GPR40 agonist ameliorates liver X receptor-induced lipid accumulation in liver by activating AMPK pathway

Abstract

Hepatic steatosis is strongly linked to insulin resistance and type 2 diabetes. GPR40 is a G protein-coupled receptor mediating free fatty acid-induced insulin secretion and thus plays a beneficial role in the improvement of diabetes. However, the impact of GPR40 agonist on hepatic steatosis still remains to be elucidated. In the present study, we found that activation of GPR40 by its agonist GW9508 attenuated Liver X receptor (LXR)-induced hepatic lipid accumulation. Activation of LXR in the livers of C57BL/6 mice fed a high-cholesterol diet and in HepG2 cells stimulated by chemical agonist caused increased expression of its target lipogenic genes and subsequent lipid accumulation. All these effects of LXR were dramatically downregulated after GW9508 supplementation. Moreover, GPR40 activation was accompanied by upregulation of AMPK pathway, whereas the inhibitive effect of GPR40 on the lipogenic gene expression was largely abrogated by AMPK knockdown. Taken together, our results demonstrated that GW9508 exerts a beneficial effect to ameliorate LXR-induced hepatic steatosis through regulation of AMPK signaling pathway.

Figure 1. Intragastric administration of GW9508 ameliorates high cholesterol diet (HCD) - induced hepatic lipid accumulation in C57BL/6 mice.

C57BL/6 mice were fed the 0.2% cholesterol diet for 3 days by gavage daily with either vehicle or GW9508 (100 mg/kg BW/day). After a 4 h fast, mice were sacrificed. Livers were collected from mice fed a control diet, a HCD supplemented with vehicle, and a HCD supplemented with GW9508. (A) LXRα and LXRβ expression was detected by western blot, data are presented as the mean ± SEM, *p < 0.05, versus control diet-fed mice. (B) Representative oil red O staining of liver cryosections, bar = 25 μm. (C) Hepatic lipid contents, data are presented as the mean ± SEM from 6–7 animals of each group. **p < 0.01, ***p < 0.001, versus control diet-fed mice; ^#p < 0.05, ^###p < 0.001, versus HCD-fed plus vehicle mice.

Figure 2. Administration of GW9508 suppresses LXR activation-induced upregulation of hepatic lipogenic gene expression and activates AMPK-ACC signal pathway in C57BL/6 mice.

Western blot analysis of protein expression and corresponding densitometric quantification of FAS, SCD1, ACC, MTP, PPARα (A) and phosphorylation of AMPKα (Thr172) and ACC (Ser79) (B) were performed. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus control diet-fed mice; ^#p < 0.05, ^##p < 0.01, versus HCD-fed plus vehicle mice.

Figure 3. GW9508 attenuates LXR activation-induced triglyceride accumulation in HepG2 cells.

HepG2 cells were pretreated with either 50 or 100 μM GW9508 for 1 h and then treated with 5 μM T0901317 for 48 h. (A) Oil red O staining showed lipid droplets, bar = 25 μm. The data was representative of three independent experiments. Oil red O was then extracted by isopropanol and quantified with spectrophotometry at 520 nm. Cellular total triglyceride (B) and cholesterol (C) were measured. Data are expressed as means ± SEM and obtained from 3 individual experiments. **p < 0.01, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with T0901317-treated group.

Figure 4. GW9508 ameliorates LXR activation-induced triglyceride accumulation through activation of GPR40.

HepG2 cells were pretreated with either 50 or 100 μM GW9508 for 1 h and then treated with 5 μM T0901317 for 24 h. (A) Measurement of mRNA levels of lipogenic genes including SREBP1c, FAS, and SCD1 by real-time PCR. (B) Western blot analysis of FAS and SCD1 protein levels. Data are expressed as means ± SEM and obtained from 3 individual experiments. ***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with T0901317-treated group. (C) Protein expression of FAS and SCD1 when GPR40 was knocked down. HepG2 cells were transfected with siRNA of GPR40 or scrambled siRNA for 24 h and pretreated with 100 μM GW9508 for 1 h before 5 μM T0901317 treatment for 24 h.

Figure 5. GPR40 reduces LXR activation-induced lipid accumulation by AMPK pathway.

(A) Western blot analysis and quantitative measurements of phosphorylation of AMPK and ACC in HepG2 cells treated with GW9508 and T0901317. Data are expressed as means ± SEM and obtained from 3 individual experiments. (B) Western blot analysis of FAS and SCD1 when AMPK pathway is inhibited. HepG2 cells were transfected with duplexed RNA oligonucleotides of AMPK or scrambled siRNA and pretreated with 100 μM GW9508 for 1 h before 5 μM T0901317 treatment for 24 h. (C) Western blot analysis and quantitative measurements of phosphorylation of AMPK and ACC in HepG2 cells pretreated with a CaMKK inhibitor, STO-609, for 1 h prior to the treatment with GW9508 and T0901317. Data are expressed as means ± SEM and obtained from 3 individual experiments. (D) The molecular mechanism by which GPR40 participates in LXR-induced hepatic lipid accumulation.