Resveratrol alleviates alcoholic fatty liver in mice

Abstract

Alcoholic fatty liver is associated with inhibition of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), two critical signaling molecules regulating the pathways of hepatic lipid metabolism in animals. Resveratrol, a dietary polyphenol, has been identified as a potent activator for both SIRT1 and AMPK. In the present study, we have carried out in vivo animal experiments that test the ability of resveratrol to reverse the inhibitory effects of chronic ethanol feeding on hepatic SIRT1-AMPK signaling system and to prevent the development of alcoholic liver steatosis. Resveratrol treatment increased SIRT1 expression levels and stimulated AMPK activity in livers of ethanol-fed mice. The resveratrol-mediated increase in activities of SIRT1 and AMPK was associated with suppression of sterol regulatory element binding protein 1 (SREBP-1) and activation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha). In parallel, in ethanol-fed mice, resveratrol administration markedly increased circulating adiponectin levels and enhanced mRNA expression of hepatic adiponectin receptors (AdipoR1/R2). In conclusion, resveratrol treatment led to reduced lipid synthesis and increased rates of fatty acid oxidation and prevented alcoholic liver steatosis. The protective action of resveratrol is in whole or in part mediated through the upregulation of a SIRT1-AMPK signaling system in the livers of ethanol-fed mice. Our study suggests that resveratrol may serve as a promising agent for preventing or treating human alcoholic fatty liver disease.

Fig. 1.

Resveratrol ameliorated alcoholic liver steatosis in mice. Oil red O staining of liver sections (A), hematoxylin and eosin (H&E) stain ×20 (B), hepatic triglyceride (TG) content (C), and plasma alanine aminotransferase level (D) of mice fed a low-fat control diet (C), 200 mg·kg⁻¹·day⁻¹ resveratrol (R200), or 400 mg·kg⁻¹·day⁻¹ resveratrol (R400) with (E+R200, E+R400) or without ethanol (E). All data are expressed as means ± SD; n = 5–8 animals. Means without a common letter differ, P < 0.05.

Fig. 2.

Resveratrol upregulated sirtuin 1 (SIRT1) in the livers of ethanol-fed mice. Hepatic SIRT1 mRNA (A), SIRT1 protein levels (B), acetylation of peroxisome proliferator-activated receptor γ (PPARγ) coactivator α (PGC-1α; C), and relative PGC-1α acetylation (D) of mice fed a low-fat diet, 200 mg·kg⁻¹·day⁻¹ resveratrol, or 400 mg·kg⁻¹·day⁻¹ resveratrol with or without ethanol. Hepatic nuclear SIRT1 protein levels were determined by use of an anti-SIRT1 antibody. A nonspecific nuclear protein band was used to confirm equal loading and to normalize the data. PGC-1α was immunoprecipitated from liver extracts then immunoblotted with either an anti-acetylated lysine (Ac-Lys) antibody to determine the extent of PGC-1α acetylation or with a PGC-1α antibody to determine the total amount of PGC-1α. All data are expressed as means ± SD; n = 4–8 animals. Means without a common letter differ, P < 0.05.

Fig. 3.

Resveratrol stimulated hepatic AMPK activity in ethanol-fed mice. A: Western blots were performed using anti-phosphorylated-AMPKα (anti-p-AMPKα), anti-AMPKα, anti-p-AMPKβ, anti-AMPKβ, and anti-phosphorylated acetyl CoA carboxylase (P-ACC) antibodies from liver extracts of mice fed a low-fat diet, 200 mg·kg⁻¹·day⁻¹ resveratrol, or 400 mg·kg⁻¹·day⁻¹ resveratrol with or without ethanol. β-Actin protein band was used to confirm equal loading and to normalize the data. B: relative levels of p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, and P-ACC. Western blots were quantified by a PhosphorImager and MultiAnalyst (Bio-Rad) software analysis. All data are expressed as means ± SD; n = 4–8 animals. Means without a common letter differ, P < 0.05.

Fig. 4.

Resveratrol suppressed sterol regulatory element-binding protein 1 (SREBP-1) activity and reduced the mRNA levels of SREBP-1 regulated genes encoding lipogenic enzymes in the livers of ethanol-fed animals. Representative Western blots of nuclear SREBP-1c (nSREBP-1c) protein (A) and relative mRNA levels of SREBP-regulated lipogenic enzymes (B) in the livers of mice fed a low-fat control diet, 200 mg·kg⁻¹·day⁻¹ resveratrol, or 400 mg·kg⁻¹·day⁻¹ resveratrol with or without ethanol. A nonspecific nuclear protein band in nuclear extracts was used to confirm equal loading and to normalize the data. All data are expressed as means ± SD; n = 5–8 animals. Means without a common letter differ, P < 0.05.

Fig. 5.

Resveratrol increased PGC-1α activity and enhanced expression of genes regulated by PGC-1α in the livers of ethanol-fed mice. Relative mRNA levels of PGC-1α (A), relative mRNA levels of PGC-1α-regulated fatty acid oxidation enzymes (B), and relative mRNA levels of PPARγ (C) in the livers of mice fed a low-fat control diet, 200 mg·kg⁻¹·day⁻¹ resveratrol, or 400 mg·kg⁻¹·day⁻¹ resveratrol with or without ethanol. All data are expressed as means ± SD; n = 5–8 animals. Means without a common letter differ, P < 0.05.

Fig. 6.

Resveratrol upregulated adiponectin and enhanced hepatic AdipoR1/R2 mRNA expression in ethanol-fed mice. Plasma adiponectin concentrations (A); relative adipose mRNA levels of adiponectin, TNF-α, SIRT1, and FOXO1; B); relative mRNA levels of AdipoR1 (C); and relative mRNA levels of AdipoR2 (D) of mice fed a low-fat control diet, 200 mg·kg⁻¹·day⁻¹ resveratrol, or 400 mg·kg⁻¹·day⁻¹ resveratrol with or without ethanol. All data are expressed as means ± SD; n = 4–6 animals. Means without a common letter differ, P < 0.05.

Fig. 7.

Proposed mechanism for the protective effects of resveratrol against alcoholic fatty liver in mice. AdipoR, adiponectin receptor; FA, free fatty acids.