Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice

Abstract

The effects of exendin-4 on Sirt1 expression as a mechanism of reducing fatty liver have not been previously reported. Therefore, we investigated whether the beneficial effects of exendin-4 treatment on fatty liver are mediated via Sirt1 in high-fat (HF) diet-induced obese C57BL/6J mice and related cell culture models. Exendin-4 treatment decreased body weight, serum free fatty acid (FA), and triglyceride levels in HF-induced obese C57BL/6J mice. Histological analysis showed that exendin-4 reversed HF-induced hepatic accumulation of lipids and inflammation. Exendin-4 treatment increased mRNA and protein expression of Sirt1 and its downstream factor, AMPK, in vivo and also induced genes associated with FA oxidation and glucose metabolism. In addition, a significant increase in the hepatic expression of Lkb1 and Nampt mRNA was observed in exendin-4-treated groups. We also observed increased expression of phospho-Foxo1 and GLUT2, which are involved in hepatic glucose metabolism. In HepG2 and Huh7 cells, mRNA and protein expressions of GLP-1R were increased by exendin-4 treatment in a dose-dependent manner. Exendin-4 enhanced protein expression of Sirt1 and phospho-AMPKα in HepG2 cells treated with 0.4 mM palmitic acid. We also found that Sirt1 was an upstream regulator of AMPK in hepatocytes. A novel finding of this study was the observation that expression of GLP-1R is proportional to exendin-4 concentration and exendin-4 could attenuate fatty liver through activation of Sirt1.

Figure 1. Change of body weight, food intake, and serum lipid levels by exendin-4 in HF-induced obese mice.

Mice divided into 3 groups were fed low fat diet, high fat diet, or high fat diet with 1 nmol/kg/day exendin-4 ip injection for 10 weeks. (A) Body weight and (B) food intake was checked twice per week. (C–D) When sacrificed at 11 weeks, serum samples were obtained for analysis of free fatty acid (FFA) and triglyceride (TG) after overnight starvation. All values are expressed as the mean ± SE for n = 8 mice. * p<0.05, ** p<0.01 compared with a control and # p<0.05, ## p<0.01 compared with a HF.

Figure 2. Change of Sirt1 and AMPK pathway and GLP-1R expression by exendin-4 in mouse livers.

Total RNA and protein were extracted from liver tissue and (A) Nampt, Sirt1, Lkb1, AMPKα1 and AMPKα1 mRNA and (B) GLP-1R, Sirt1, phospho-AMPKα, AMPK and β-actin protein expression were measured by quantitative real-time RT-PCR and western blot, respectively. Data were normalized to the β-actin of each sample. All values are expressed as the mean ± SE for n = 5–7 mice. * p<0.05, ** p<0.01 compared with a control and # p<0.05, ## p<0.01 compared with a HF.

Figure 3. Effect of exendin-4 on expression of genes associated with fatty acid oxidation, lipogenesis, and glucose homeostasis.

(A) Adiponectin (Adipoq), adiponectin receptor 1 (AdipoR1), adiponectin receptor 1 (AdipoR2), peroxisome proliferator-activated receptor- α (Ppara), acyl-CoA oxidase (Acox), and medium chain acyl-Coenzyme A dehydrogenase (MCAD); (B) sterol regulatory element binding protein 1c (SREBP-1c), stearoyl-Coenzyme A desaturase 1 (Scd-1), fatty acid synthase (Fasn), and acetyl-Coenzyme A carboxylase alpha (Acaca) mRNA expression; and (C) GLUT2, phosphorylated Foxo1 at serine 349, Foxo1 and β-actin protein expression in mouse livers were measured by quantitative real-time RT-PCR and western blot and were normalized to the β-actin of each sample. All values are expressed as the mean ± SE for n = 5–7 mice. * P<0.05, ** P<0.01 compared with a control and # p<0.05, ## p<0.01 compared with a HF.

Figure 4. Histopathology of mice fed control (low fat), HF, HF+exendin-4 diet for 10 wks.

Sections were stained with H&E. Arrows indicates lipid droplets. (A–C) Liver sections of mice fed control, HF and HF+Ex-4, respectively. (magnification, ×200). Scale bars = 500 µm. (D) NAFLD activity scores were evaluated semi-quantitatively: steatosis (0–3), lobular inflammation (0–2), and hepatocellular ballooning (0–2). N.D., not detected (E) Hepatic triglycerides were extracted from frozen tissue and measured by enzymatic assays. Values of TG were normalized to protein concentration. * p<0.05, ** p<0.01 compared with a control and # p<0.05, ## p<0.01 compared with a HF.

Figure 5. Effect of exendin-4 on PA-induced lipid accumulation in human hepatocytes.

HepG2 and Huh7cells were treated with PA (0.4 mM) with or without Ex-4 (100 nM) for 24 h and lipid accumulation in cells was observed by Oil-red O staining. (A) HepG2 and Huh7 cells stained with Oil-red O were examined by light microscopy (magnification, ×400). Cells treated with 10% BSA, 0.4 mM PA and 0.4 mM PA+100 nM Ex-4; (B) intracellular lipid droplets were quantified using a spectrophotometer at 540 nm. * p<0.05, ** p<0.01 compared with a control and # p<0.05, ## p<0.01 compared with PA. Scale bars = 100 µm.

Figure 6. Regulation of GLP-1R, Sirt1 and AMPK by exendin-4 in HepG2 and Huh7 cells.

(A) Cells were treated with 50 nM, 100 nM, or 500 nM Ex-4 for 24 h. GLP-1R and β-actin were measured by western blot and real time PCR. GLP-1R was normalized to β-actin. (B) Cells given 0.4 mM palmitic acid (PA) were treated with either vehicle or 50 nM to 100 nM exendin-4 for 24 h. (C) Cells given 0.4 mM palmitic acid were treated with 100 nM exendin-4 in the absence or presence of 10 mM nicotinamice (NAM) or 10 uM compound C (CC) for 24 h. (B–C) Sirt1, phosphorylated AMPKα at threonine 172, AMPK and β-actin were measured by western blot in HepG2 cells. Sirt1 and phosphorylated AMPKα were normalized to the β-actin and total AMPK of each sample, respectively. * p<0.05, ** p<0.01 compared with control, # p<0.05, ## p<0.01 compared with PA, and † p<0.05, †† p<0.01 compared with Ex-4.