The presence of p47phox in liver parenchymal cells is a key mediator in the pathogenesis of alcoholic liver steatosis

Abstract

Background: Reactive oxygen species contribute to steatosis and inflammation in alcoholic liver disease (ALD). Here, we evaluated the selective contribution of p47phox, a critical subunit of nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase complex, in liver parenchymal cells and in bone marrow (BM)-derived cells.

Methods: Female C57Bl/6 wild type [WT], total body p47phox-deficient knockout [KO] or p47phox chimera mice generated by BM transplantation of p47phox-KO-BM into irradiated WT mice (WT/p47phox-KO-BM mice) received 5% Lieber-DeCarli alcohol or control (pair feeding) diet for 4 weeks.

Results: Alcohol-induced liver steatosis as measured by Oil Red O staining and serum triglyceride up-regulation were prevented in p47phox-KO mice but not in WT/p47phox-KO-BM chimeras compared to WT controls. Serum alanine aminotransferase (ALT) was significantly increased in alcohol-fed WT mice but not in p47phox-KO mice compared to pair-fed controls. There was no protection from alcohol-induced increase in ALT and liver damage in the WT/p47phox-KO-BM mice. Alcohol-induced liver steatosis was accompanied by up-regulation of the lipid droplet-stabilizing protein, adipocyte differentiation-related protein (ADRP), and the fatty acid synthesis-associated genes, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACACA). Total body deficiency in p47phox but not selective absence of p47phox in BM-derived cells prevented alcohol-induced up-regulation of ADRP, FASN, and ACACA in the liver. Finally, alcohol-induced activation and DNA binding of nuclear factor κB (NF-κB), a master regulator of inflammation, were significantly increased after alcohol feeding in WT but not in p47phox-KO mice. Selective deficiency of p47phox in BM-derived cells (WT/p47phox-KO-BM chimera) failed to prevent NF-κB induction after alcohol feeding.

Conclusions: Total body deficiency in p47phox subunit of NADPH oxidase complex protects mice from alcohol-induced liver steatosis via mechanisms involving ADRP, FASN, and ACACA as well as from alcohol-induced NF-κB activation. In contrast, selective absence of p47phox in BM-derived cells fails to provide protection via these mechanisms. These results suggest that p47phox in parenchymal cells plays a critical role in the pathogenesis of ALD.

Fig. 1

Total body p47phox knockout (p47phox-KO) but not cell-specific deficiency of p47phox protects mice from alcohol-induced liver steatosis. C57Bl/6 (wild type [WT]) female mice received Lieber–DeCarli alcohol or pair-fed diet for 4 weeks. (A) Photomicrographs prepared from H&E-stained liver sections from ethanol (EtOH)-fed or pair-fed mice. (B) Photomicrographs prepared from Oil Red O-stained frozen liver sections from EtOH-fed or pair-fed mice. (C, D) Oil Red O–positive area was quantified for the comparison of steatotic changes between mouse groups. There were 6 to 8 mice/group. PF, pair-fed; BM, bone marrow.

Fig. 2

Total body p47phox deficiency more efficiently reduced alcohol-induced liver injury than selective lack of p47phox expression in bone marrow (BM)-derived cells. Mice received alcohol or pair-fed diet as described in Fig. 1. (A, B) Liver triglycerides were measured using the L-Type Triglyceride H kit. (C, D) Serum alanine aminotransferase (ALT) was quantified as an index of liver injury in sera from ethanol (EtOH)-fed or pair-fed mice. There were 6 to 8 mice/group. KO, knockout; PF, pair-fed; WT, wild type.

Fig. 3

Chronic ethanol (EtOH) feeding stimulates stabilization of lipid droplets in the liver in a p47phox-dependent manner. Whole-liver lysates were prepared and used for the measurement of adipocyte differentiation–related protein (ADRP) in (A) WT and p47phox-KO groups or (C) in WT/WT-BM and WT/p47phox-KO-BM groups. Western blot (WB) ADRP band densitometry is shown in the corresponding graphs (B, D). BM, bone marrow; KO, knockout; PF, pair-fed; WT, wild type.

Fig. 4

Chronic ethanol (EtOH) feeding stimulates fatty acid synthesis in the liver in a p47phox-dependent manner. (A, B) Fatty acid synthase (FASN) and (C, D) acetyl-CoA carboxylase (ACACA) mRNA expression in livers of mice after 4 weeks of 5% (vol/vol) EtOH or pair feeding. There were 6 to 8 mice/group. BM, bonemarrow; KO, knockout; PF, pair-fed; WT, wild type.

Fig. 5

Chronic ethanol (EtOH) feeding in mice leads to the activation of NADPH oxidase. (A, B) gp91phox or (C, D) p40phox subunit mRNA in EtOH-fed or pair-fed mouse livers was quantified by real-time polymerase chain reaction. (E, F) p47phox mRNA expression in livers of mice after 4 weeks of 5% (vol/vol) EtOH or pair feeding. There were 6 to 8 mice/group. BM, bone marrow; KO, knockout; PF, pair-fed; WT, wild type.

Fig. 6

Chronic ethanol (EtOH) feeding in mice leads to the increased nuclear factor κB (NF-κB) binding in liver and occurs despite inactivation of p47phox in bone marrow (BM)-derived cells. (A) NF-κB DNA-binding activity was measured after equal loading of liver nuclear extracts using the electrophoretic mobility shift assay (EMSA) and a NF-κB-specific probe; 10-fold access of unlabeled NF-κB-oligo was used for competition (cold comp); densitometry results of EMSA are shown as mean ± SE in the graph below (B). BM, bone marrow; KO, knockout; PF, pair-fed; WT, wild type.