CYP2E1 is a central mediator for the synergistic effects of alcohol and free fatty acids on hepatocellular steatosis and inflammation

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are the most frequent conditions leading to elevated liver enzymes and liver cirrhosis, respectively, in the Western world. However, despite strong epidemiological evidence for combined effects on the progression of liver injury, the mutual interaction of the pathophysiological mechanisms is incompletely understood.

The aim of this study was to establish an in vitro model for joint effects of alcohol and lipids on hepatic steatosis and inflammation. Herein, we particularly wanted to assess the role of CYP2E1, which is activated by several hepatotoxins and contributes to alcoholic as well as non-alcoholic liver injury.

Methods and Results: Initially, we established the dose range in which neither alcohol nor incubation with the free fatty acid oleate affected viability or mitochondrial activity in primary human hepatocytes (PHH). Subsequently, we assessed the combined effect of alcohol (1 – 2%) and oleate (0.2mM) on hepatocellular lipid accumulation in PHH. Under these conditions, alcohol significantly enhanced oleate induced expression of genes regulating lipogenesis (FASN, SCD-1) and lipid peroxidation (CPT-1) as well as cellular triglyceride content and free fatty acid (FFA) levels, while alcohol alone had only a minimal effect. Analysis of heme oxygenase-1 (HMOX-1) expression and malondialdehyde levels revealed that the combination of alcohol and oleate caused significantly higher oxidative stress and lipid peroxidation than either of the two substances alone. Further, we observed a synergistic effect of alcohol and FFA on pro-inflammatory gene expression (IL-8 and ICAM-1) as well as CYP2E1 activity. HepG2 hepatoma cells stably overexpressing CYP2E1 but not control transfected HepG2 cells lacking CYP2E1 revealed similar synergistic effects of alcohol and FFA as PHH. Conversely, inhibition of CYP2E1 activity by chlormethiazole or incubation with ROS scavengers blunted these synergistic effects on lipogenesis, TG accumulation, lipid peroxidation and inflammatory response in PHH.

Conclusion: In summary, our new model allows the investigation of isolated or joint effects of alcohol and FFA on hepatocellular lipid metabolisms and inflammatory signaling. Our present findings indicate that CYP2E1 and CYP2E1-derived reactive oxygen species, respectively, play an important role in mediating synergistic effects of alcohol and lipids on hepatic steatosis and inflammation.