Zinc supplementation reverses alcohol-induced steatosis in mice through reactivating hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor-alpha

Abstract

Alcoholic steatosis is a fundamental metabolic disorder in the progression of alcoholic liver disease. Zinc deficiency is one of the most consistently observed biochemical/nutritional manifestations of alcoholic liver disease. The purpose of this study is to determine whether dietary zinc supplementation to mice previously exposed to alcohol could reverse alcoholic steatosis. Male 129S mice were pair-fed an alcohol or isocaloric maltose dextrin liquid diet for 16 weeks with or without dietary zinc supplementation for the last 4 weeks. Zinc supplementation significantly attenuated alcohol-mediated increases in hepatic triglyceride, cholesterol, and free fatty acids in association with accelerated hepatic fatty acid oxidation and very low density lipoproteins (VLDL) secretion. Hepatic genes related to fatty acid oxidation and VLDL secretion were up-regulated by zinc supplementation, which was accompanied by restoring activity of hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferators activated receptor-alpha (PPAR-alpha). Zinc supplementation enhanced alcohol metabolism and attenuated oxidative stress and liver injury. Zinc supplementation also normalized alcohol-mediated increases in plasma triglycerides and partially reversed decrease in gonadal adipose depot mass. Studies in HepG2 cells showed that zinc deprivation significantly suppressed the DNA-binding activities of HNF-4alpha and PPAR-alpha, and reduced HNF-4alpha and PPAR-alpha target proteins. Consequently, zinc deprivation caused cellular accumulation of lipid droplets, triglycerides and free fatty acids in the HepG2 cells.

Conclusion: Zinc supplementation reverses alcoholic steatosis, and reactivation of HNF-4alpha and PPAR-alpha by increasing zinc availability and inhibiting oxidative stress are potential mechanisms underlying these beneficial effects of zinc on hepatic lipid homeostasis.

Figure 1

Hepatic lipid accumulation in mice fed alcohol for 16 weeks with or without zinc supplementation for the last 4 weeks. A. Oil Red O staining of the neutral lipid in the liver. B. Hepatic Triglyceride. C. Hepatic cholesterol. D. Hepatic free fatty acids (FFAs). Results are means ± SD (n=4–6). Significant differences (p<0.05) among a, b and c are determined by ANOVA. Ctrl: control. EtOH: Ethanol. Zn: Zinc.

Figure 2

Hepatic fatty acid oxidation and VLDL secretion in mice fed alcohol for 16 weeks with or without zinc supplementation for the last 4 weeks. A. Hepatic fatty acid β-oxidation. B. Hepatic VLDL-TG (triglyceride) secretion rate measured by Triton WR1339 method. C. Real time RT-PCR assay of genes related to fatty acid β-oxidation and VLDL secretion. Results are means ± SD (n=4–6 in A and B; n=4 in C). Significant differences (p<0.05) among a and b are determined by ANOVA. Ctrl: control. EtOH: Ethanol. Zn: Zinc.

Figure 3

Hepatic HNF-4α and PPAR-α status in mice fed alcohol for 16 weeks with or without zinc supplementation for the last 4 weeks. Protein and mRNA levels and activation of HNF-4α (A) and PPAR-α (B) measured by real time RT-PCR, immunoblotting and DNA binding ELISA, respectively. The immunoblotting bands were quantified by densitometry analysis and the ratio to GAPDH was calculated by setting the value of controls as one. Results are means ± SD (n=4-6). Significant differences (p<0.05) among a, b and c are determined by ANOVA. Ctrl: control. EtOH: Ethanol. Zn: Zinc.

Figure 4

Hepatic antioxidant capacity and alcohol metabolic genes in mice fed alcohol for 16 weeks with or without zinc supplementation for the last 4 weeks. Protein and/or activities of Cu/Zn-SOD (A), GPx (B) and catalase (C). The bands in A and B were quantified by densitometry analysis and the ratio to GAPDH was calculated by setting the value of controls as one. * Significantly different from other groups. D. MDA concentrations. E. Real time RT-PCR assay of alcohol metabolic genes. Results are means ± SD (n=4–6). Significant differences (p<0.05) among a, b and c are determined by ANOVA. Ctrl: control. EtOH: Ethanol. Zn: Zinc.

Figure 5

Effect of zinc deprivation on HNF-4α and PPAR-α function in HepG2 cell culture. HepG2 cells were treated with TPEN at 2 µM with or without zinc supplementation at 25 or 50 µM for 4 days. Immunoblotting analysis of HNF-4α (A) and PPAR-α (B) protein. DNA binding activity of HNF-4α (C) and PPAR-4α (D). E. Immunoblotting of HNF-4α and PPAR-α target proteins. The bands were quantified by densitometry analysis and the ratio to GAPDH was calculated by setting the value of controls as one. Results in C and D are means ± SD (n=4). Significant differences (p<0.05) among a and b are determined by ANOVA. Ctrl: control. EtOH: Ethanol. Zn: Zinc.

Figure 6

Effect of zinc deprivation on cellular lipid accumulation in HepG2 cell culture. HepG2 cells were treated with TPEN at 2 µM with or zinc supplementation at 25 or 50 µM for 4 days. A. Oil Red O staining of neutral lipid. B. Triglyceride concentrations. C. Free fatty acids (FFAs) concentrations. Results are means ± SD (n=4). Significant differences (p<0.05) between a and b are determined by ANOVA. Ctrl: control. T: TPEN.