3A). Ethanol-fed HIF1dPA mice had the highest LW/BW ratios (P < 0.05 versus HIF1dPA pair-fed mice). Examination of liver triglycerides in whole-liver extracts revealed that alcohol caused an up-regulation of triglyceride in hepatic extracts in

control mice at 4 weeks (Fig. 3B). Triglyceride levels were higher in pair-fed HIF1dPA mice versus pair-fed control mice (P < 0.05, buy MLN2238 HIF1dPA pair-fed versus Alb-Cre pair-fed) indicating an effect of constitutive HIF-1α on lipid accumulation in the absence of any other stimulus. Alcohol-fed HIF1dPA mice had the highest average hepatic triglyceride content (P < 0.05 versus all other groups). The presence of HIF1dPA transgene also led to serum ALT levels comparable to Alb-Cre ethanol-fed mice (Fig. 3C). Histopathology analysis also confirmed that ethanol-fed HIF1dPA mice had more lipid vacuolization than ethanol-fed Alb-Cre mice (Fig. 3D). These results suggested that constitutive HIF1 activation in hepatocytes (HIF1dPA IDH inhibitor mice) results in liver abnormalities reminiscent of ALD and that alcohol feeding and constitutive HIF-1 activation cooperatively up-regulated

hepatic steatosis. Because our findings suggested an effect of hepatocyte-specific HIF-1α expression on lipid accumulation, we sought to test whether elimination of HIF-1α activity in hepatocytes could ameliorate the pathology associated with chronic ethanol feeding. We used a mouse engineered by Johnson and coworkers11 where native HIF-1α is flanked by LoxP sites, and coexpression of Cre recombinase results in tissue-specific deletion of HIF-1α. Analysis of mice with hepatocyte-specific deletion of HIF-1α and controls maintained on the ethanol diet revealed increased LW/BW ratios in WT ethanol-fed mice versus control mice at 4 weeks. In contrast, HIF-1α(Hep−/−) mice showed no significant difference in LW/BW ratio between pair-fed and ethanol-fed groups (Fig. 4A). Consistent with the role of HIF-1α in hepatocyte steatosis, HIF-1α(Hep−/−) mice were MCE protected from the increase in liver triglyceride content observed in WT mice after

alcohol feeding (Fig. 4B). WT mice showed a robust cooperative up-regulation of serum ALT with chronic ethanol and LPS challenge (P < 0.02, WT ethanol/LPS versus WT pair-fed). In contrast, HIF1α(Hep−/−) mice were protected against serum ALT increase, even in the presence of chronic ethanol and LPS (Fig. 4C). Next, we performed immunoblotting on nuclear extracts from WT and HIF-1α(Hep−/−) mice. Ethanol feeding resulted in a significant increase in HIF-1α expression in nuclear extracts prepared from WT mice (Fig. 4D). In contrast, nuclear extracts from HIF-1α(Hep−/−) mice had very low levels of HIF-1α expression, and no further up-regulation with ethanol feeding was observed, confirming suppression of HIF-1α signaling in our mouse model (Fig. 4D,E).