Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis

Abstract

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.

Keywords: cytochrome P450 2E1; ethanol; fatty acid-binding protein-4; sirtuin-1; steatosis.

Figure 1.

Cytochrome P4502E1 (CYP2E1) dependent ethanol metabolism alters FABP4 mRNA expression and FABP4 protein secretion. HepG2 and HuH7 cells were exposed to 0, 50, or 100mM ethanol (EtOH) in the presence of an acetaldehyde generating system and A) Fatty acid binding protein 4 (FABP4) mRNA expression and B) FABP4 protein secretion into culture medium measured. N=3 independent experiments. Effect of 0 or 50mM EtOH on HepG2 and HuH7 cells transfected to express CYP2E1 (E47 and HuH7^CYP+) in the absence or presence of chlormethiazole (CMZ; 100μM) on C) FABP4 mRNA expression and D) FABP4 protein secretion into culture medium. N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 CMZ + 50mM EtOH versus 50mM EtOH. E) Representative images of E47 and HuH7^CYP+ cells exposed to 0 or 50mM EtOH in the absence or presence of CMZ (100μM) following Oil red-O staining to detect neutral lipids. F) Quantification of Oil red-O staining by optical density (OD) absorption (492nm). N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 CMZ + 50mM EtOH versus 50mM EtOH.

Figure 2.. Cytochrome P4502E1-dependent ethanol metabolism inhibits sirtuin-1 expression.

A) Representative Western blot analysis of amount of sirtuin-1 (SIRT1) detected in HepG2 cells transfected to express CYP2E1 (E47) following exposure to ethanol (EtOH; 0-100mM) or exposure to EtOH (50mM) in the absence or presence of chlormethiazole (CMZ; 100μM). Equal protein loading was assessed by Ponceau-S (PS) membrane stain. B) Representative Western blot analysis of amount of SIRT1 detected in HuH7 cells transfected to express CYP2E1 (HuH7^CYP+) following exposure to EtOH (0-100mM) or exposure to EtOH (50mM) in the absence or presence of CMZ (100μM). Equal protein loading was assessed by PS membrane stain. C) Cumulative densitometric analysis of relative SIRT1 protein detected in E47 cells following exposure to EtOH (0-100mM), or exposure to EtOH (50mM) in the absence or presence of CMZ (100μM). N=3 independent experiments, *p<0.05 versus 0mM EtOH). D) Cumulative densitometric analysis of relative SIRT1 protein detected in HuH7^CYP+ cells following exposure to EtOH (0-100mM), or exposure to EtOH (50mM) in the absence or presence of CMZ (100μM). N=3 independent experiments, *p<0.05 versus 0mM EtOH).

Figure 3.. Altering sirtuin 1 activity/expression alters FABP4 mRNA expression and FABP protein secretion.

A) Effect of EtOH (50mM) or Ex547 (SIRT1 inhibitor, 50μM) on FABP4 mRNA expression in HepG2 and HuH7 cells transfected to express CYP2E1 (E47/HuH7^CYP+). N=3 independent experiments, *p<0.05 versus 0mM EtOH). B) Effect of EtOH (50mM) or Ex547 (50μM) on amount of FABP4 protein detected in culture medium in E47 and HuH7^CYP+ cells. N=3 independent experiments, *p<0.05 versus 0mM EtOH). C) Effect of EtOH (50mM) on FABP4 mRNA expression in E47 and HuH7^CYP+ cells and E47 and HuH7^CYP+ cells transfected to overexpress SIRT1 (SIRT1⁺). N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 SIRT1⁺ + 50mM EtOH versus 50mM EtOH. D) Effect of EtOH (50mM) on FABP4 protein secretion into culture medium from E47 and HuH7^CYP+ cells and SIRT1⁺ E47 and HuH7^CYP+ cells. N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 SIRT1⁺ + 50mM EtOH versus 50mM EtOH.

Figure 4.. Cytochrome P4502E1-dependent ethanol metabolism induces dephosphorylation of FOXO1/nuclear localization.

A) Representative Western blot analysis of phosphorylated and dephosphorylated FOXO1 (pFOXO1/FOXO1) protein abundance in cytoplasmic and nuclear cell fractions in HepG2 cells and HepG2 cells transfected to express CYP2E1 (E47) following exposure to ethanol (EtOH; 50mM). B) Representative Western blot analysis of pFOXO1 and FOXO1 protein abundance in cytoplasmic and nuclear cell fractions in HuH7 cells and HuH7 cells transfected to express CYP2E1 (HuH7^CYP+) following exposure to EtOH (50mM). C) Cumulative densitometric analysis of relative pFOXO1 and FOXO1 protein detected in cytoplasmic and nuclear cell fractions from HepG2 and E47 cells following exposure to EtOH (50mM). N=3 independent experiments, *p<0.05 versus 0mM EtOH. D) Cumulative densitometric analysis of relative pFOXO1 and FOXO1 protein detected in cytoplasmic and nuclear cell fractions from HuH7 and HuH7^CYP+cells following exposure to EtOH (50mM). N=3 independent experiments, *p<0.05 versus 0mM EtOH.

Figure 5.. Inhibition of FOXO1 abrogates the effect of ethanol on FABP4 mRNA expression, FABP4 secretion and neutral lipid accumulation in CYP2E1 expressing cells.

A) Effect of ethanol (EtOH, 50mM) on FABP4 mRNA expression in HepG2 and HuH7 cells transfected to express CYP2E1 (E47/HuH7^CYP+) in the absence or presence of the FOXO1 inhibitor AS184 3856 (AS; 1μM). N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 AS + 50mM EtOH versus 50mM EtOH. B) Effect of EtOH (50mM) on FABP4 protein detected in culture medium from E47 and HuH7^CYP+ cells in the absence or presence of AS (1μM). N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 AS + 50mM EtOH versus 50mM EtOH. C) Representative images of E47 and HuH7^CYP+ cells exposed to 0 or 50mM EtOH in the absence or presence of AS (1μM) following Oil red-O staining to detect neutral lipids. D) Relative quantification of Oil red-O staining by optical density (OD absorption, 492nm). N=3 independent experiments, *p<0.05 50mM EtOH versus 0mM EtOH, ^#p<0.05 AS + 50mM EtOH versus 50mM EtOH.

Figure 6.. Potential mechanisms by which CYP2E1-catalyzed ethanol metabolism alters hepatic FABP4 mRNA expression and FABP4 protein synthesis.

Chronic ethanol (EtOH) exposure induces CYP2E1 induction which utilizes NADPH as a cofactor to oxidize EtOH to acetaldehyde and generate reactive oxygen species (ROS). Increased acetaldehyde/ROS decreases sirtuin 1 (SIRT1) via decreased intracellular NAD⁺ availability. Decreased SIRT1 leads to reduced pFOXO1 translocation to the cytoplasm and increased nuclear FOXO1 protein levels Elevated nuclear FOXO1 leads to decreased fatty acid β-oxidation/increased lipogenesis and intracellular neutral lipid accumulation resulting in increased fatty acid binding protein-4 (FABP4) mRNA expression and FABP4 protein secretion.