Ablation of cytochrome P450 omega-hydroxylase 4A14 gene attenuates hepatic steatosis and fibrosis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is characterized by simple hepatic steatosis (SS), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and cirrhosis. Dysregulated fatty acid metabolism in the liver plays a critical role in the pathogenesis of NAFLD. Cytochrome P450 omega-hydroxylase 4A14 (CYP4A14) is a homolog of human CYP4A hydroxylase that catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid in mice. The goal of this study was to determine the role of CYP4A14 in the development and the progression of NAFLD. Here, we showed that hepatic CYP4A expression was up-regulated in the livers of patients and three murine models of NAFLD. Adenovirus-mediated overexpression of CYP4A14 in the livers of C57BL/6 mice resulted in a fatty liver phenotype with a significant increase in hepatic fatty acid translocase (FAT/CD36) expression. In contrast, CYP4A14 gene-deficient mice fed a high-fat diet or a methionine and choline-deficient (MCD) diet exhibited attenuated liver lipid accumulation and reduced hepatic FAT/CD36 expression. In addition, hepatic inflammation and fibrosis was markedly ameliorated in MCD diet-fed CYP4A14-deficient mice. Collectively, CYP4A14 plays an important role in the pathogenesis of both SS and NASH and may represent a potential therapeutic target for the treatment of NAFLD.

Keywords: NAFLD; arachidonic acid; fatty acid; hepatic fibrosis; inflammation.

Fig. 1.

Up-regulation of CYP4A14 expression in the livers of three murine models of NAFLD. (A–C) Western blot analysis showing a significant increase in hepatic CYP4A14 protein expression in HFD-fed mice (A), db/db mice (B), and MCD-fed mice (C), respectively. *P < 0.05, **P < 0.01, n = 3. (D–F) Real-time PCR assay demonstrating that CYP4A14 mRNA expression was significantly increased in HFD-fed mice (D), db/db mice (E), and MCD-fed mice (F), respectively. **P < 0.01, n = 6. Data are presented as mean ± SEM. ND, normal diet.

Fig. S1.

Increased CYP4A protein expression in the livers of patients with NAFLD. (A) Western blot assay demonstrating increased CYP4A protein levels in the livers of patients with NAFLD. **P < 0.01, n = 4. (B) Immunohistochemistry analysis showing that NAFLD patients showed stronger hepatic CYP4A immunoreactivity than controls. (Magnification: 200×.) Negative control, immunostaining without primary antibody against CYP4A. Data are presented as mean ± SEM.

Fig. 2.

Hepatic CYP4A14 overexpression increased hepatic triglycerides accumulation. Male C57BL/6 mice were i.v. injected with 1.5 × 10⁹ pfu control adenovirus (Ad-GFP) (n = 7) or a CYP4A14 (Ad-cyp4a14) expressing adenovirus (n = 7) for 7 d. (A) Western blot analysis of CYP4A14 protein expression in the liver. **P < 0.01, n = 4. (B) Quantitative RT-PCR analysis of CYP4A14 mRNA levels in the liver. **P < 0.01, n = 7. (C) Oil Red O staining showing increased neutral lipid accumulation in Ad-CYP4A14–infected livers. (Magnification: 200×.) (D) Levels of hepatic triglycerides. *P < 0.05, n = 7. (E) Quantitative RT-PCR analysis of mRNA levels of genes involved in hepatic lipid metabolism. *P < 0.05, n = 7. (F) Western blot analysis demonstrating increased CD36 protein expression in Ad-CYP4A14–infected livers. *P < 0.05, n = 4. Data are presented as mean ± SEM. FA, fatty acid; VLDL, very low-density lipoprotein.

Fig. 3.

CYP4A14 gene deficiency ameliorated HFD-induced hepatic steatosis. Male wild-type littermates and CYP4A14 gene knockout mice (cyp4a14^−/−) were fed a normal diet (ND) or HFD for 8 wk. (A) Oil Red O staining showing a marked attenuation in HFD-induced hepatic steatosis in cyp4a14^−/− mice. (Magnification: 200×.) (B) Cyp4a14^−/− mice exhibited a significant reduction of hepatic triglycerides levels. n = 6–7. (C) Quantitative RT-PCR analysis demonstrating that CD36 mRNA levels were significantly decreased in the livers of HFD-fed cyp4a14^−/− mice. n = 6–7. (D) Western blot analysis showing a reduced hepatic CD36 protein expression in the livers of HFD-fed cyp4a14^−/− mice. n = 3. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. WT on ND; ^#P < 0.05 vs. WT on HFD.

Fig. S2.

Hepatic CYP4A10 and CYP4A12 mRNA levels were down-regulated in CYP4A14 gene-deficient male mice. Quantitative RT-PCR analysis of mRNA levels of hepatic CYP4A10 and CYP4A12 in the livers. Data are presented as mean ± SEM, *P < 0.05, n = 10.

Fig. S3.

CYP4A14 gene deficiency attenuated palmitic acid-induced lipid accumulation in primary cultured hepatocytes. (A) Fluorescence images of cellular BODIPY-C16 uptake in primary cultured mouse hepatocytes from wild-type and CYP4A14 gene knockout mice. (Magnification: 200×.) The cells were treated with vehicle [control (CON)] or palmitic acid (PA) at 0.5 mmol/L. (B) Quantification of BODIPY-C16 uptake in A. (C) Quantitative RT-PCR analysis of mRNA levels of FAT/CD36. (D) Western blot analysis of protein levels of FAT/CD36. *P < 0.05, n = 3.

Fig. S4.

CYP4A14 gene deficiency ameliorated HFD-induced hepatic steatosis. Female wild-type (fWT) littermates and CYP4A14 gene knockout mice (CYP4A14^−/−) were fed a ND or HFD for 8 wk. (A) Oil Red O staining showing a marked attenuation in HFD-induced hepatic steatosis in CYP4A14^−/− mice. (Magnification: 200×.) (B) CYP4A14^−/− mice exhibited a significant reduction of hepatic triglycerides levels. (C) Quantitative RT-PCR analysis demonstrating that CD36 mRNA levels were significantly decreased in the livers of HFD-fed CYP4A14^−/− mice. (D) Western blot analysis showing a reduced hepatic CD36 protein expression in the livers of HFD-fed CYP4A14^−/− mice. *P < 0.05, **P < 0.01 vs. fWT-ND; ^#P < 0.05 vs. fWT-HFD; n = 5–7. Data are presented as mean ± SEM.

Fig. 4.

CYP4A14 deficiency ameliorated MCD-induced steatohepatitis and liver injury. Male wild-type (WT) littermates and cyp4a14^−/− mice were fed with an ND (normal diet) or MCD diet for 4 wk. (A) Oil Red O staining showing that cyp4a14^−/− mice were resistant to MCD-induced lipid accumulation in the livers. (Magnification: 200×.) (B) Biochemical assay showing that hepatic triglycerides levels were significantly lower in MCD-fed cyp4a14^−/− mice than that in MCD-fed WT mice, n = 8–9. (C) Quantitative RT-PCR analysis showing that hepatic CD36 mRNA levels were significantly reduced in MCD-fed cyp4a14^−/− mice compared with MCD-fed WT mice, n = 8–9. (D) Western blot analysis demonstrating that hepatic CD36 protein levels were much lower in MCD-fed cyp4a14^−/− mice than that in MCD-fed WT mice, n = 3. (E and F) Cyp4a14^−/− mice exhibited reduced serum ALT and AST levels after receiving a MCD diet, n = 8–9. (G) Hepatic levels of malonaldehyde (MDA) in the liver, n = 8–9. (H) Quantitative RT-PCR analysis of mRNA levels of proinflammatory genes in the livers, n = 8–9. *P < 0.05, **P < 0.01 vs. WT on ND, ^#P < 0.05 vs. WT on MCD. Data are presented as mean ± SEM.

Fig. S5.

Inhibition of CYP4A in Sprague–Dawley rats ameliorated MCD diet-induced steatosis and liver injury. Male Sprague–Dawley rats were fed with MCD diet in the presence or absence of TS-011 for 2 wk. (A) Representative images of Oil Red O staining of livers of mice receiving ND, MCD diet alone, and MCD diet plus TS-011, respectively. (Magnification: 200×.) (B) Levels of hepatic triglycerides. (C) Levels of serum ALT. *P < 0.05, n = 10.

Fig. S6.

CYP4A14 deficiency ameliorated MCD-induced steatohepatitis and liver injury. Female wild-type (fWT) littermates and CYP4A14^−/− mice were fed with an ND or MCD diet for 4 wk. (A) Oil Red O staining showing that CYP4A14^−/− mice were resistant to MCD-induced lipid accumulation in the livers. (Magnification: 200×.) (B) Biochemical assay showing that hepatic triglycerides levels were significantly lower in MCD-fed CYP4A14^−/− mice than that in MCD-fed WT mice. (C) Quantitative RT-PCR analysis showing that hepatic CD36 mRNA levels were significantly reduced in MCD-fed CYP4A14^−/− mice compared with MCD-fed WT mice. (D) Western blot analysis demonstrating that hepatic CD36 protein levels were much lower in MCD-fed CYP4A14^−/− mice than that in MCD-fed WT mice. (E and F) CYP4A14^−/− mice exhibited reduced serum ALT and AST levels after receiving an MCD diet. (G) Hepatic levels of malonaldehyde (MDA) in the liver. (H) Quantitative RT-PCR analysis of mRNA levels of proinflammatory genes in the livers. *P < 0.05, **P < 0.01 vs. fWT-ND; ^#P < 0.05 vs. fWT-MCD; n = 5–7. Data are presented as mean ± SEM. TG, triglyceride.

Fig. 5.

CYP4A14 deficiency ameliorated MCD-induced hepatic fibrosis. Male wild-type (WT) and cyp4a14^−/− mice were fed with an ND (normal diet) or MCD diet for 8 wk. (A) Masson’s staining indicating that MCD-induced hepatic fibrosis was significantly attenuated in cyp4a14^−/− mice. a, WT on ND; b, WT on MCD; c, CYP4A14^−/− on ND; d, CYP4A14^−/− on MCD. (Magnification: 200×.) (B) CYP4A14 gene deficiency markedly attenuated MCD-induced α-SMA protein expression in the livers as assessed by an immunostaining analysis. n = 5–7. a, WT on ND; b, WT on MCD; c, CYP4A14^−/− on ND; d, CYP4A14^−/− on MCD. (Magnification: 200×.) (C) Western blot assay demonstrating reduced protein levels of COL1A2 and αSMA in the livers of cyp4a14^−/− mice. n = 3. (D) Quantitative RT-PCR analysis showing reduced mRNA levels of collagen 1a1, collagen 1a2, α-SMA, and TGFβ1 in the livers of cyp4a14^−/− mice. mRNA levels of related genes of hepatic fibrosis, n = 5–7. *P < 0.05 vs. WT on ND; ^#P < 0.05 vs. WT on MCD. Data are presented as mean ± SEM.

Fig. S7.

CYP4A14 deficiency ameliorated MCD-induced hepatic fibrosis. Female wild-type (fWT) and CYP4A14^−/− mice (fCYP4A14^−/−) were fed with an ND or MCD diet for 8 wk. (A) Masson’s staining indicating that MCD-induced hepatic fibrosis was significantly attenuated in CYP4A14^−/− mice. a, fWT on ND; b, fWT on MCD; c, fCYP4A14^−/− on ND; d, fCYP4A14^−/− on MCD. (Magnification: 200×.) (B) CYP4A14 gene deficiency markedly attenuated MCD-induced α-SMA protein expression in the livers as assessed by an immunostaining analysis. a, fWT on ND; b, fWT on MCD; c, fCYP4A14^−/− on ND; d, fCYP4A14^−/− on MCD; Semiquantitative analysis was shown in the bar graph (Right). (Magnification: 200×.) (C) Western blot assay demonstrating reduced protein levels of COL1A2 and α-SMA in the livers of CYP4A14^−/− mice. (D) Quantitative RT-PCR analysis showing reduced mRNA levels of profibrotic genes, collagen 1a1, collagen 1a2, α-SMA, and TGFβ1 in the livers of CYP4A14^−/− mice. *P < 0.05, **P < 0.01 vs. fWT-ND; ^#P < 0.05 vs. fWT-MCD; n = 5–7. Data are presented as mean ± SEM.