Susceptibility of L-FABP-/- mice to oxidative stress in early-stage alcoholic liver

Abstract

Chronic ethanol consumption is a prominent cause of liver disease worldwide. Dysregulation of an important lipid uptake and trafficking gene, liver-fatty acid binding protein (L-FABP), may contribute to alterations in lipid homeostasis during early-stage alcoholic liver. We have reported the detrimental effects of ethanol on the expression of L-FABP and hypothesize this may deleteriously impact metabolic networks regulating fatty acids. Male wild-type (WT) and L-FABP(-/-) mice were fed a modified Lieber-DeCarli liquid diet for six weeks. To assess the response to chronic ethanol ingestion, standard biochemical indicators for alcoholic liver disease (ALD) and oxidative stress were measured. Ethanol ingestion resulted in attenuation of hepatic triglyceride accumulation and elevation of cholesterol in L-FABP(-/-) mice. Lipidomics analysis validated multiple alterations in hepatic lipids resulting from ethanol treatment. Increased immunohistochemical staining for the reactive aldehydes 4-hydroxynonenal and malondialdehyde were observed in WT mice ingesting ethanol; however, L-FABP(-/-) mice displayed prominent protein adducts in liver sections evaluated from pair-fed and ethanol-fed mice. Likewise, alterations in glutathione, thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyl content all indicated L-FABP(-/-) mice exhibit high sustained oxidative stress in the liver. These data establish that L-FABP is an indirect antioxidant protein essential for sequestering FFA and that its impairment could contribute to in the pathogenesis of ALD.

Fig. 1.

Chronic ethanol consumption alters lipid content in WT and L-FABP^−/− mice. Ethanol-consuming animals exhibit early-stage ALD following the six-week ethanol-feeding regimen as demonstrated with H&E staining and Plin2 immunohistochemistry. WT mice accumulate triglyceride in the midzonal region, and L-FABP^−/− mice display mild attenuation in zones 2 and 3 of the lobule. 400× magnification.

Fig. 2.

Evaluation of the oxidative stress response in WT and L-FABP^−/− mice following the Lieber-DeCarli diet regimen. Immunohistochemistry reveals ethanol- and genotype-induced elevations in CYP2E1 and the lipid peroxidation markers 4-HNE and MDA (A), 400× magnification. Immunoblot analysis (B) and relative quantification (C) of CYP2E1, HO-1, GST A4/Pi, and GPx. Different letters (a, b) indicate statistical significance of P < 0.05 or greater.

Fig. 3.

Elevated biotin tagging of protein carbonyls in L-FABP^−/− mice. Anti-biotin immunoblot of pooled cytosolic fractions from pair- and ethanol-fed WT and L-FABP^−/− mice (A). Densitometry represented as percentage of the WT control (B). N = 6. Different letters (a, b, c) indicate statistical significance of P < 0.05 or greater.

Fig. 4.

Immunoblot and coimmunoprecipitation analysis of pair- and ethanol-fed WT and L-FABP^−/− mice. Immunoblots reveal alterations in genes involved in hepatic lipid uptake and trafficking (A), metabolic regulation (B), and downstream PPARα genes (C) with relative densitometry quantification (D). PPARα and HNF-4α binding activities (E) are altered, which may be further explained by the reduced ligand activation facilitated by L-FABP as demonstrated by coimmunoprecipitation analysis of PPARα and L-FABP (F). Different letters (a, b) indicate statistical significance of P < 0.05 or greater.