Nuclear Factor Erythroid 2-Related Factor 2 Deficiency Results in Amplification of the Liver Fat-Lowering Effect of Estrogen

Abstract

Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple biologic processes, including hepatic lipid metabolism. Estrogen exerts actions affecting energy homeostasis, including a liver fat-lowering effect. Increasing evidence indicates the crosstalk between these two molecules. The aim of this study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice fed a high-fat diet and the liver fat-lowering effect of exogenous estrogen was subsequently assessed. We found that exogenous estrogen eliminated 49% and 90% of hepatic triglycerides in wild-type and Nrf2-null mice with NAFLD, respectively. This observation demonstrates that Nrf2 signaling is antagonistic to estrogen signaling in hepatic fat metabolism; thus, Nrf2 absence results in striking amplification of the liver fat-lowering effect of estrogen. In addition, we found the association of trefoil factor 3 and fatty acid binding protein 5 with the liver fat-lowering effect of estrogen. In summary, we identified Nrf2 as a novel and potent inhibitor of estrogen signaling in hepatic lipid metabolism. Our finding may provide a potential strategy to treat NAFLD by dually targeting Nrf2 and estrogen signaling.

Fig. 1.

Liver-to-body weight ratios. Adult male wild-type (Nrf2^+/+) and Nrf2-null (Nrf2^−/−) mice were fed a HFD for 16 weeks. Afterward, vehicle (sesame oil), E2 (, 625 µg/kg), progesterone (P, 25 mg/kg), or a combination of these two steroids (625 µg/kg E2 and 25 mg/kg progesterone) was injected subcutaneously once daily for 3 weeks. At 4 hours after the last dosing, mice were weighed and then euthanized for sample collection. Nrf2^+/+ and Nrf2^−/− mice fed a normal chow served as homeostasis controls. The liver-to-body weight ratios were calculated and are shown. *P < 0.05, compared with normal chow-fed Nrf2^+/+ mice; ^#P < 0.05, compared with normal chow-fed Nrf2^−/− mice; ^¥P < 0.05, compared as indicated. n = 5.

Fig. 2.

Histologic assessments of liver sections. Livers were collected from the experiment described in Fig. 1. Formalin-fixed and paraffin-embedded liver sections were stained with hematoxylin and eosin and were visualized at low magnification (A) and high magnification (B). Original magnification, ×100 in A; ×400 in B.

Fig. 3.

Liver fat assessments. Livers were harvested from the experiment indicated in Fig. 1. (A) Oil Red O staining of liver fat. Frozen liver sections were stained with Oil Red O. Liver fat was stained red. (B) Quantification of liver fat contents. Frozen livers were used for measuring the concentrations of hepatic triglycerides, free fatty acids, and total cholesterol with various commercially available kits. *P < 0.05. n = 5.

Fig. 4.

(A) mRNA expression of hepatic Nrf2, NQO-1, ERα, and TFF3. Total RNA was extracted from each liver collected from the experiment illustrated in Fig. 1. Hepatic mRNA expression of the genes indicated was quantified with quantitative real-time PCR. The hepatic mRNA level of each gene in normal chow-fed Nrf2^+/+ mice was set to 1. *P < 0.05, compared with normal chow-fed Nrf2^+/+ mice; ^#P < 0.05, compared with normal chow-fed Nrf2^−/− mice; ^¥P < 0.05, compared between Nrf2^+/+ and Nrf2^−/− mice. n = 5. (B) Protein expression of hepatic NQO-1, ERα, and FABP5. Total liver lysates were prepared from the livers collected from the experiment described in Fig. 1. Western blotting was performed with the antibodies against the proteins indicated. The lanes represent samples prepared from individual mice. (C) Hepatic expression and distribution of TFF3 protein. Formalin-fixed and paraffin-embedded liver sections prepared from the experiment indicated in Fig. 1 were subjected to immunostaining with a TFF3 antibody. TFF3 protein was stained dark brown.

Fig. 5.

Nrf2- and estrogen-dependent induction of FABP5 in the liver. (A) Hepatic expression of FABP5 mRNA. Total RNA samples prepared from the experiment described in Fig. 4 were used for evaluating the transcript levels of hepatic FABP5 with quantitative real-time PCR. Hepatic mRNA expression of the gene in normal chow-fed Nrf2^+/+ mice was set to 1. *P < 0.05, compared with normal chow-fed Nrf2^+/+ mice; ^#P < 0.05, compared with normal chow-fed Nrf2^−/− mice; ^¥P < 0.05, compared between Nrf2^+/+ and Nrf2^−/− mice. n = 5. (B) Hepatic expression and distribution of FABP5 protein. Formalin-fixed and paraffin-embedded liver sections prepared from the experiment indicated in Fig. 1 were subjected to immunostaining with a FABP5 antibody. FABP5-expressing cells were stained dark brown.