Cytochrome P450-2E1 promotes fast food-mediated hepatic fibrosis

Abstract

Cytochrome P450-2E1 (CYP2E1) increases oxidative stress. High hepatic cholesterol causes non-alcoholic steatohepatitis (NASH) and fibrosis. Thus, we aimed to study the role of CYP2E1 in promoting liver fibrosis by high cholesterol-containing fast-food (FF). Male wild-type (WT) and Cyp2e1-null mice were fed standard chow or FF for 2, 12, and 24 weeks. Various parameters of liver fibrosis and potential mechanisms such as oxidative and endoplasmic reticulum (ER) stress, inflammation, and insulin resistance (IR) were studied. Indirect calorimetry was also used to determine metabolic parameters. Liver histology showed that only WT fed FF (WT-FF) developed NASH and fibrosis. Hepatic levels of fibrosis protein markers were significantly increased in WT-FF. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in FF-fed WT. Serum endotoxin, TLR-4 levels, and inflammatory markers were highest in WT-FF. FAS, PPAR-α, PPAR-γ, and CB1-R were markedly altered in WT-FF. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in FF-fed WT. Indirect calorimetry showed that Cyp2e1-null-mice fed FF exhibited consistently higher total energy expenditure (TEE) than their corresponding WT. These results demonstrate that CYP2E1 is important in fast food-mediated liver fibrosis by promoting nitroxidative and ER stress, endotoxemia, inflammation, IR, and low TEE.

Figure 1. Increased histopathological hepatic injury and weight gain in WT-FF mice.

(a) Representative H&E staining images for the livers of the indicated mouse groups fed for 2, 12, or 24 wks are presented. (b) NAFLD activity score, (c) caloric intake, (d) percentage of body weight gain at 24 wks, (e) serum ALT, (f) serum leptin, (g) hepatic TG, (h) liver index, and (i) fat index are shown (n = 4–5/group). D, diet; G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 2. Development of hepatic fibrosis in WT-FF mice.

(a,b) Representative Sirius red staining for collagen in hepatic sections (n = 4–5/group) at 24 wks of feeding and percentage of fibrotic areas, respectively, following 2–24 wks of feeding. Immunoreactive levels and densitometric analyses for (c) collagen, (d) α-SMA, (e) MMP-9, (f) TGF-β and (g) the loading control β-actin are shown. (h) Images and densitometric analysis of P-SMAD-3 and (i) the loading control SMAD-3. Values of WT-STD were set at 1 as controls (n = 3–4/group). D, diet; G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 3. Increased hepatic iNOS protein levels, but not CYP2E1 in WT-FF mice.

(a,b) Immunoblot images and densitometric analyses for CYP2E1 and iNOS, respectively, normalized to β-actin (c). Values for WT-STD were set at 1 as controls. G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 4. Increased serum endotoxin and hepatic markers of inflammation in WT-FF.

(a) Tabulated serum endotoxin levels. Immunoblot images and densitometric analyses for (b) TLR-4, (c) TNF-α, (d) F4/80, and (e) cleaved osteopontin, normalized to β-actin (f). Values for WT-STD were set at 1 as controls (n = 3–4/group). D, diet; G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 5. Alteration of hepatic proteins involved in lipid homeostasis in FF-fed mice.

Immunoblot images and densitometric analyses for (a) FAS, (b) PPAR-α, (c) PPAR-γ, and (d) CB1-R, normalized to β-actin (e) are shown. Values for WT-STD were set at 1 as controls (n = 3–4/group). D, diet; G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 6. Increased levels of ER stress proteins in WT-FF.

Transmission EM analysis demonstrates (a) regularly organized ER (ER) (STD) and irregularly arranged and/or disrupted ER in FF (FF), which are more prominent in WT-FF (n = 3–4/group). (b) Nuclei are shown for all groups. Immunoblot images and densitometric analyses for (c) P-ATF-2, (F, upper) P-ELF-2, and (G, upper) P-PERK, normalized to their corresponding protein as indicated. Values for WT-STD were set at 1 as controls (n = 3–4/group). D, diet; G, genotype. Columns without a common letter are significantly different from each other, P < 0.05.

Figure 7. Impaired GT and increased IR in WT-FF.

(a,b) Tail blood was collected following glucose injection (i.p., 2 g/kg) or (C and D) insulin injection (i.p., 0.75 U/kg; Eli Lilly) at indicated times, and total AUC values were tabulated (n = 4/group). *Significantly different from control, P < 0.05.

Figure 8. Schematic diagram for the role of CYP2E1 in western FF-mediated liver fibrosis.

The first and second hits for the development of NASH and fibrosis by western fast food are presented. CYP2E1 seems to play a critical role in elevating the first hit, fat accumulation, and the development of advanced stages of NASH with liver fibrosis through multiple second hits such as increased nitroxidative stress, ER stress, gut derived endotoxin levels, inflammation, and insulin resistance (IR). The dotted arrows refer to delayed or little development.