Synthetic FXR agonist GW4064 prevents diet-induced hepatic steatosis and insulin resistance

Abstract

Purpose: To examine the effect of farnesoid X receptor (FXR) activation by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) on diet-induced obesity and hepatic steatosis.

Methods: Fifteen week-old C57BL/6 mice fed with high-fat diet (HFD) or high-fat, high-cholesterol diet were treated by twice weekly injection of GW4064 (50 mg/kg) intraperitoneally or DMSO (carrier solution) for 6 weeks. Body weight, body composition and food intake were monitored weekly. Serum glucose and insulin levels and lipid content in the liver were measured at the end of study. Additionally, genes involved in lipogenesis, gluconeogenesis and inflammation were analyzed by real time PCR. CD36 protein level was detected by western blot.

Results: Activation of FXR by GW4064 suppressed weight gain in C57BL/6 mice fed with either HFD or high-fat and high-cholesterol diet. GW4064 treatment of mice significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter Cd36 gene expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis.

Conclusions: The results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders.

Figures

Figure 1. GW4064 treatment delays HFD-induced obesity in mice

Fifteen-week-old C57BL/6 mice (male) were fed with HFD with or without inclusion of 0.2% cholesterol for 6 weeks. Mice were intraperitoneally injected by GW4064 (50 mg/kg, twice weekly) or carrier solution (DMSO) as the control. A, B, growth curve; C, D, fat and lean mass; E, F, food intake; G, H, H&E staining of white and brown adipose tissue. Each data point represents the average ± SD of 4 animals. *P<0.05 compared to DMSO-treated control animals. HFD-DMSO, HFD-GW4064: mice were fed with high-fat diet and treated with DMSO or GW4064; HFD-Chol-DMSO, HFD-Chol-GW4064: mice were fed with high-fat diet with 0.2% cholesterol and treated with DMSO or GW4064.

Figure 2. GW4064 repressed HFD-induced hepatic steatosis

At the end of the 6-week treatment, animals were sacrificed and liver weights were measured (A, B). Liver histology was evaluated by H&E staining (C-F, original magnifications 100×). Lipid accumulation was evaluated by Oil Red O staining (G-J, original magnifications 200×). HFD-DMSO, HFD-GW4064: mice were fed with high-fat diet and treated with DMSO or GW4064; HFD-Chol-DMSO, HFD-Chol-GW4064: mice were fed with HFD containing 0.2% cholesterol and treated with DMSO or GW4064. *P<0.05, **P<0.01 compared to DMSO-treated groups.

Figure 3. Reduction of hepatic and plasma lipid levels by GW4064 treatment in HFD-induced obese mice

(A-C) Triglyceride, free fatty acid and cholesterol content in the liver; (D-F) Serum levels of triglyceride, free fatty acid and cholesterol after 6-week treatment. Each data point represents the average ± SD of 4 animals in each group. *P<0.05, **P<0.01 compared to DMSO-treated groups

Figure 4. GW4064 attenuated HFD-induced hepatic inflammation

At the end of the sixth week, mice were treated with GW4064 or DMSO (carrier solution) and sacrificed 4 h later. Liver and WAT tissues were harvested and total RNA was extracted. Relative mRNA levels of selected genes were determined by real-time PCR. Macrophage markers F4/80, Cd68, Cd11b, and Cd11c in liver (A) and WAT (C); Cytokine gene expression for Tnfα, Il-1β and chemotactic factor genes Mcp-1 in liver (B) and WAT (D); Each data point represents the average ± SD of 4 animals. *P<0.05, **P<0.01 compared to DMSO-treated groups; #P<0.05, ##P<0.01 compared to HFD-DMSO group

Figure 5. Effect of GW4064 treatment on expression of genes involved in lipogenesis and CD36 mRNA and protein expression

At the end of the 6-week treatment, mice were sacrificed and livers were harvested. The mRNA levels of Srebp-1, Acc1, Scd-1, Cd36 and Ppar-α were examined by real-time PCR (A). CD36 protein level was determined by Western blot (B). Each data point represents the average ± SD of 4 animals. *P<0.05, **P<0.01 compared to DMSO-treated group.

Figure 6. GW4064 reduced the oleic acid-induced lipids accumulation and CD36 protein level in liver cell line (BNL CL.2)

Mouse liver BNL-CL2 cells were incubated with 500 μM oleic acid and treated at different concentration of GW4064 for 24 h. Lipid accumulation in the cells was evaluated by Oil Red O staining (A). CD36 protein expression was detected by Western blot (B).

Figure 7. Effect of GW4064 treatment on glucose tolerance, serum concentration of insulin, and mRNA levels of G6pase and Pepck

Animals at the end of the 6-week treatment were fasted overnight for glucose tolerance tests. A, C, time-dependent blood concentration of glucose upon IP injection of glucose (2 g/kg); B, D, area under the curve from glucose tolerance test; E, insulin levels at the end of the 6-week feeding with or without GW4064 treatment; F, HOMA-IR values calculated based on formula: (fasting insulin [μU/ml] × fasting glucose [mmol/l]) / 22.5. G, relative mRNA levels of G6pase and Pepck in mouse liver at the end of animal feeding and GW4064 treatment. Each data point represents the average ± SD of 4 animals. *P<0.05, **P<0.01 compared to DMSO-treated groups.