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

ABSTRACT: OBJECTIVE: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. METHODS: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 PKC?-GFP and PKC?-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 ?-helix of the homeodomain of Antennapedia. RESULTS: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 PKC? translocation was rarely observed, PKC? translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKC? and sustained translocation as well as transient translocation of PKC?. While the inhibitors (75 ?M) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKC? inhibitor had a more potent effect. CONCLUSION:GW9508 activated PKC? but not PKC? 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.

SUBMITTER: Hashimoto T

PROVIDER: S-EPMC6733457 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Hashimoto Takuya T Mogami Hideo H Tsuriya Daisuke D Morita Hiroshi H Sasaki Shigekazu S Kumada Tatsuro T Suzuki Yuko Y Urano Tetsumei T Oki Yutaka Y Suda Takafumi T

PloS one 20190909 9

<h4>Objective</h4>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.<h4>Methods</h4>Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of ...[more]

PMID: 31498851

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Project description:BACKGROUND:Diminished growth hormone (GH) is associated with impaired endothelial function and fibrinolysis. GH-releasing hormone is the primary stimulus for GH secretion and a substrate of dipeptidyl peptidase-4. We tested the hypothesis that dipeptidyl peptidase-4 inhibition with sitagliptin increases stimulated GH secretion, vasodilation, and tissue plasminogen activator (tPA) activity. METHODS AND RESULTS:Healthy adults participated in a 2-part double-blind, randomized, placebo-controlled, crossover study. First, 39 patients (29 women) received sitagliptin or placebo on each of 2 days separated by a washout. One hour after study drug, blood was sampled and then arginine (30 g IV) was given to stimulate GH. Vasodilation was assessed by plethysmography and blood sampled for 150 minutes. Following a washout, 19 of the original 29 women received sitagliptin alone versus sitagliptin plus antagonist to delineate GH receptor (GHR)- (n=5), nitric oxide- (n=7), or glucagon-like peptide-1 receptor- (n=7) dependent effects. Sitagliptin enhanced stimulated GH secretion (P<0.01 versus placebo, for 30 minutes) and free insulin-like growth factor-1 (P<0.001 versus placebo, after adjustment for baseline) in women. Vasodilation and tPA increased in all patients, but sitagliptin enhanced vasodilation (P=0.01 versus placebo) and increased tPA (P<0.001) in women only. GHR blockade decreased free insulin-like growth factor-1 (P=0.04 versus sitagliptin alone) and increased stimulated GH (P<0.01), but decreased vascular resistance (P=0.01) such that nadir vascular resistance correlated inversely with GH (rs=-0.90, P<0.001). GHR blockade suppressed tPA. Neither nitric oxide nor glucagon-like peptide-1 receptor blockade affected vasodilation or tPA. CONCLUSIONS:Sitagliptin enhances stimulated GH, vasodilation, and fibrinolysis in women. During sitagliptin, increases in free insulin-like growth factor-1 and tPA occur via the GHR, whereas vasodilation correlates with GH but occurs through a GHR-independent mechanism. CLINICAL TRIAL REGISTRATION:URL: http://www.clinicaltrials.gov. Unique identifier: NCT01701973.

Dataset Information

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

Publications

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

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