Fasiglifam (TAK-875) has dual potentiating mechanisms via Gαq-GPR40/FFAR1 signaling branches on glucose-dependent insulin secretion

Abstract

Fasiglifam (TAK-875) is a free fatty acid receptor 1 (FFAR1)/G-protein-coupled receptor 40 (GPR40) agonist that improves glycemic control in type 2 diabetes with minimum risk of hypoglycemia. Fasiglifam potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells glucose dependently, although the precise mechanism underlying the glucose dependency still remains unknown. Here, we investigated key cross-talk between the GSIS pathway and FFAR1 signaling, and Ca(2+) dynamics using mouse insulinoma MIN6 cells. We demonstrated that the glucose-dependent insulinotropic effect of fasiglifam required membrane depolarization and that fasiglifam induced a glucose-dependent increase in intracellular Ca(2+) level and amplification of Ca(2+) oscillations. This differed from the sulfonylurea glimepiride that induced changes in Ca(2+) dynamics glucose independently. Stimulation with cell-permeable analogs of IP3 or diacylglycerol (DAG), downstream second messengers of Gαq-FFAR1, augmented GSIS similar to fasiglifam, indicating their individual roles in the potentiation of GSIS pathway. Intriguingly, the IP3 analog triggered similar Ca(2+) dynamics to fasiglifam, whereas the DAG analog had no effect. Despite the lack of an effect on Ca(2+) dynamics, the DAG analog elicited synergistic effects on insulin secretion with Ca(2+) influx evoked by an L-type voltage-dependent calcium channel opener that mimics glucose-dependent Ca(2+) dynamics. These results indicate that the Gαq signaling activated by fasiglifam enhances GSIS pathway via dual potentiating mechanisms in which IP3 amplifies glucose-induced Ca(2+) oscillations and DAG/protein kinase C (PKC) augments downstream secretory mechanisms independent of Ca(2+) oscillations.

Keywords: Ca2+ oscillation; FFAR1/GPR40; fasiglifam (TAK‐875); glucose‐stimulated insulin secretion (GSIS); type 2 diabetes mellitus (T2DM).

Figure 1

Membrane depolarization is essential and sufficient for fasiglifam to exert its insulinotropic effect. (A) Effects of diazoxide on the glucose‐stimulated insulin secretion (GSIS)‐potentiating effect of fasiglifam in mouse MIN6 cells. Data are normalized to the vehicle (0 mmol/L glucose) group and shown as means ± SEM (n = 3). ***P < 0.001 versus 0 mmol/L glucose by Dunnett's test. ^$ P < 0.05 by Welch's t‐test. (B) Effect of fasiglifam on KCl‐induced depolarization‐mediated insulin secretion in MIN6 cells. Data are normalized to the vehicle (no KCl) group and shown as means ± SEM (n = 3). ***P < 0.001 versus vehicle group by Dunnett's test. ^$$ P < 0.01 by Welch's t‐test.

Figure 2

Both IP₃‐mediated Ca²⁺ release from ER, and protein kinase C (PKC) activation downstream of FFAR1 contribute to the glucose‐stimulated insulin secretion (GSIS) potentiating effect of fasiglifam. (A) Potentiating effects of fasiglifam and γ‐linolenic acid on GSIS in MIN6 cells. Data are normalized to the vehicle (0 mmol/L glucose) group and shown as means ± SEM (n = 3). **P < 0.01, *P < 0.05, by Dunnett's test. (B) Effect of IP₃ receptor activation by Bt₃IP₃ on GSIS in MIN6 cells. Data are normalized to the vehicle (0 mmol/L glucose) group and shown as means ± SEM (n = 3). Two‐way ANOVA: main effect of glucose: P < 0.01; main effect of Bt₃IP₃: P < 0.01; interaction of glucose and Bt₃IP₃: P < 0.01. *P < 0.05 by Welch's t‐test. (C) Effect of PKC activation by OAG on GSIS in MIN6 cells. Data are normalized to the vehicle (0 mmol/L glucose group) and shown as means ± SEM (n = 3). Two‐way ANOVA: main effect of glucose: P < 0.01; main effect of OAG: P < 0.01; interaction of glucose and OAG: P < 0.01. **P < 0.01 by Welch's t‐test.

Figure 3

Effect of fasiglifam and glimepiride on glucose‐dependent and ‐independent increases in intracellular Ca²⁺ level and Ca²⁺ oscillations. Ca²⁺ dynamics were determined in MIN6 cells loaded with fura‐2 AM and stimulated with (A–C) fasiglifam or (D–E) glimepiride. (A), (D) Representative traces in 21 regions of interest in one experiment at the indicated glucose concentrations. (B), (E) Changes in the intracellular Ca²⁺ response quantified by the area under the curve 1 min before and 1 min after stimulation. Data are presented as means ± SEM of 63 regions of interest within three experiments. (C), (F) Power spectrum density profiles describing the power of the oscillations at different frequencies generated by fast Fourier transform. Power is proportional to amplitude squared. The results were subclassified into three groups: low (1–100 mHz), middle (101–200 mHz), and high (201–1000 mHz). The Nyquist frequency in these experiments is about 1000 mHz. Data are expressed as means ± SEM of 63 regions of interest within three experiments. #P < 0.025 versus 0 mmol/L glucose by one‐tailed Williams’ test. *P < 0.05, **P < 0.01 by Welch's t‐test.

Figure 4

Effects of Bt₃IP₃ and OAG on glucose‐dependent intracellular Ca²⁺ dynamics. Detailed analyzes of Ca²⁺ dynamics were performed in MIN6 cells loaded with fura‐2 AM and stimulated with (A–C) Bt₃IP₃ or (D–E) OAG. (A), (D) Representative traces in 21 regions of interest in one experiment at the indicated glucose concentrations. (B), (E) Changes in the intracellular Ca²⁺ response quantified by the area under the curve 1 min before and 1 min after stimulation. Data are presented as means ± SEM of 63 regions of interest within three experiments. (C), (F) Power spectrum density profiles describing the power of the oscillations at different frequencies generated by fast Fourier transform. The mathematical conditions are the same as in Figure 3. Data are expressed as means ± SEM of 63 regions of interest within three experiments. ^# P < 0.025 versus 0 mmol/L glucose by one‐tailed Williams’ test. *P < 0.05, **P < 0.01 by Welch's t‐test.

Figure 5

Synergistic effects on insulin secretion elicited by the induction of Ca²⁺ influx and activation of protein kinase C. (A) Effects of nifedipine on the glucose‐stimulated insulin secretion potentiating effect of fasiglifam. Data are normalized to the vehicle (0 mmol/L glucose) group and shown as means ± SEM (n = 3). ***P < 0.001 versus 0 mmol/L glucose by Dunnett's test, ^$ P < 0.05 by Welch's t‐test. (B) Dose–response effect of (S)‐(‐)‐Bay K 8644 (Bay) on insulin secretion in the absence of glucose in MIN6 cells. Data are normalized to the vehicle group and shown as means ± SEM (n = 3). *P < 0.025 versus vehicle by one‐tailed Williams’ test. (C) Dose–response effect of OAG, with and without 1 μmol/L Bay, on insulin secretion in the absence of glucose in MIN6 cells. Data are normalized to the vehicle (neither OAG nor Bay) and shown as means ± SEM (n = 3). *P < 0.025 versus vehicle by one‐tailed Williams’ test. ^# P < 0.025 versus 1 μmol/L Bay alone by one‐tailed Williams’ test. Two‐way ANOVA: main effect of OAG: P < 0.01; main effect of Bay: P < 0.01; interaction of OAG and Bay: P < 0.01.

Figure 6

Schematic dual potentiating model for fasiglifam‐induced FFAR1 signaling. Fasiglifam, a selective FFAR1 ago‐allosteric modulator, potentiates glucose‐stimulated insulin secretion in depolarized β‐cells via at least two distinct Gαq‐mediated mechanisms: (1) fasiglifam‐induced IP₃ production amplifies glucose‐induced Ca²⁺ oscillations, and (2) fasiglifam‐induced protein kinase C (PKC)/(PKD) protein kinase D activation augments downstream secretory mechanisms independent of Ca²⁺ oscillations.