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Background Hepatic steatosis is an early pathology of alcohol‐associated liver disease (ALD). Fatty acid‐binding protein‐4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. Methods Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1‐inhibitor; CMZ) and/or EX‐527 (a sirtuin‐1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4 mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], β‐catenin, peroxisome proliferator‐activated receptor‐α [PPARα], and lipin‐1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. Results CYP2E1‐dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased β‐catenin, PPARα, and lipin‐1α protein levels in CYP2E1‐expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1 mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1‐expressing cells. Pharmacological inhibition of β‐catenin, PPARα, or lipin‐1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. Conclusion CYP2E1‐dependent ethanol metabolism inhibits SIRT1‐FOXO1 signaling, which leads to increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.

Fatty acid binding protein-4 (FABP4) is not normally expressed in the liver but is induced in alcohol-dependent liver disease (ALD)). This study sought to identify mechanisms whereby ethanol (EtOH) metabolism alters triglyceride accumulation and FABP4 production. Human hepatoma cells which were stably transfected to express alcohol dehydrogenase (ADH) or cytochrome P4502E1 (CYP2E1) were exposed to EtOH in the absence/presence of inhibitors of ADH (4-methylpyrazole) or CYP2E1 (chlormethiazole). Cells were analyzed for free fatty acid (FFA) content and FABP4 mRNA, then culture medium assayed for FABP4 levels. Cell lysates were analyzed for AMP-activated protein kinase-α (AMPKα), Acetyl-CoA carboxylase (ACC), sterol regulatory element binding protein-1c (SREBP-1c), and Lipin-1β activity and localization in the absence/presence of EtOH and pharmacological inhibitors. CYP2E1-EtOH metabolism led to increased FABP4 mRNA/protein expression and FFA accumulation. Analysis of signaling pathway activity revealed decreased AMPKα activation and increased nuclear-SREBP-1c localization following CYP2E1-EtOH metabolism. The role of AMPKα-SREBP-1c in regulating CYP2E1-EtOH-dependent FFA accumulation and increased FABP4 was confirmed using pharmacological inhibitors and over-expression of AMPKα. Inhibition of ACC or Lipin-1β failed to prevent FFA accumulation or changes in FABP4 mRNA expression or protein secretion. These data suggest that CYP2E1-EtOH metabolism inhibits AMPKα phosphorylation to stimulate FFA accumulation and FABP4 protein secretion via an SREBP-1c dependent mechanism.

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