+ ].sub.i . Although PKC[alpha] translocation was rarely observed, PKC[epsilon] translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKC[alpha] and sustained translocation as well as transient translocation of PKC[epsilon]. While the inhibitors (75 [mu]M) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKC[epsilon] inhibitor had a more potent effect. GW9508 activated PKC[epsilon] but not PKC[alpha] 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.