Potentiation of Glucose-stimulated Insulin Secretion by the GPR40-PLC-TRPC Pathway in Pancreatic β-Cells

Abstract

G protein-coupled receptors (GPCRs) are expressed in pancreatic beta-cells. G protein-coupled receptor 40 (GPR40) contributes to medium- or long-chain fatty acid-induced amplification of glucose-stimulated insulin secretion (GSIS), and GPR40 agonists are promising therapeutic targets in type 2 diabetes. Recently, we demonstrated that glucagon-like peptide 1, a ligand of pancreatic GPCR, activates a class of nonselective cation channels (NSCCs) and enhances GSIS. The aim of the current study was to determine whether the GPR40 signal interacts with NSCCs. A GPR40 agonist (fasiglifam) potentiated GSIS at 8.3 and 16.7 mM glucose but not 2.8 mM glucose. The NSCC current was activated by fasiglifam at 5.6 mM glucose with 100 μM tolbutamide (-70 mV), and this activation was prevented by the presence of pyrazole-3 (transient receptor potential canonical; a TRPC3 channel blocker). Inhibitors of phospholipase C or protein kinase C (PKC) inhibited the increases in GSIS and the NSCC current induced by GPR40 stimulation. The present study demonstrates a novel mechanism for the regulation of insulin secretion by GPR40 agonist in pancreatic beta-cells. The stimulation of the GPR40-PLC/PKC-TRPC3 channel pathway potentiates GSIS by the depolarization of the plasma membrane in pancreatic beta-cell.

Figure 1. Fasiglifam increased nonselective cation channel (NSCC) current and depolarized the resting membrane potential in pancreatic rat single beta-cell.

(a) Effect of fasiglifam on the holding current recorded from a beta-cell. The cell was superfused with 5.6 mM glucose and 100 μM tolbutamide at the holding potential of −70 mV. The holding current was increased for the inward direction in the presence of 10 μM fasiglifam. (b) The current density that was expressed as pA/pF (amplitude of the holding current was divided by membrane capacitance that was measured on the establishment of whole-cell clamp) was shown, and fasiglifam significantly increased the current density. (c) Fasiglifam-induced increase in NSCC current was observed in the absence of tolbutamide at a holding potential of −80 mV. (d) The current–voltage relationship showed a reversal potential of −12 mV (arrow). The line was drawn using linear regression fit to the data points. (e) The membrane potential was recorded at 2.8 mM glucose. The membrane was depolarized upon superfusion with 10 μM fasiglifam in a reversible manner. (f ) Comparison of resting membrane potentials in the absence and presence of 10 μM fasiglifam. The glucose concentration was 2.8 mM. Membrane potentials in the absence and presence of 10 μM fasiglifam were −66.5 ± 2.9 and −50.5 ± 3.7 mV, respectively. Data were expressed as mean ± standard error of the mean. The number of data points was five. *P < 0.05 vs. control by paired t-test.

Figure 2. Fasiglifam potentiated insulin secretion at supra-threshold concentration of glucose.

Each batch containing 10 size-matched rat islets was incubated for 1 h to evoke insulin secretion. Fasiglifam potentiated insulin secretion at 8.3 and 16.7 mM glucose. However, fasiglifam did not potentiate insulin secretion at 2.8 mM glucose. The number of data points was five to seven. *P < 0.05 vs. 8.3 mM glucose; **P < 0.01 vs. 16.7 mM glucose by unpaired t-test.

Figure 3. Abolition of fasiglifam-induced increase in nonselective cation channel (NSCC) current by inhibitors of the phospholipase (PLC)/protein kinase C (PKC) and TRP channels.

(a–c) Effects of the nonselective transient receptor potential (TRP) channel blocker 2-aminoethyl diphenylborinate (2-APB; 10 μM) (a), TRP canonical (TRPC) channel blocker 3,5-bis(trifluoromethyl)pyrazole derivative 2 (BTP2; 10 μM) (b) and selective TRPC3 channel blocker pyrazole-3 (Pyr3; 10 μM) (c) on the NSCC current. (d–f) Effects of the PLC inhibitor U73122 (2 μM) (d) and PKC inhibitors Gö6983 (1 μM) (e) and Gö6976 (1 μM) (f) on the NSCC current. Fasiglifam-induced current increases were inhibited by these inhibitors. Rat single beta cells were voltage-clamped at −70 mV in the presence or absence of 10 μM fasiglifam, under the condition of 5.6 mM glucose and 100 μM tolbutamide throughout the experiments. The number of data points was five.

Figure 4. Transient receptor potential canonical 3 (TRPC3) channel blocker inhibited fasiglifam-induced [Ca²⁺]_i increase.

(a) Fasiglifam (10 μM) increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and pyrazole-3 (Pyr3, 10 μM) inhibited fasiglifam-induced [Ca²⁺]_i increase at 8.3 mM glucose. At the end of the experiment, 100 μM tolbutamide (Tolb) was added to confirm that the responsive cells were beta-cell. (b) Pyr3 at 10 μM attenuated fasiglifam-induced [Ca²⁺]_i increases in single mouse beta-cell. (n = 16, number of single mice beta-cells examined in each group); ^∗P < 0.01 vs. fasiglifam.

Figure 5. Phospholipase C (PLC) inhibitor and transient receptor potential canonical (TRPC) channel blocker inhibited fasiglifam-induced insulin secretion.

(a) Batches containing 10 rat islets in each were incubated for 1 hour to evoke insulin secretion at 2.8 or 16.7 mM glucose. Addition of fasiglifam (10 μM) potentiated insulin secretion at 16.7 mM glucose. The PLC inhibitor U73122 inhibited fasiglifam-induced insulin secretion at 16.7 mM glucose. U73443, a negative control for PLC inhibition, did not affect fasiglifam-mediated insulin secretion. (b) TRPC channel blocker 3,5-bis(trifluoromethyl)pyrazole derivative 2 (BTP2) and TRPC3 channel selective blocker pyrazole-3 (Pyr3) inhibited fasiglifam-induced insulin secretion at 16.7 mM glucose. The number of data points was four to 14. *P < 0.05 vs. 10 μM fasiglifam at 16.7 mM glucose; ^†P < 0.01 vs. 10 μM fasiglifam at 16.7 mM glucose ; ^#P < 0.05 vs. 16.7 mM glucose; **P < 0.05 vs. 10 μM fasiglifam at 16.7 mM glucose with 2 μM U73122 by unpaired t-test. ns; not significant between two groups.

Figure 6. The cAMP–PKA pathway was not involved in fasiglifam-evoked insulin secretion.

(a) The cAMP production induced by 5.6 mM glucose was potentiated by exendin-4 (10 nM) in isolated islets. Fasiglifam (10 μM) did not significantly affect the cAMP production induced by 5.6 mM glucose. Batches containing 10 rat islets in each were incubated for 1 hour at 5.6 mM glucose. The number of data points was three. (b) The protein kinase A (PKA) inhibitor H89 (10 μM) did not affect fasiglifam-induced insulin secretion. Batches containing 10 rat islets in each were incubated for 1 h to evoke insulin secretion at 16.7 mM glucose. The number of data points was five. *P < 0.05 vs. 5.6 mM glucose; ^†P < 0.01 vs. 16.7 mM glucose by unpaired t-test.