Hepatocyte-specific hypoxia-inducible factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice

Abstract

Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated hypoxia-inducible factor 1-α, (HIF-1α) may regulate liporegulatory genes, but the relationship of HIF-1 to steatosis remains unknown. We investigated HIF-1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels, and increased serum alanine aminotransferase (ALT) levels, suggesting liver injury in wild-type (WT) mice. There was increased hepatic HIF-1α messenger RNA (mRNA), protein, and DNA-binding activity in alcohol-fed mice compared with controls. Mice engineered with hepatocyte-specific HIF-1 activation (HIF1dPA) had increased HIF-1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared with WT mice. In contrast, hepatocyte-specific deletion of HIF-1α [HIF-1α(Hep(-/-) )], protected mice from alcohol- and lipopolysaccharide (LPS)-induced liver damage, serum ALT elevation, hepatomegaly, and lipid accumulation. HIF-1α(Hep(-/-) ), WT, and HIF1dPA mice had equally suppressed levels of peroxisome proliferator-activated receptor α mRNA after chronic ethanol, whereas the HIF target, adipocyte differentiation-related protein, was up-regulated in WT mice but not HIF-1α(Hep(-/-) ) ethanol-fed/LPS-challenged mice. The chemokine monocyte chemoattractant protein-1 (MCP-1) was cooperatively induced by alcohol feeding and LPS in WT but not HIF-1α(Hep(-/-) ) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF-1α protein expression as well as DNA-binding activity. Small interfering RNA inhibition of HIF-1α prevented MCP-1-induced lipid accumulation, suggesting a mechanistic role for HIF-1α in hepatocyte lipid accumulation.

Conclusion: Alcohol feeding results in lipid accumulation in hepatocytes involving HIF-1α activation. The alcohol-induced chemokine MCP-1 triggers lipid accumulation in hepatocytes via HIF-1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease.

Copyright © 2011 American Association for the Study of Liver Diseases.

Figures

Figure 1

Changes in liver histology with chronic ethanol feeding. (A) Wild-type ethanol-fed mice developed hepatomegaly as measured by liver-weight to body weight ratio after 4 weeks of ethanol feeding. (B) Liver triglyceride was quantified using a biochemical assay and normalized to protein content of liver samples. (C) Serum ALT was quantified as an index of liver injury in sera from ethanol-fed or pair-fed mice. (D) HIF1α mRNA was amplified using real-time PCR from RNA extracts from livers of ethanol-fed or pair-fed mice. (E) Photomicrographs prepared from H&E stained liver sections from ethanol-fed or pair-fed mice.

Figure 2

(A) Western blot for HIF1α protein prepared from nuclear extracts from livers of ethanol-fed or pair-fed mice, band quantification in (B). (C) HIF1α DNA-binding activity was measured using the electrophoretic mobility shift assay (EMSA) and a HIF-specific probe, (D) EMSA band densitometry.

Figure 3

Alcohol feeding and changes in the liver of mice with hepatocyte-specific, constitutively active HIF1α. (A) Liver-weight to body weight ratio from ethanol or pair-fed HIF1dPA or Alb-Cre control mice. (B) Triglycerides were quantified from whole livers of ethanol-fed or pair-fed Alb-Cre or HIF1dPA mice. (C) Serum ALT in ethanol and pair-fed Alb-Cre mice and HIF1dPA mice. (D) Microscopy of HIF1dPA and Alb-Cre ethanol- and pair-fed mice.

Figure 4

Protection from liver injury in HIF-1α(Hep-/-) mice after chronic ethanol challenge. (A) Liver-weight to body-weight ratio in HIF-1α(Hep-/-) or control mice with chronic ethanol. (B) Triglycerides were quantified from whole livers of ethanol-fed or pair-fed HIF-1α(Hep-/-) or control mice. (C) Serum ALT data from HIF-1α(Hep-/-) and wild-type mice after chronic ethanol and LPS challenge. (D) Western blot showing HIF1α protein expression with chronic ethanol in control and HIF-1α(Hep-/-) mice, Densitometry in (E).

Figure 5

Microscopy of HIF-1α(Hep-/-) and control mice with chronic ethanol feeding or control feeding.

Figure 6

Altered patterns of genes involved in lipid homeostasis in HIF-1α(Hep-/-) or control mice. (A) PPARα mRNA in ethanol-fed or pair-fed control and HIF-1α(Hep-/-) mice was quantified by real-time PCR. (B) ADRP mRNA with ethanol and LPS challenge in wild-type and HIF-1α(Hep-/-) mice, quantified by real-time PCR. (C) ADRP mRNA expression in control (Alb-Cre) and HIF1dPA mice with ethanol or pair-feeding. (D) ADRP protein expression in Alb-Cre and HIF1dPA mice with ethanol or pair feeding, Densitometry in (E).

Figure 7

Changes in MCP-1 with chronic ethanol and/or LPS challenge. (A) Serum MCP-1 quantified by low-threshold cytokine assay in ethanol or pair-fed mice. (B) Hepatic MCP-1 mRNA in ethanol or pair-fed mice. (C) Serum MCP-1 quantified using high threshold cytokine assay in control or ethanol fed HIF-1α(Hep-/-) or wild-type mice, with or without LPS injection.

Figure 8

In vitro studies on relationship of MCP-1 and HIF1α (A) HIF1α protein measured by western blot of nuclear extracts from Huh7 cells treated with MCP-1; Densitometry appears below. (B) Real-time PCR for HIF1α mRNA after HIF1dPA plasmid treatment. (C) Triglyceride content in HIF1dPA treated cells or control cells. (D) Triglyceride in scrambled siRNA but not HIF1α siRNA pretreated cells treated with MCP-1. (E) Lipid accumulation in MCP-1 treated cells and HIF1dPA plasmid treated cells, yellow arrowheads show refractile lipid droplets by Oil-red O stain. (F) Lipid accumulation in HIF1α siRNA pretreated cells treated with MCP-1, yellow arrowheads show refractile lipid droplets by Oil-red O stain.