A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis

Abstract

Background & aims: The aryl hydrocarbon receptor (AhR) also known as the dioxin receptor or xenobiotic receptor is a member of the basic helix-loop-helix/period AhR nuclear translocator single minded family. The goal of this study was to determine the endobiotic role of AhR in hepatic steatosis.

Methods: Wild-type, constitutively activated AhR transgenic, AhR null and CD36/fatty acid translocase null mice were used to investigate the role of AhR in steatosis and the involvement of CD36 in the steatotic effect of AhR. The promoters of the mouse and human CD36 genes were cloned and their regulation by AhR was analyzed.

Results: Activation of AhR induced spontaneous hepatic steatosis characterized by the accumulation of triglycerides. The steatotic effect of AhR likely is owing to the combined up-regulation of CD36 and fatty acid transport proteins, suppression of fatty acid oxidation, inhibition of hepatic export of triglycerides, increase in peripheral fat mobilization, and increased hepatic oxidative stress. Promoter analysis established CD36 as a novel transcriptional target of AhR. Activation of AhR in liver cells induced CD36 gene expression and enhanced fatty acid uptake. The steatotic effect of an AhR agonist was inhibited in CD36-/- mice.

Conclusions: Our study reveals a novel link between AhR-induced steatosis and the expression of CD36. Industrial or military exposures to dioxin and related compounds have been linked to increased prevalence of fatty liver in human beings. Results from this study may help to establish AhR and its target CD36 as novel therapeutic and preventive targets for fatty liver disease.

Figure 1. Generation of transgenic mice expressing the constitutively activated AhR (CA-AhR) in the liver

(A) Schematic representation of the “Tet-off” FABP-tTA/TetRE-CA-AhR transgenic system. DOX, doxycycline; FABP, fatty acid binding protein; P_CMV, minimal CMV promoter; TetRE, tetracycline responsive element; tTA, tetracycline transcriptional activator. (B) Hepatic and intestinal expression of CA-AhR as shown by Northern blot analysis using the AhR cDNA probe. The membranes were stripped and re-probed with Cyp1a2 for a positive control of AhR activation and Gapdh for a loading control. (C) Treatment of bi-transgenic mice with DOX for one week silenced the expression of CA-AhR and induction of Cyp1a2. (D) CA-AhR transgene is not expressed in tissues outside of the liver and intestine, as determined by semi-quantitative RT-PCR. Cyclophilin is included as a loading control. (E) Activation of AhR in CA-AhR mice was not associated with obvious hepatotoxicity. Serum levels of ALT were measured in female mice of 5-6 weeks of age. Mice in the TCDD group received a single p.o. dose of TCDD (30 μg/kg) and sacrificed 7 days after. Each group has at least five mice. *, P<0.05.

Figure 2. CA-AhR transgenic mice developed spontaneous hepatic steatosis, and showed decreased body weight and fat mass

Five to six weeks-old female mice were used. (A) The liver weight (LW) was measured as percentage of the total body weight (BW). (B) Liver sections of wild type (WT) and CA-AhR mice were stained with H&E (a and b) or Oil-red O (c-e). Mouse in (e) was treated with DOX for two weeks. The original magnification of all panels is 200x. (C-E) Measurements of liver lipid contents (C, n=4 for each group), plasma triglyceride content (D), and body weight (E). (F) Fat mass and lean mass were measured by MRI and the results are presented as percentage of BW. *, P<0.05; **, P<0.01, compared to the wild type, or the comparisons are labeled.

Figure 3. Activation of AhR in transgenic mice induced hepatic expression of CD36 and uptake of fatty acids

(A) Real-time PCR analysis on the hepatic expression of CD36 mRNA in two-month old female mice. When applicable, mice were treated with DOX for 2 weeks before sacrificing. N=4 for each group. (B) Western blot analysis on the protein expression of CD36. Lanes represent individual mice. (C) Expression of CD36 and Cyp1a2 in WT and AhR-/- mice as determined by real-time PCR. (D) Real-time PCR analysis on the hepatic expression of Fatps, VLDLR, LDLR, SR-A, and SR-B. N=6 for each group. (E) Free fatty acid uptake in primary mouse hepatocytes was monitored by the uptake of BODIPY-C16. Top and bottom panels are fluorescence and phase contrast images of the cells, respectively. DOX concentration is 1 μg/ml. (F) Quantification of BODIPY-C16 uptake in (E). AU, arbitrary unit. *, P<0.05; **, P<0.01, compared to control animals, or the comparisons are labeled.

Figure 4. The mouse and human CD36 gene promoters are transcriptional targets of AhR

(A) Top: The sequences of mouse and human CD36 dioxin responsive elements (DREs) and their mutant variants. Underlined are DREs and their mutants. Bottom: EMSA results. (B) Chromatin immunoprecipitation assays to show the recruitment of AhR onto CD36 promoter. Huh-7 cells were treated with vehicle (Veh) or 10nM of TCDD (T) for 24 hrs. CYP1A1/DRE and CYP7B1/non-DRE were included as a positive control and negative control, respectively. (C and D) Activation of the mouse (C) and human (D) CD36 promoter reporter genes by AhR in the presence of 3-MC, or by CA-AhR without an exogenously added ligand. HepG2 cells were co-transfected with indicated reporters and receptors. Transfected cells were treated with vehicle (DMSO) or 3-MC (2 μM) for 24 hrs before luciferase assay. (E) Activation of mCD36 and hCD36 promoter reporter genes by endogenous AhR agonists Indigo (10 μM) and FICZ (200 nM). *, P<0.05; **, P<0.01.

Figure 5. Loss of CD36 in mice inhibited the steatotic effect of an AhR agonist

(A and B) Free fatty acid uptake in primary mouse hepatocytes from WT and CD36^-/- mice treated with vehicle (Veh) or TCDD is monitored by the uptake of BODIPY-C16. (A) Top and bottom panels are fluorescence and phase contrast images of the cells, respectively. (B) Quantification of BODIPY-C16 uptake in (A). TCDD concentration is 10 nM. (C and D) Two-month old female mice were gavaged with a single dose of TCDD (30 μg/kg). The mice were sacrificed 7 days later and subjected to the measurements of hepatic (C, n=4 for each group) and plasma (D, n=5 for each group) triglyceride levels. Mice were fasted for 16 hrs before sacrificing. *, P<0.05.

Figure 6. Treatment with AhR agonist inhibited VLDL-triglyceride secretion

(A) Two-month old WT female mice were treated with a single dose of TCDD (30 μg/kg) for 7 days before measured for VLDL-triglyceride (TG) secretion. (B and C) Measurements of ApoB protein (B, shown is the Western blot result on plasma) and ApoB100 mRNA (C, shown is the real-time PCR result on liver) levels. (D-F) VLDL-TG secretion (D), plasma ApoB protein (E), and liver ApoB100 mRNA (F) levels in CA-AhR transgenic mice. *, P<0.05; **, P<0.01, compared to the vehicle control.

Figure 7. Activation of AhR in transgenic mice inhibited hepatic fatty acid β-oxidation, decreased white adipose tissue (WAT) adiposity, and increased hepatic stress

(A) Suppression of PPARa and its target genes involved in fatty acid oxidation in two-month old female CA-AhR transgenic mice, as shown by real-time PCR analysis. N=6 for each group. (B) Inhibition of peroxisomal β-oxidation in the liver extracts of transgenic mice. (C) Representative appearance of the omental fat, H&E staining of epididymal fat, and quantification of adipocyte size. (D) Omental fat tissue weight (WAT) was measured as percentage of total body weight (BW) in two-month old male WT and CD36^-/- mice gavaged with vehicle (Veh) or TCDD (30 μg/kg) 7 days prior to being sacrificed (n=5 for each group). (E) Abdomen fat triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) mRNA expression as measured by real-time PCR analysis (N=5 for each group). (F) Hepatic concentrations of malondialdehyde (MDA). Liver lipids were extracted from 5-6 week old female mice and subjected to MDA measurement. When necessary, transgenic mice were treated with DOX for 2 weeks. N=3 for each group. *, P<0.05; **, P<0.01.