Inhibition of soluble epoxide hydrolase attenuates high-fat-diet-induced hepatic steatosis by reduced systemic inflammatory status in mice

Abstract

Non-alcoholic fatty liver disease is associated with obesity and considered an inflammatory disease. Soluble epoxide hydrolase (sEH) is a major enzyme hydrolyzing epoxyeicosatrienoic acids and attenuates their cardiovascular protective and anti-inflammatory effects. We examined whether sEH inhibition can protect against high-fat (HF)-diet-induced fatty liver in mice and the underlying mechanism. Compared with wild-type littermates, sEH-null mice showed lower diet-induced lipid accumulation in liver, as seen by Oil-red O staining and triglycerides levels. We studied the effect of sEH inhibition on diet-induced fatty liver by feeding C57BL/6 mice an HF diet for 8 weeks (short-term) or 16 weeks (long-term) and administering t-AUCB, a selective sEH inhibitor. sEH inhibition had no effect on the HF-diet-increased body and adipose tissue weight or impaired glucose tolerance but alleviated the diet-induced hepatic steatosis. Adenovirus-mediated overexpression of sEH in liver increased the level of triglycerides in liver and the hepatic inflammatory response. Surprisingly, the induced expression of sEH in liver occurred only with the long-term but not short-term HF diet, which suggests a secondary effect of HF diet on regulating sEH expression. Furthermore, sEH inhibition attenuated the HF-diet-induced increase in plasma levels of proinflammatory cytokines and their mRNA upregulation in adipose tissue, which was accompanied by increased macrophage infiltration. Therefore, sEH inhibition could alleviate HF-diet-induced hepatic steatosis, which might involve its anti-inflammatory effect in adipose tissue and direct inhibition in liver. sEH may be a therapeutic target for HF-diet-induced hepatic steatosis in inhibiting systemic inflammation.

Figure 1. sEH deficiency ameliorated high-fat (HF)-diet–induced hepatic steatosis in mice.

Wild type (WT) littermates and sEH-null mice were fed a regular chow or HF diet for 8 weeks. (WT Chow, WT HF: n=6; sEH-null Chow, sEH-null HF: n=8) (A) Body weight, liver weight and epididymal fat weight. (B) Plasma levels of triglycerides and cholesterol. (C) Oil-red O staining in liver sections. (D) Levels of cholesterol and triglycerides in liver. Data are mean ± SEM (* P<0.05, ** P<0.01).

Figure 2. 4-week sEH inhibitor administration attenuated 8-week HF-diet–induced triglycerides accumulation in mouse liver.

Male C57BL/6 mice were fed regular chow or a HF diet for 8 weeks with or without sEH inhibitor (sEHI) t-AUCB administration in drinking water from week 5 (n=8 mice/group). (A) Body weight, liver weight and epididymal fat weight. (B) Plasma levels of triglycerides and cholesterol. (C) Oil-red O staining in liver sections. (D) Levels of triglycerides and cholesterol in liver. (E) Western blot analysis of protein levels of sEH and β-actin as a normalization control in liver. (F) sEH activity in liver. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 3. sEH inhibition or deficiency could block 8-week HF-diet–induced inflammation.

(A) Proinflammatory cytokine levels in plasma of mice fed an 8-week chow or HF diet with or without 4-week t-AUCB. (B) Proinflammatory cytokine levels in plasma of WT and sEH-null mice fed an 8-week regular chow or HF diet. (C) mRNA levels of macrophage marker F4/80 in epididymal fat tissue. mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), interferon γ (IFN-γ), monocyte chemoattractant protein 1 (MCP-1) in epididymal fat tissue of (D) mice fed an 8-week chow or HF diet with or without 4-week sEH inhibitor and (E) WT or sEH-null mice fed a chow or HF diet. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 4. Full-term sEH inhibitor administration attenuated 16-week HF-diet–induced hepatic steatosis.

Male C57BL/6 mice were fed a regular chow or HF diet for 16 weeks with or without sEHI t-AUCB in drinking water starting from 3 days before diet (Chow 16W: n=10, HF 16W: n=15, HF 16W+sEHI 16W: n=11). (A) Systolic and diastolic blood pressure. (B) Body weight and ratio of liver weight compared to tibia length. (C) Plasma levels of triglycerides and cholesterol. (D) Oil-red O staining in liver sections. (E) Levels of triglycerides and cholesterol in liver. (F) Proinflammatory cytokine levels in plasma. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 5. sEH inhibition decreased 16-week HF-diet–induced activation of inflammatory pathways in the liver.

Male C57BL/6 mice were fed a regular chow or HF diet for 16 weeks with or without sEHI t-AUCB in drinking water starting 3 days before diet. (A) Western blot analysis of protein levels of sEH and β-actin as a normalization control in liver. (B) sEH activity in liver. (C) Quantitative RT-PCR analysis of mRNA levels of TNF-α and IL-6 in liver. (D) Western blot analysis of protein levels of phosphorylated Jun N-terminal kinase (p-JNK), JNK1, p-p38, p38 and β-actin and relative protein content compared to that of JNK1 or p38. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 6. Hepatic sEH overexpression increased hepatic triglycerides accumulation.

Male C57BL/6 mice were intravenously infected with 1×10⁹ PFU adenovirus (Ad)-sEH (n=7) or Ad-GFP (n=6) for 7 days. (A) Western blot analysis of sEH and β-actin in liver. (B) qRT-PCR analysis of mRNA levels of human sEH and mouse sEH. (C) sEH activity in liver. (D) Plasma levels of cholesterol and triglycerides. (E) Oil-red O staining in liver. (F) Levels of triglycerides and cholesterol in liver. (G) qRT-PCR analysis of mRNA levels of TNF-α and IL-6 in liver. (H) Western blot analysis of protein levels of p-JNK, JNK1, p38, p–p38 and β-actin and relative protein content compared to that of JNK1 or p38. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 7. sEH inhibition attenuated 16-week HF-diet–induced macrophage infiltration in adipose tissue.

(A) Ratio of epididymal fat weight compared to tibia length, and mean size of adiopocytes in epididymal fat of 16-week HF-diet–fed mice with or without full-term sEHI. (B) Immnohistochemical staining for macrophage markers CD68 and Mac3 in epididymal fat. (C) mRNA levels of CD68 and F4/80 in epididymal adipose tissue. (D) mRNA levels of TNF-α, IL-6, IL-1β and MCP-1 in epididymal adipose tissue. Data are mean ± SEM. (* P<0.05, ** P<0.01).

Figure 8. Proposed model for the regulation of hepatic steatosis by sEH in mice.

Long-term HF diet increases sEH protein level in the liver and leads to hepatic lipid accumulation and obesity with increased macrophage infiltration in white adipose tissue. sEH inhibition or deficiency reduces HF-diet–induced hepatic steatosis by inhibiting systemic inflammation, especially in adipose tissue and liver (long term) as one possible mechanism. sEH overexpression in the liver with adenovirus injection increased inflammation and hepatic lipid accumulation.