High-fat diet induces fibrosis in mice lacking CYP2A5 and PPARα: a new model for steatohepatitis-associated fibrosis

Abstract

Obesity is linked to nonalcoholic steatohepatitis. Peroxisome proliferator-activated receptor-α (PPARα) regulates lipid metabolism. Cytochrome P-450 2A5 (CYP2A5) is a potential antioxidant and CYP2A5 induction by ethanol is CYP2E1 dependent. High-fat diet (HFD)-induced obesity and steatosis are more severe in CYP2A5 knockout (cyp2a5^-/-) mice than in wild-type mice although PPARα is elevated in cyp2a5^-/- mice. To examine why the upregulated PPARα failed to prevent the enhanced steatosis in cyp2a5^-/- mice, we abrogate the upregulated PPARα in cyp2a5^-/- mice by cross-breeding cyp2a5^-/- mice with PPARα knockout (pparα^-/-) mice to create pparα^-/-/cyp2a5^-/- mice. The pparα^-/-/cyp2a5^-/- mice, pparα^-/- mice, and cyp2a5^-/- mice were fed HFD to induce steatosis. After HFD feeding, more severe steatosis was developed in pparα^-/-/cyp2a5^-/- mice than in pparα^-/- mice and cyp2a5^-/- mice. The pparα^-/-/cyp2a5^-/- mice and pparα^-/- mice exhibited comparable and impaired lipid metabolism. Elevated serum alanine transaminase and liver interleukin-1β, liver inflammatory cell infiltration, and foci of hepatocellular ballooning were observed in pparα^-/-/cyp2a5^-/- mice but not in pparα^-/- mice and cyp2a5^-/- mice. In pparα^-/-/cyp2a5^-/- mice, although redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 and its target antioxidant genes were upregulated as a compensation, thioredoxin was suppressed, and phosphorylation of JNK and formation of nitrotyrosine adduct were increased. Liver glutathione was decreased, and lipid peroxidation was increased. Interestingly, inflammation and fibrosis were all observed within the clusters of lipid droplets, and these lipid droplet clusters were all located inside the area with CYP2E1-positive staining. These results suggest that HFD-induced fibrosis in pparα^-/-/cyp2a5^-/- mice is associated with steatosis, and CYP2A5 interacts with PPARα to participate in regulating steatohepatitis-associated fibrosis.

Keywords: 3-NT; CYP2E1; IL-1β; choline; lipid peroxidation.

Graphical abstract

Fig. 1.

High-fat diet (HFD)-induced hepatic steatosis is more severe in peroxisome proliferator-activated receptor-α knockout (pparα^−/−)/cytochrome P-450 2A5 knockout (cyp2a5^−/−) mice (P⁻/A⁻) than in mice expressing cyp2a5 but not expressing pparα (P⁻/A⁺) and mice expressing pparα but not expressing cyp2a5 (P⁺/A⁻). A: liver nuclear peroxisome proliferator-activated receptor α (PPARα) DNA-binding activity (n = 4–5 mice). *P < 0.05 compared with P⁻/A⁻ control diet (CD) group; $P < 0.05 compared with P⁻/A⁻ CD group; #P < 0.05 compared with P⁻/A⁺ CD group; &P < 0.05 compared with P⁻/A⁻ HFD group; ^P < 0.05 compared with P⁻/A⁺ HFD group. B: liver expression of cytochrome P-450 2A5 (CYP2A5). C: body weight (n = 4–5 mice). *P < 0.05 compared with P⁻/A⁻ HFD group. D: liver contents of triglyceride (TG, n = 4–5 mice). *P < 0.05 compared with corresponding CD group; #P < 0.05 compared with P⁺/A⁻ HFD group; &P < 0.05 compared with P⁻/A⁺ HFD group. E: liver sections with H&E staining. F: adipose differentiation-related protein (ADRP) IHC staining.

Fig. 2.

High-fat diet (HFD) induces steatohepatitis in peroxisome proliferator-activated receptor-α knockout (pparα^−/−)/cytochrome P-450 2A5 knockout (cyp2a5^−/−) mice (P⁻/A⁻) rather than in mice expressing cyp2a5 but not expressing pparα (P⁻/A⁺) and mice expressing pparα but not expressing cyp2a5 (P⁺/A⁻). A: neutrophil [naphthol AS-D chloroacetate esterase (NASDCA)] staining. Top, negative staining in the foci; arrows on bottom show isolated positive staining. B: H&E staining in liver sections from HFD-fed P⁻/A⁻ mice. Black arrows show inflammatory infiltration; a green arrow shows a lipid droplet surrounded by inflammatory cells; yellow arrows show foci of hepatocellular ballooning fibrosis. C: IHC for CD3, F4/80, and CD335 in liver sections from HFD-fed P⁻/A⁻ mice. Red arrows show positive staining, and green arrows show negative staining. D: IHC for adipose differentiation-related protein (ADRP), Ki-67, α-fetoprotein (AFP), and collagen I in liver sections from HFD-fed P⁻/A⁻ mice. Red arrows show positive staining; yellow arrows show foci of hepatocellular ballooning. Note that foci of hepatocellular ballooning with negative staining have unspecific orange staining. E: serum alanine transaminase (ALT) level (n = 4–5 mice). *P < 0.05 compared with P⁻/A⁻ control diet (CD) group; #P < 0.05 compared with P⁺/A⁻ HFD group; &P < 0.05 compared with P⁻/A⁺ HFD group. F: liver contents of interleukin-1β (IL-1β, n = 3–5 mice). *P < 0.05 compared with P⁺/A⁻ CD group; ^P < 0.05 compared with P⁻/A⁺ CD group; #P < 0.05 compared with P⁺/A⁻ HFD group; &P < 0.05 compared with P⁻/A⁺ HFD group.

Fig. 3.

High-fat diet (HFD) induces steatofibrosis in peroxisome proliferator-activated receptor-α knockout (pparα^−/−)/cytochrome P-450 2A5 knockout (cyp2a5^−/−) mice (P⁻/A⁻) but not in mice expressing cyp2a5 but not expressing pparα (P⁻/A⁺) and mice expressing pparα but not expressing cyp2a5 (P⁺/A⁻). A: fibrosis was visualized by Sirius red/fast green staining (top) and Masson’s trichrome staining (bottom). Black arrows show positive staining, and yellow arrows show foci of hepatocellular ballooning. B: α-smooth muscle actin (α-SMA) staining. A black arrow shows positive staining, a yellow arrow shows foci of hepatocellular ballooning, and red arrows show blood vessel walls with positive staining. C: Sirius red/fast green staining in liver sections from HFD-fed P⁻/A⁻ mice. Bottom, higher magnification of top. Yellow arrows show foci of hepatocellular ballooning. D: comparison of different fibrosis models. Left, HFD-induced steatofibrosis in P⁻/A⁻ mice (squared area is shown in A with a higher magnification); middle, thioacetamide (TAA)-induced necrofibrosis in P⁺/A⁻mice (TAA was injected ip at 200 mg/kg two times a week for 30 days); right, necrofibrosis induced by binge-on-chronic ethanol in humanized cytochrome P-450 2E1 (CYP2E1) knock-in mice (cyp2e1^−/− KI mice). The cyp2e1^−/− KI mice were fed liquid ethanol diet for 5 wk, and binge ethanol was administrated by gavage at 5 g/kg one time per week for 5 times during the chronic ethanol feeding.

Fig. 4.

Lipid metabolism is comparable in peroxisome proliferator-activated receptor-α knockout (pparα^−/−)/cytochrome P-450 2A5 knockout (cyp2a5^−/−) mice (P⁻/A⁻) and mice expressing cyp2a5 but not expressing pparα (P⁻/A⁺) mice. A: serum glycerol. B: serum triglyceride (TG, n = 4–5 mice). C: serum free fatty acids (FFAs, n = 4–5 mice). D: serum β-hydroxylbutyrate (n = 4–5 mice). E: liver expression of liver fatty acid-binding protein (L-FABP), fatty acid synthase (FAS), and lipogenic regulators. SREBP-1, sterol regulatory element-binding protein-1. *P < 0.05 compared with group of mice expressing pparα but not expressing cyp2a5 (P⁺/A⁻) and given control diet (CD); $ P < 0.05 compared with P⁻/A⁻ CD group; #P < 0.05 compared with P⁻/A⁺ CD group; &P < 0.05 compared with P⁻/A⁻ high-fat diet (HFD) group; ^P < 0.05 compared with P⁻/A⁺ HFD group.

Fig. 5.

Antioxidant capacity is compensated and oxidative stress is induced in peroxisome proliferator-activated receptor-α knockout (pparα^−/−)/cytochrome P-450 2A5 knockout (cyp2a5^−/−) mice (P⁻/A⁻). A: nuclear factor erythroid 2-related factor 2 (Nrf2)-binding activity (n = 4–5 mice). *P < 0.05 compared with corresponding control diet (CD) group. B: hepatic expression of glutamylcysteine synthetase (GCS), catalase, thioredoxin, and JNK. Trx, thioredoxin. C: total glutathione (GSH) levels in liver (n = 4–5 mice). *P < 0.05 compared with corresponding CD group; #P < 0.05 compared with mice expressing pparα but not expressing cyp2a5 (P⁺/A⁻) and given a high-fat diet (HFD); &P < 0.05 compared with mice expressing cyp2a5 but not expressing pparα (P⁻/A⁺) and given HFD. F: IHC nitrotyrosine (3-NT) staining was positive in the HFD-fed P⁻/A⁻ mice. PV, portal vein; CV, central vein. D: foci of hepatocellular ballooning were positive with 3-NT but negative for cytochrome P-450 2E1 (CYP2E1). The arrows show the foci of hepatocellular ballooning. Black arrows show positive staining. E: IHC staining for CYP2E1 in the HFD-fed P⁻/A⁻ mice. Red arrows show foci of hepatocellular ballooning. G: thiobarbituric acid reactive substances (TBARS) levels in liver (n = 3–5 mice). *P < 0.05 compared with P⁻/A⁻ CD group; #P < 0.05 compared with P⁺/A⁻ HFD group; &P < 0.05 compared with P⁻/A⁺ HFD group.

Fig. 6.

Schematic hypothesized mechanism of peroxisome proliferator-activated receptor-α (PPARα) and cytochrome P-450 2A5 (CYP2A5) interaction in the development of steatofibrosis. ROS, reactive oxygen species; LPO, lipid peroxidation; HSC, hepatic stellate cells; Nrf2, nuclear factor erythroid 2-related factor 2.