Atorvastatin promotes AMPK signaling to protect against high fat diet-induced non-alcoholic fatty liver in golden hamsters

Abstract

Non-alcoholic fatty liver disease (NAFLD) is characterized by diffuse fatty acid degeneration and excess fat accumulation in the liver. Notably, the currently available medications used to treat NAFLD remain limited. The aim of the present study was to investigate the protective role of atorvastatin (Ato) against NAFLD in golden hamsters fed a high fat diet (HFD) and in HepG2 cells treated with palmitate, and identify the underlying molecular mechanism. Ato (3 mg/kg) was administered orally every day for 8 weeks to the hamsters during HFD administration. Hamsters in the model group developed hepatic steatosis with high serum levels of triglyceride, cholesterol, insulin and C-reactive protein, which were effectively reduced by treatment with Ato. Additionally, the relative liver weight of hamsters treated with Ato was markedly lower compared with that of the model group. Hematoxylin and eosin, and oil red O staining indicated that the livers of the animals in the model group exhibited large and numerous lipid droplets, which were markedly decreased after Ato treatment. Western blot analysis indicated that Ato inhibited fat accumulation in the liver through the AMP-activated protein kinase (AMPK)-dependent activation of peroxisome proliferator activated receptor α (PPARα), peroxisome proliferator-activated receptor-γ coactivator 1 α and their target genes. Furthermore, in vitro, Ato inhibited PA-induced lipid accumulation in HepG2 cells. This inhibitory effect was attenuated following Compound C treatment, indicating that AMPK may be a potential target of Ato. In conclusion, the increase in AMPK-mediated PPARα and its target genes may represent a novel molecular mechanism by which Ato prevents NAFLD.

Keywords: AMP-activated protein kinase; atorvastatin; lipid accumulation; lipolysis; non-alcoholic fatty liver.

Figure 1.

Effects of Ato on weight gain and serum lipid levels of golden hamsters fed a HFD. (A) Summary of the experimental design. (B) Body weight of golden hamsters in each group from 0 to 8 weeks of treatment. (C and D) CHO, TG, LDL-c and HDL-c in the serum of golden hamsters following Ato treatment for (C) 4 and (D) 8 weeks. Data are presented as the mean ± SEM (n=8/group). ##P<0.01 vs. control group; *P<0.05, **P<0.01 vs. model group. Ato, atorvastatin; HFD, high fat diet; CHO, cholesterol; TG, triglyceride; LDL, low density lipoprotein; HDL, high density lipoprotein.

Figure 2.

Effect of Ato on the serum levels of INS and CRP. (A) Serum INS and CRP levels of golden hamsters following Ato treatment for 4 weeks. (B) Serum INS and CRP levels of golden hamsters following Ato treatment for 8 weeks. Data are presented as the mean ± SEM (n=8/group). ##P<0.01 vs. control group; *P<0.05, **P<0.01 vs. model group. Ato, atorvastatin; INS, insulin; CRP, C-reactive protein.

Figure 3.

Effect of Ato on liver weight and hepatic TG in HFD-fed golden hamsters. (A) Representative images of livers from different groups. (B) Relative liver weight in each group. (C) Hepatic TG level in golden hamsters following Ato treatment for 8 weeks. Data are presented as the mean ± SEM (n=8/group). #P<0.05, ##P<0.01 vs. control group; *P<0.05, **P<0.01 vs. model group. Ato, atorvastatin; TG, triglyceride; HFD, high fat diet; BW, body weight.

Figure 4.

Effect of Ato on lipid accumulation in livers of HFD-fed golden hamsters. (A-C) Hematoxylin and eosin staining of liver tissues from each group under a light microscope (magnification, ×200). (D-F) Oil red O staining of liver tissues from each group under a light microscope (magnification, ×200). Ato, atorvastatin; HFD, high fat diet.

Figure 5.

Effect of Ato on the mRNA levels of genes involved in lipid metabolism. (A) mRNA levels of genes associated with lipolysis and β-oxidation. (B) mRNA levels of genes associated with fatty acid synthesis. Data are presented as the mean ± SEM (n=8/group). #P<0.05, ##P<0.01 vs. control group; *P<0.05, **P<0.01 vs. model group. Ato, atorvastatin; ATGL, adipose triglyceride lipase; HSL, lipase E, hormone sensitive type; PDK4, pyruvate dehydrogenase kinase 4; CPT1a/CPT1b, carnitine palmitoyltransferase 1A/B; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase; DGAT1, diacylglycerol O-acyltransferase 1.

Figure 6.

Ato inhibits lipid accumulation by promoting the AMPK signaling pathway. (A) Representative images of p-AMPK, AMPK, p-Akt, Akt, PPARα and PGC1α western blots. (B) Relative p-AMPK/AMPK, p-Akt/Akt and (C) PPARα/GAPDH and PGC1α/GAPDH protein levels are presented. #P<0.05, ##P<0.01 vs. control group; *P<0.05 vs. model group. (D) Lipid accumulation in HepG2 cells, quantified by the absorbance value of oil red O reagent at 490 nm. Data are presented as the mean ± SEM (n=6). (E) Representative images of p-AMPK and AMPK in PA-treated HepG2 cells with or without Ato and Compound C treatment. (F) Relative p-AMPK/AMPK protein levels. Data are presented as the mean ± SEM (n=3). ##P<0.01 vs. control group; *P<0.05 vs. PA group; ^ΔP<0.05 vs. PA + Ato group. (G) mRNA levels of PPARα and PPARGC1A in each group. Data are presented as the mean ± SEM (n=3/group). ##P<0.01 vs. control group; *P<0.05 vs. model group. (H) Schematic illustration showing the suggested mechanisms underlying the protective effect of Ato against non-alcoholic fatty liver disease. Ato, atorvastatin; p-, phosphorylated; PPARα, peroxisome proliferator activated receptor α; AMPK, AMP-activated protein kinase; PGC1α, peroxisome proliferator-activated receptor-γ coactivator 1 α; PPARGC1A, PPARG coactivator 1 α; PA, palmitate.