Activation of the aryl hydrocarbon receptor sensitizes mice to nonalcoholic steatohepatitis by deactivating mitochondrial sirtuin deacetylase Sirt3

Abstract

Nonalcoholic steatohepatitis (NASH) is a liver disorder that still demands improved treatment. Understanding the pathogenesis of NASH will help to develop novel approaches to prevent or treat this disease. In this study, we revealed a novel function of the aryl hydrocarbon receptor (AhR) in NASH. Transgenic or pharmacological activation of AhR heightened animal sensitivity to NASH induced by the methionine- and choline-deficient (MCD) diet, which was reasoned to be due to increased hepatic steatosis, production of reactive oxygen species (ROS), and lipid peroxidation. Mechanistically, the increased ROS production in AhR-activated mouse liver was likely a result of a lower superoxide dismutase 2 (SOD2) activity and compromised clearance of ROS. Activation of AhR induced tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP) gene expression, depleted NAD(+), deactivated the mitochondrial sirtuin deacetylase 3 (Sirt3), increased SOD2 acetylation, and thereby decreased SOD2 activity. We also showed that Sirt3 ablation sensitized mice to NASH, whereas adenoviral overexpression of Sirt3 alleviated the NASH phenotype in AhR-transgenic mice. We conclude that activation of AhR sensitizes mice to NASH by facilitating both the "first hit" of steatosis and the "second hit" of oxidative stress. Our results suggest that the use of AhR antagonists might be a viable approach to prevent and treat NASH. Manipulation of the expression or activity of Sirt3 may also represent a novel approach to manage NASH.

Fig 1

Transgenic activation of AhR sensitized mice to MCD diet-induced NASH. (A) Top, schematic representation of the liver-specific “Tet-off” CA-AhR transgenic system. DOX, doxycycline; FABP, fatty acid binding protein; P_CMV, minimal CMV promoter; TetRE, tetracycline responsive element; tTA, tetracycline transcriptional activator. Bottom, the expression of CA-AhR was confirmed by transgene-specific real-time PCR. The cycle numbers are labeled. (B to G) WT and TG mice were fed with the MCD diet for 6 weeks in the presence or absence of DOX before being analyzed for hepatic triglyceride level (B); liver section Picro-sirius red staining, with the arrowhead indicating the collagen deposition (C); liver section H&E staining, with the arrows indicating the neutrophil infiltration (D); hepatic expression of fibrogenic genes (E) and inflammatory genes (F); and serum ALT levels (G). n = 6 for each group. *, P < 0.05.

Fig 2

Pharmacological activation of AhR sensitized mice to MCD diet-induced NASH. (A to E) WT mice were injected with a single dose of TCDD (10 μg/kg) or vehicle and then fed with the MCD diet for 2 weeks before being analyzed for serum ALT level (A); hepatic triglyceride content (B); liver section H&E staining, with the arrows indicating the neutrophil infiltration (C); and hepatic expression of fibrogenic genes (D) and inflammatory genes (E). n = 5 for each group. *, P < 0.05; **, P < 0.01. TCDD versus vehicle.

Fig 3

Activation of AhR increased ROS production, compromised SOD2 activity, and increased lipid peroxidation. Mice were the same as described in the legends to Fig. 1 and 2. (A and B) Hepatic H₂O₂ levels in WT and TG mice treated without or with DOX (A) or WT mice treated with vehicle or TCDD (B). (C) Superoxide (O₂⁻) levels in primary hepatocytes treated with vehicle or TCDD. (D and E) Total SOD, SOD2, and SOD1 activities in WT and TG mice treated without or with DOX (D) or WT mice treated with vehicle or TCDD (E). (F and G) Hepatic concentrations of malondialdehyde (MDA) in WT and TG mice treated without or with DOX (F) or WT mice treated with vehicle or TCDD (G). *, P < 0.05.

Fig 4

AhR activation increased SOD2 acetylation and decreased Sirt3 activity. (A and B) SOD2 acetylation in TG mice (A) and TCDD-treated WT mice (B), as determined by immunoprecipitation and immunoblotting. The bottom rows in panels A and B are the quantifications of the Western blot results. (C) Hepatic Sirt3 mRNA expression in TG mice (left) and TCDD-treated WT mice (right); (D) NAD⁺ level in TG mice and TCDD-treated WT mice; (E) hepatic TiPARP mRNA expression in TG mice and TCDD-treated WT mice; (F) hepatic Sirt3 activity in TG mice (left) and TCDD-treated WT mice (right). *, P < 0.05; **, P < 0.01.

Fig 5

Sirt3 ablation sensitized mice to MCD diet-induced NASH. (A to E) WT and Sirt3^−/− mice were fed the MCD diet for 4 weeks before being analyzed for serum ALT level (A); hepatic fibrogenic gene expression (B); inflammatory gene expression (C); hepatic triglyceride content (D); liver section H&E staining, with the arrows indicating the neutrophil infiltration (E); SOD activities (F); and SOD2 acetylation (G). The right section in panel G is the quantification of the Western blot results. The inset in panel A shows the lack of Sirt3 protein expression in Sirt3^−/− mice, in which the composite image was derived from a single original image. *, P < 0.05. Sirt3^−/− versus WT.

Fig 6

Overexpression of Sirt3 alleviated CA-AhR transgenic mice from NASH. Transgenic mice infected with the control virus (Ad-Ctrl) or Ad-Sirt3 were fed with the MCD diet for 2 weeks. (A) Western blotting to confirm the expression of Flag-Sirt3. The top and bottom panels were blotted with anti-Flag and anti-Sirt3 antibodies, respectively. The composite image of the top panel was derived from a single original image. (B to I) Serum ALT level (B); hepatic triglyceride content (C); liver section H&E staining, with the arrows indicating the neutrophil infiltration (D); hepatic fibrogenic gene expression (E) and inflammatory gene expression (F); SOD activities (G); hepatic H₂O₂ level (H); and SOD2 acetylation (I). The right section in panel I is the quantification of the Western blot results. *, P < 0.05. Ad-Sirt3 versus Ad-Ctrl.

Fig 7

Summary of the effects of AhR on Sirt3 activity and NASH. We propose that activation of AhR sensitizes mice to NASH by facilitating both “hits”: the first hit of steatosis, and the second hit of oxidative stress.