Nrf2 inhibits LXRα-dependent hepatic lipogenesis by competing with FXR for acetylase binding

Abstract

Aims: The nuclear receptor liver X receptor-α (LXRα) stimulates lipogenesis, leading to steatosis. Nuclear factor erythroid-2-related factor-2 (Nrf2) contributes to cellular defense mechanism by upregulating antioxidant genes, and may protect the liver from injury inflicted by fat accumulation. However, whether Nrf2 affects LXRα activity is unknown. This study investigated the inhibitory role of Nrf2 in hepatic LXRα activity and the molecular basis.

Results: A deficiency of Nrf2 enhanced the ability of LXRα agonist to promote hepatic steatosis, as mediated by lipogenic gene induction. In hepatocytes, Nrf2 overexpression repressed gene transactivation by LXR-binding site activation. Consistently, treatment of mice with sulforaphane (an Nrf2 activator) suppressed T0901317-induced lipogenesis, as confirmed by the experiments using hepatocytes. Nrf2 activation promoted deacetylation of farnesoid X receptor (FXR) by competing for p300, leading to FXR-dependent induction of small heterodimer partner (SHP), which was responsible for the repression of LXRα-dependent gene transcription. In human steatotic samples, the transcript levels of LXRα and SREBP-1 inversely correlated with those of Nrf2, FXR, and SHP.

Innovation: Our findings offer the mechanism to explain how decrease in Nrf2 activity in hepatic steatosis could contribute to the progression of NAFLD, providing the use of Nrf2 as a molecular biomarker to diagnose NAFLD. As certain antioxidants have the abilities to activate Nrf2, clinicians might utilize the activators of Nrf2 as a new therapeutic approach to prevent and/or treat NAFLD.

Conclusion: Nrf2 activation inhibits LXRα activity and LXRα-dependent liver steatosis by competing with FXR for p300, causing FXR activation and FXR-mediated SHP induction. Our findings provide important information on a strategy to prevent and/or treat steatosis.

FIG. 1.

Increase in T090-induced lipogenesis by Nrf2 deficiency. (A) Treatment schedules of LXRα agonist (T090). WT or Nrf2 knockout mice were treated with T090 (50 mg/kg/day) for either 3 times per week (Exp #1) or 7 times per week (Exp #2). Oil Red O staining. (B) Hepatic TG contents. (C) Liver weight to body weight ratio. (D) Real-time PCR assays and immunoblottings for SREBP-1c. SREBP-1c levels were assessed in the livers of WT or Nrf2 knockout mice treated with T090. (E) Real-time PCR assays for FAS and ACC mRNAs. Data represent the mean±S.E. of 7 mice per each group; the statistical significance of differences between each knockout treatment group and the respective WT treatment group (*p<0.05, **p<0.01) were determined.

FIG. 2.

Increase in T090-induced steatohepatitis by Nrf2 deficiency. (A) Histopathology of hepatic central and portal areas. The liver sections of WT or Nrf2 knockout mice treated as described in Figure 1A were subjected to H&E staining. Microphotographs show views of the liver sections: arrows and arrowheads indicate necrosis and vacuole formation, respectively. (B) Blood biochemical parameters. Plasma transaminase activities were monitored in WT or Nrf2 knockout mice treated with T090. (C) Real-time PCR assays for TNFα and iNOS mRNAs. Data represent the mean±S.E. of 7 mice per each group; the statistical significance of differences between each knockout treatment group and the respective WT treatment group (*p<0.05, **p<0.01) were determined.

FIG. 3.

Nrf2 repression of LXRE-mediated gene induction. (A) LXRE luciferase activities. The relative luciferase activities were measured on the lysates of HepG2 cells treated with 3 μM T090 for 24 h following transfection with TK-LXREx3 luciferase construct and Nrf2 plasmids. (B) PXRE and SRE luciferase activities. The relative luciferase activities were measured on the lysates of HepG2 cells treated with 3 μM T090 for 24 h following transfection with TK-PXREx3 luciferase construct and Nrf2 plasmids. To measure SRE luciferase activity, HepG2 cells were treated with 3 μM T090 for 24 h after transfection with pGL-FAS luciferase construct, Nrf2 and/or SREBP-1c. (C) Real-time PCR assays and immunoblottings for SREBP-1c. HepG2 cells were treated with T090 for 12 h after Nrf2 transfection. (D) Immunoblottings for SREBP-1c. Cells were treated with 10 μM GW3965 or 100 nM insulin for 12 h after Nrf2 transfection. Data represent the mean ± S.E. of 4 separate experiments; the statistical significance of differences between each treatment group and the control (**p < 0.01) or T090 alone were determined.

FIG. 4.

Inhibition by sulforaphane of T090-mediated lipogenesis in vivo. (A) Serum TG contents. Mice were exposed to a single dose of 50 mg/kg T090 after sulforaphane (SFN) treatment (90 mg/kg/day, for 2 days). (B) Immunoblottings for hepatic SREBP-1c. Immunoblottings were performed 24 h after T090 treatment. (C) Real-time PCR assays for hepatic lipogenic gene transcripts. (D) Real-time PCR assays for hepatic NQO1 and HO-1 transcripts. Data represent the mean±S.E. of 5 mice per each treatment group; the statistical significance of differences between each treatment group and the vehicle-treated control (*p<0.05, **p<0.01) or T090 alone were determined.

FIG. 5.

Inhibition by sulforaphane of T090-mediated induction of SREBP-1c and lipogenic genes in hepatocytes. (A) LXRE reporter activity. Luciferase expression from TK-LXREx3 luciferase construct was measured on the lysates of HepG2 cells that had been treated with 10 μM SFN for 1 h and continuously exposed to 3 μM T090 for 24 h. (B) Effect of SFN on SREBP-1c expression. The levels of SREBP-1c mRNA and protein were measured in HepG2 cells treated as described in (A). (C) Real-time PCR assays for SREBP-1c mRNA. Primary hepatocytes were treated as described above. (D) Effects of NAC and SFN on DCFH oxidation and SREBP-1c induction. HepG2 cells were treated with 1 mM NAC or 10 μM SFN for 1 h and continuously exposed to T090 for 12 h. For DCFH oxidation assay, 10 μM of T090 was used to increase hydrogen peroxide generation. Data represent the mean±S.E. of four separate experiments. The statistical significance of differences between each treatment group and the control (*p<0.05, **p<0.01) or T090 alone were determined. DCFH, dichlorofluorescein; NAC, N-acetylcysteine; N.S., not significant; SFN, sulforaphane.

FIG. 6.

Effects of Nrf2 on FXR deacetylation and FXR-dependent SHP induction. (A) Real-time PCR assays for FXR mRNA. The mRNA levels were determined in the livers of WT or Nrf2 knockout mice treated with vehicle or SFN (left), or in primary hepatocytes treated with vehicle or 10 μM SFN for 12 h (right). (B) FXRE reporter gene induction by Nrf2. The luciferase reporter gene activity was measured in cells transfected with Mock or Nrf2 plasmid after siRNA knockdown of FXR. (C) Reversal by FXR knockdown of Nrf2′s repression of LXRE reporter activity (left) or SREBP-1c mRNA increase (right). Luciferase expression from TK-LXREx3 construct was measured on the lysates of cells treated with T090 for 12 h after transfection with the plasmid of Nrf2, and/or control siRNA (nontargeting) or FXR siRNA. SREBP-1c mRNA was assessed by real-time PCR assays. (D) Decrease in FXR acetylation by the binding of Nrf2 with p300. Immunoblottings for acetylated lysine, RXRα, or p300 were performed on FXR immunoprecipitates prepared from HepG2 cells transfected with Nrf2. Nrf2 immunoprecipitate was immunoblotted with anti-p300 antibody. (E) ChIP assays. DNA–protein complexes were precipitated with anti-FXR antibody, and were subjected to PCR amplifications using the flanking primers for the FXRE or an irrelevant region. One tenth of cross-linked lysates served as the input control. (F) Reversal by p300 transfection of FXR- and/or Nrf2-induced FXRE reporter activity (left) or SHP mRNA increase (right). Luciferase expression from human SHP promoter was determined on the lysates of cells following different transfection combinations of FXR, Nrf2, and p300. SHP mRNA was assessed by real-time PCR assays. Data represent the mean±S.E. of four separate experiments; the statistical significance of differences between each treatment group and the control (*p<0.05, **p<0.01) were determined. N.S., not significant.

FIG. 7.

SHP-dependent inhibition of LXRα activation by Nrf2. (A) Real-time PCR assays for SHP mRNA. The mRNA levels were determined on the livers of WT or Nrf2 knockout mice treated with vehicle or SFN (left), or in primary hepatocytes treated with vehicle or 10 μM SFN for 12 h (right). (B) The induction of SHP by Nrf2. SHP mRNA and protein levels were assessed in HepG2 cells transfected with Mock or the plasmid encoding Nrf2 for 24 h. (C) FXR-mediated SHP induction by Nrf2. SHP mRNA levels were measured in cells transfected with Mock or Nrf2 after FXR knockdown. (D) Inhibition of the interaction between LXRα and RXRα by SFN. Cells were treated with 10 μM SFN for 1 h and continuously exposed to T090 for 12 h. (E) LXRE reporter activity. Luciferase activity was measured on the lysates of HepG2 cells transfected with LXRE reporter construct following different transfection combinations with LXRα/RXRα and Nrf2 plasmids for 24 h. The statistical significance of differences between each treatment group and the control (**p<0.01) or LXRα/RXRα (^##p<0.01) were determined. (F) Reversal by SHP knockdown of Nrf2's repression of LXRE reporter activity (left) or SREBP-1c mRNA increase (right). Luciferase expression from TK-LXREx3 construct was measured on the lysates of cells treated with T090 for 12 h after transfection with Nrf2 and/or control (nontargeting) or SHP siRNA. SREBP-1c mRNA was assessed by real-time PCR assays. Data represent the mean±S.E. of four separate experiments; the statistical significance of differences between each treatment group and the control (*p<0.05, **p<0.01) were determined.

FIG. 8.

Repression of Nrf2, FXR, and SHP by hepatic steatosis. (A) Real-time PCR assays. The mRNA levels were determined on the livers of normal subjects and patients with hepatic steatosis (n=10 each). (B) Nrf2, SHP, and FXR mRNA levels in the liver of mice fed on either a normal diet (ND) or 60% high-fat diet (HFD) for 11 weeks. (C) A scheme illustrating the signaling pathway by which Nrf2 negatively regulates the activity of LXRα in the liver.