(Fig. 5). Because peroxisome proliferator-associated receptor α (PPARα) is associated with lipid accumulation, we examined PPARα mRNA levels in ethanol- and pair-fed control and HIF-1α(Hep−/−) mice. To amplify the effect of ethanol feeding, we also applied an LPS challenge. LPS has been identified in the portal circulation after chronic alcohol intake in mice and men, and it contributes to the development of ALD.20 To our surprise, we found that PPARα was similarly suppressed by ethanol feeding in each of these experimental groups, indicating that the HIF-1α effect on lipid accumulation

was independent selleckchem of PPARα (Fig. 6A). Next, we examined adipocyte differentiation-related protein (ADRP), which has been associated with HIF-1α expression.21

We found that ADRP mRNA was significantly up-regulated with ethanol feeding alone (P < 0.05) (Fig. 6B). Although no cooperative effect of LPS injection and chronic ethanol was observed in ADRP mRNA expression 2 hours after LPS injection (Fig. 6B), by 18 hours there was a robust cooperative up-regulation of ADRP mRNA with chronic ethanol and LPS injection (P < 0.05) (Supporting Fig. 2). HIF-1α(Hep−/−) mice were protected from any up-regulation of ADRP with chronic ethanol alone or with chronic ethanol and LPS challenge (Fig. 6B; Supporting Fig. 2). These results indicated that ADRP may be implicated in the differential effect of HIF-1α on lipid accumulation. Thus, we examined the effect of constitutive HIF activation on the expression of ADRP in Selleck Dabrafenib HIF1dPA and in control Alb-Cre mice (Fig. 6C). We found a significant increase in ADRP expression with ethanol feeding in Alb-Cre mice, similar to that observed in WT mice (P < 0.02). Furthermore, we found that the presence of the HIF1dPA transgene up-regulated hepatic ADRP protein expression to a similar extent as ethanol feeding (P < 0.01) (Fig. 6D,E).

In order to further dissect the mechanism of HIF-1α regulation in hepatic lipid accumulation, we supplemented our in vivo work with an in vitro model of hepatic lipid accumulation. The chemokine MCP-1 has recently been demonstrated to result in lipid accumulation in the hepatocyte cell line Huh7.8 First, we examined MCP-1 expression levels in ethanol-fed control and HIF-1α(Hep−/−) Rutecarpine mice. We found that alcohol feeding alone resulted in a small, but significant up-regulation in MCP-1 serum levels (Fig. 7A). This corresponded to increased MCP-1 hepatic mRNA with chronic ethanol (Fig. 7B). LPS stimulation and ethanol cooperatively up-regulated MCP-1 in WT mice (Fig. 7C). LPS induced MCP-1 in HIF1α(Hep−/−) mice to an extent comparable to WT, but there was no further increase in HIF-1α(Hep−/−) with alcohol feeding (Fig. 7C). To evaluate mechanistic events, we next treated Huh7 cells with recombinant MCP-1 or with a plasmid containing the degradation-resistant HIF1dPA mutant.