Estrogen-related receptor γ controls sterol regulatory element-binding protein-1c expression and alcoholic fatty liver

BBA - Molecular and Cell Biology of Lipids 1864 (2019) 158521

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Estrogen-related receptor γ controls sterol regulatory element-binding protein-1c expression and alcoholic fatty liver

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Don-Kyu Kima,1, Yong-Hoon Kimb,c,1, Jae-Ho Leed, Yoon Seok Junge, Jina Kimf, Rilu Fengg, Tae-Il Jeonh, In-Kyu Leei,j, Sung Jin Chof,j, Seung-Soon Imd, Steven Dooleyg, Timothy F. Osbornek, ⁎ ⁎⁎ Chul-Ho Leeb,c, , Hueng-Sik Choie, a

Department of Molecular Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea c University of Science and Technology (UST), Daejeon 34113, Republic of Korea d Department of Physiology, Keimyung University School of Medicine, Daegu 42601, Republic of Korea e National Creative Research Initiatives Center for Nuclear Receptor Signals, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea f New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea g Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim 105760, Germany h Department of Animal Science, College of Agriculture & Life Science, Chonnam National University, Gwangju 61186, Republic of Korea i Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu 41944, Republic of Korea j Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Republic of Korea k Institute for Fundamental Biomedical Research, Departments of Medicine and Biological Chemistry, Johns Hopkins University School of Medicine, St. Petersburg, FL 33701, USA b

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A B S T R A C T

Keywords: ERRγ SREBP-1c CB1 receptor Gene regulation Hepatic lipogenesis Alcoholic fatty liver

Although SREBP-1c regulates key enzymes required for hepatic de novo lipogenesis, the mechanisms underlying transcriptional regulation of SREBP-1c in pathogenesis of alcoholic fatty liver is still incompletely understood. In this study, we investigated the role of ERRγ in alcohol-mediated hepatic lipogenesis and examined the possibility to ameliorate alcoholic fatty liver through its inverse agonist. Hepatic ERRγ and SREBP-1c expression was increased by alcohol-mediated activation of CB1 receptor signaling. Deletion and mutation analyses of the Srebp-1c gene promoter showed that ERRγ directly regulates Srebp-1c gene transcription via binding to an ERR-response element. Overexpression of ERRγ significantly induced SREBP-1c expression and fat accumulation in liver of mice, which were blocked in Srebp-1c-knockout hepatocytes. Conversely, liver-specific ablation of ERRγ gene expression attenuated alcohol-mediated induction of SREBP-1c expression. Finally, an ERRγ inverse agonist, GSK5182, significantly ameliorates fatty liver disease in chronically alcohol-fed mice through inhibition of SREBP-1c-mediated fat accumulation. ERRγ mediates alcohol-induced hepatic lipogenesis by upregulating SREBP-1c expression, which can be blunted by the inverse agonist for ERRγ, which may be an attractive therapeutic strategy for the treatment of alcoholic fatty liver disease in human.

1. Introduction Alcoholic fatty liver disease, a common response of the liver to acute

or chronic alcohol exposure, is characterized by increased accumulation of lipid droplets containing triglycerides (TGs) and cholesterols in the liver. A number of studies have revealed that it can progress to more

Abbreviations: SREBP-1c, sterol regulatory element-binding protein-1c; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; 2-AG, 2-arachidonyl glycerol; NR, nuclear receptor; ERRγ, estrogen-related receptor γ; PA, phosphatidate; DAG, diacylglycerol; TG, triglyceride; ROS, reactive oxygen species; ERRE, ERR-response element ⁎ Correspondence to: Chul-Ho Lee: Korea Research Institute of Bioscience and Biotechnology, Daejeon 24141, Republic of Korea. ⁎⁎ Correspondence to: H.-S. Choi, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. E-mail addresses: [email protected] (C.-H. Lee), [email protected] (H.-S. Choi). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbalip.2019.158521 Received 24 May 2019; Received in revised form 22 August 2019; Accepted 28 August 2019 Available online 31 August 2019 1388-1981/ © 2019 Elsevier B.V. All rights reserved.

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harmful stages of liver injury, such as steatohepatitis, fibrosis and cirrhosis. The adverse events behind disease progression are alcoholmediated oxidative stress and hepatic inflammation, both causing liver damage [1]. The mechanisms through which alcohol causes a fatty liver are multifactorial and still incompletely understood. It is proposed that enhanced de novo lipogenesis and reduced fatty acid oxidation are critical biochemical steps that trigger alcoholic fatty liver generation [2]. Considerable evidence from in vitro and in vivo studies supports the hypothesis that induction of sterol regulatory element-binding protein (SREBP)-1 by alcohol contributes to hepatic de novo lipogenesis through transcriptional regulation of key lipogenic enzymes, such as acetyl-CoA carboxylase (ACC, encoded by Acaca), fatty acid synthase (FAS, encoded by Fasn) and stearoyl-CoA desaturase, which are mainly responsible for fatty acid synthesis and TG accumulation in liver [2–5]. SREBPs belong to the basic helix-loop-helix-leucine zipper family that consists of two Srebp genes: Srebp-1 (encoding SREBP-1a and -1c) that primarily regulates fatty acid metabolism, and Srebp-2 (encoding SREBP-2) mediating cholesterol metabolism [6]. SREBPs are synthesized as precursors bound to endoplamic reticulum. Upon activation, the precursor membrane-bound forms (pSREBPs) are translocated into nucleus as mature nuclear forms (nSREBPs) [6]. SREBP-1a is primarily expressed at high levels in cells of the immune system, while SREBP-1c and SREBP-2 are the predominant forms in lipogenic tissues, including the liver [7,8]. Although the three SREBPs differ in their tissue distribution and response to a variety of regulatory stimuli, their expression is commonly regulated at the transcriptional level. Ablation of SREBP-1c in mice decreased alcohol-induced fatty liver, supporting its critical role in alcoholic liver disease [9]. 2-arachidonyl glycerol (2AG), an endogenous endocannabinoid, is produced in and secreted from hepatic stellate cells in a setting of chronic alcohol consumption. 2-AG then activates CB1 receptor signaling in hepatocytes, which induces SREBP-1c and FAS expression, as well as fat accumulation [10]. These findings suggested that SREBP-1c is a major contributor to the pathogenesis of alcoholic fatty liver disease. Thus, delineating the mechanism of SREBP-1c transcriptional regulation by alcohol is of major interest to set up potential therapeutic approaches. The estrogen-related receptor (ERR) subfamily of nuclear receptor (NR), which consists of three members, ERRα (NR3B1), ERRβ (NR3B2), and ERRγ (NR3B3), is known to be constitutively active due to active conformation of the ligand-binding domain in the absence of a ligand. This is in contrast to most NRs, whose activation and function mainly depends on ligand binding [11]. The ligand-independent transcriptional activity of ERRγ (encoded by Esrrg) is dynamically regulated by interaction of co-regulators and post-transcriptional modifications in response to different endocrine and metabolic signals [11–16]. Interestingly, ERRγ gene expression is highly inducible in the liver and tightly regulated by multiple membrane receptors that recognize extracellular stimuli such as nutrients, hypoxia, and inflammation [17–20]. Recent findings from in vitro and in vivo studies suggest that ERRγ participates in hepatic lipid and alcohol metabolism. For example, ERRγ elicits ectopic accumulation of diacylglycerol (DAG) through induction of Lipin-1, which contributes to the biosynthesis of TG and phospholipids by catalyzing phosphatidate (PA) conversion to DAG [21]. In addition, ERRγ mediates alcohol-induced oxidative stress and liver injury through CYP2E1-mediated production of reactive oxygen species (ROS) [22]. Based on these recent findings, we hypothesize that ERRγ is directly involved in alcohol-mediated hepatic lipogenesis and alcoholic fatty liver disease. In this study, we investigated the role of ERRγ in alcohol-mediated hepatic lipogenesis and further examined the possibility to ameliorate alcoholic fatty liver disease using its inverse agonist.

2. Experimental procedures 2.1. Chemicals GSK5182 was synthesized and dissolved for in vitro and in vivo experiments as previously described [22,23]. 2-arachidonyl glyceryl ether (2-AG ether) (noladin ether, Ellisville, MO, USA) and AM251 (Ellisville, MO, USA) were purchased from Tocris Bioscience and dissolved in the recommended solvents. Insulin (St Louis, MO, USA) and SP600125 (Danvers, MA, USA) were purchased from Sigma-Aldrich and Cell Signaling Technology, respectively and dissolved in the recommended solvents. 2.2. Plasmid DNAs and recombinant adenoviruses The plasmid carrying human Srebp-1c gene promoter fused to luciferase (Srebp-1c-luc) and encoding mouse ERRγ (pcDNA3-FLAG-ERRγ) were previously described [19,24]. The plasmid carrying human Srebp1c ERR-response element (ERRE) mutant (mut) gene promoter fused to luciferase (Srebp-1c ERRE-luc) was generated by site-directed mutagenesis using the following primers: 5′-CTAGAAGGGGGAAATCCCTGC ACCC -3′ and 5′-GGGTGCAGGGATTTCCCCCTTCTAG-3′. Adenoviruses (Ad) expressing green fluorescent protein (Ad-GFP), Flag-ERRγ (AdFlag-ERRγ), unspecific short hairpin (sh) RNA (Ad-US), and shERRγ (Ad-shERRγ) were previously described [19] and all viruses were purified using CsCl2 gradient method. 2.3. Animal experiments Male 8-week old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used for this study unless mentioned. Wild-type (WT) and Srebp-1c knockout (KO) mice were purchased from the Jackson Laboratory. These mice were mixed background strain with 129S6/SvEvTac. Srebp-1c KO mice were backcrossed with C57BL/6 J mice for over 10 times to generate the Srebp-1c KO/C57BL/6 J mice used in current study. Prior to the experiments, all mice were housed in a specific pathogen-free facility with a 12 hours light/dark cycle at 22 ± 2 °C with free access to water and a standard chow diet in specific pathogen free animal facility unless mentioned. To examine the effect of ERRγ in hepatic lipogenesis, Ad- GFP (5 × 109 pfu) or Ad-Flag-ERRγ (5 × 109 pfu) were injected via tail-vein of C57BL/6 J mice and the mice were sacrificed at day 8 after the injection. Ad-US (5 × 109 pfu) or Ad-shERRγ (5 × 109 pfu) was injected via tail-vein of C57BL/6 J mice and the mice were administered vehicle or alcohol (6 g/kg) for 24 h at day 5 after the injection. For an animal mouse model of chronic alcoholic fatty liver, four groups of five mice each were treated for four weeks: (a) alcohol-containing Lieber-DeCarli formulation based liquid (Dyets, Bethlehem, PA) diet (27.5% of total calories), (b) pair-fed control diet in which alcohol was replaced isocalorically with carbohydrate, (c) control diet supplemented with GSK5182 (40 mg/kg, p.o.), and (d) alcohol-containing diet supplemented with GSK5182. In the latter two groups, GSK5182 was given by oral gavage once-daily for the last 2 weeks of the study. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology, Chonnam National University and Keimyung University School of Medicine. All experimental procedures in mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.4. Mouse primary hepatocytes Primary hepatocytes were isolated from liver of male 8-week old C57BL/6 and Srebp-1c KO mice by collagenase perfusion method as described previously [25]. Infection of adenoviruses and treatments of as 2-AG ether, SP600125, insulin and GSK5182 were performed as 2

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described in the figure legends. A minimum of three different batches of the primary hepatocytes were used for infection of adenoviruses.

(Cell signaling, cat.2368S; Stratagene, anti-FLAG M2), anti-α-tubulin antibody (Ab frontier, cat.LF-MA0117A), anti-β-actin antibody (SigmaAldrich, cat.A5441) and anti-FAS antibody (Cell signaling, cat.3180). Anti-SREBP-1 antibody (Abcam, cat.ab3259; Santa Cruz Biotechnology, cat.sc-13551X) was used for detection of SREBP-1 in AML12 cell or primary hepatocytes, and anti-SR1 antibody [26] for detection of SREBP-1 in liver of mice. Primary antibodies were detected using HRPconjugated secondary antibodies (Ab frontier). Images were acquired using an X-ray film or ChemiDoc™ XRS+ (Bio-Rad, CA, USA).

2.5. Cell culture and transient transfection HepG2 (human hepatocellular carcinoma cell), 293 T (human embryonic kidney cell) and AML12 (immortalized mouse hepatocyte) cell were obtained from ATCC (Manassas, VA, USA). HepG2 and 293 T cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) (Welgene, Gyeongsan-si, South Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, GE Healthcare Life Science, UK). AML12 cell were cultured in DMEM/F-12 medium supplemented with 10% FBS, insulin-transferrinselenium mixture (Welgene), dexamethasone (40 ng/ml) and 1% penicillin-streptomycin. All cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C and routinely examined for mycoplasma contamination. AML12 cells were infected with Ad-GFP, Ad-ERRγ, Ad-US or Ad-shERRγ as described in the figure legends. HepG2 and 293 T cells were transiently co-transfected with a reporter and indicated expression vector in Figures using Lipofectamine 2000 (Invitrogen, Carlsbad, CA USA). pCMV-β-galactosidase, an internal control, was also co-transfected along with the vectors and a luciferase activity was normalized to the β-galactosidase activity. Data are representative of at least three independent experiments.

2.9. Histopathology Liver samples were fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 5-μm-thick sections, and stained with hematoxylin and eosin (H&E). For in vivo oil red O staining, liver tissues were embedded in a Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and sectioned at a thickness of 8-μm using a cryotome (Sakura Finetek). Cryostat sections of liver tissue were fixed in 10% neutral buffered formalin. After fixation, liver tissue sections were stained with 0.3% oil-red O solution and counterstained with hematoxylin. Images were captured using a light microscope (BX51; Olympus Corporation, Tokyo, Japan). For in vitro oil red O staining, primary mouse hepatocytes were isolated from WT and Srebp-1c KO mice. Briefly, Oil red O was dissolved in 99% 2-propanol and filtered using Whatman #4 filter paper. The media was removed from the dishes gently and the plates were rinsed by ice-cold PBS. Five ml of 10% formalin was added to each well for over 30 min at RT to fix the cells. Each well was then removed fixation solution and added 1 ml of oil red O stain per well for 1 h, and discarded. The wells were rinsed with distilled H2O several times to remove excess stain.

2.6. Chromatin immunoprecipitation assay The chromatin immunoprecipitation (ChIP) assay was carried out according to the manufacturer's protocol (EMD Millipore, Billerica, MA, USA). Briefly, HepG2 cells and mouse primary hepatocytes were exposed to 2-AG ether or AM251 as described in the figure legends were fixed using a final concentration of 1% formaldehyde and then harvested. Soluble chromatins isolated from the cells were immunoprecipitated with anti-ERRγ antibody (Perseus Proteomics Inc., Tokyo, Japan). After recovering DNA, polymerase chain reaction (PCR) was carried out using primers encompassing the ERRγ-binding region on the human or mouse Srebp-1c gene promoter (5′-GAAGTGAGGTGT TTTAGGAG-3′ and 5′-CTCCAACCCTCCGTTTACTCC-3′ for human; 5′-CAACTAGGCTGCCAGACCT-3′ and 5′-TTCTCTTATAGCACTCTGC-3′ for mouse). The size of the amplified PCR product was 249 base pairs (human) and 244 base pairs (mouse).

2.10. Statistical analysis Data are expressed as means ± S.D. Statistical analysis was carried out using the two-tailed Student's t-test (GraphPad Prism 8 software, San Diego, CA, USA). Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Hepatic ERRγ and SREBP-1c expression is increased by alcohol feeding

2.7. Quantitative real-time PCR analysis

In an attempt to elucidate the function of nuclear receptor ERRγ in hepatic de novo lipogenesis, we first examined ERRγ and lipogenic gene expression in the liver of mice fed an alcohol-containing diet for 4 weeks. As expected, Oil-Red O and H&E staining showed that lipid accumulation and lipid droplet formation are markedly increased in liver of alcohol-fed mice (Fig. 1A). In addition, expression of hepatic ERRγ, Srebp-1c, Fasn and Cyp2e1 was significantly induced by alcohol feeding (Fig. 1B). To further investigate the pattern of ERRγ and Srebp1c expression, we analyzed mRNA levels in the liver of mice administered with either alcohol or 2-AG in a time-resolved manner. Interestingly, mRNA levels of ERRγ was significantly induced 1 h after alcohol feeding and further increased until 12 h, while those of Srebp-1c were significantly increased at 3 h and were further enhanced until 24 h (Fig. 1C). It was reported that activation of hepatic CB1 receptors by alcohol-mediated induction of 2-AG stimulates hepatic lipogenesis through induction of SREBP-1c [10]. Therefore, we analyzed the patterns of ERRγ and Srebp-1c gene expression in liver of mice administered with 2-AG at different time points. mRNA levels of ERRγ were significantly induced 1 h after 2-AG injection further increasing until 6 h, while those of Srebp-1c were significantly increased at 3 h and further enhance until 24 h (Fig. 1D), exactly as upon alcohol feeding. These results suggest that CB1 receptor-mediated induction of ERRγ expression precedes that of Srebp-1c expression in liver of mice exposed to alcohol.

Total RNAs were extracted from AML12 cell, mouse primary hepatocytes and mouse livers using TRIzol regent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Quantity and purity of RNAs were confirmed using a Biophotometer D30 (Eppendorf, Hamburg, Germany). RNAs were reversed-transcribed into cDNAs using the Maxime RT PreMix Kit (iNtRON Biotechnology, Seoul, South Korea) following the manufacturer's recommendations. Quantitative PCRs were performed on an Applied Biosystems StepOnePlus real-time PCR system (Applied Biosystems, CA, USA) by using the Power SYBR Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized to the L32 or beta-actin gene expression, and variations and relative gene expression was analyzed using a cycle threshold (delta-delta Ct) method. 2.8. Protein extraction and western blot analysis Whole-cell extracts of cell line, primary hepatocytes or mouse liver were prepared using RIPA buffer (Elpis-Biotech). The extracted proteins (50–100 μg) were separated by 10–12% SDS-PAGE, and then transferred to nitrocellulose or PVDF membranes (EMD Millipore). The membranes were probed with following primary antibodies: anti-ERRγ antibody (Perseus Proteomics Inc., cat.PP-6812), anti-FLAG antibody 3

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Fig. 1. Alcohol feeding induces ERRγ and Srebp-1c expression via hepatic CB1 receptor signaling. (A-B) Male C57BL/6J mice (n = 5 per group) were fed an alcoholcontaining diet for 4 weeks. H&E (scale bar, 100 μm) and Oil-Red O (scale bar, 100 μm) staining of liver (A). Q-PCR analysis showing mRNA levels of ERRγ, Cyp2e1 and lipogenic genes in the liver of mice (B). (C) Q-PCR analysis showing mRNA levels of ERRγ and Srebp-1c in the liver of mice (n = 4 per group) administered with liquid alcohol (6 g/kg) for the indicated time. (D) Q-PCR analysis showing mRNA levels of ERRγ and Srebp-1c in the liver of mice (n = 4 per group) administered with 2-AG ether (5 mg/kg, i.p.) for the indicated time. (E) ERRγ and Srebp-1c gene expression in the liver of mice (n = 4 per group) administered with vehicle, 2-AG ether (5 mg/kg, i.p.) or AM251 (5 mg/kg, i.p.) for 12 h. (F) ERRγ and Srebp-1c gene expression in mouse primary hepatocytes treated with vehicle, 2-AG ether (10 μM) or SP600125 (20 μM) for 12 h. Equal amounts of RNA from each mouse in the same group was pooled for analysis. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 by two-tailed Student t-test.

hepatocytes significantly induced both precursor and nuclear forms of SREBP-1c (Fig. 2B), indicating that ERRγ is a transcriptional regulator of hepatic SREBP-1c expression. Indeed, we identified a conserved ERR response element (ERRE) in human and mouse Srebp-1c gene promoters, and confirmed its functional significance using site-specific mutagenesis and promoter stimulation experiments (Fig. 2C). Moreover, ChIP assays performed in HepG2 cells and mouse primary hepatocytes showed that 2-AG-mediated occupancy of ERRγ on the ERRE of the Srebp-1c gene promoter is completely blunted by AM251 treatment (Fig. 2D). These findings suggest that ERRγ directly regulates hepatic Srebp-1c gene expression.

To examine involvement of CB1 receptor signaling in induction of ERRγ and Srebp-1c expression, mice were administered either 2-AG or 2-AG plus AM251, a specific antagonist of the CB1 receptor. As expected, 2-AG-mediated induction of hepatic ERRγ and Srebp-1c gene expression was significantly diminished by treatment with CB1 receptor antagonist, AM251 (Fig. 1E). We also examined the association of 2-AG signaling with ERRγ and Srebp-1c gene expression in primary mouse hepatocytes. 2-AG-mediated induction of ERRγ and Srebp-1c mRNA was significantly attenuated by treatment with SP600125, a specific c-Jun N-terminal kinase inhibitor (Fig. 1F). We concluded from these data that hepatic ERRγ and Srebp-1c expression is mediated by 2-AG receptor signaling upon alcohol feeding.

3.3. ERRγ modulates SREBP-1c expression and de novo lipogenesis in liver 3.2. ERRγ is a transcriptional regulator of SREBP-1c gene expression We next tested if ERRγ could contribute to hepatic de novo lipogenesis through induction of SREBP-1c expression in vivo. Adenovirusmediated expression of ERRγ in the liver of normal mice elicited a marked increase in hepatic lipid accumulation and lipid droplet formation as well as hepatic TG contents compared to controls (Fig. 2E, F). In addition, mRNA levels of Srebp-1c, Fasn and Acaca were increased in the liver of mice infected with Ad-ERRγ (Fig. 2G). These results indicated that ERRγ expression elicits induction of SREBP-1c and hepatic lipogenesis. We then examined if hepatic ERRγ deficiency would lead to reduced alcohol-mediated SREBP-1c expression. Hepatic ERRγ expression was depleted by Ad-shERRγ, which significantly decreases alcoholmediated induction of Srebp-1c and Fasn mRNA as well as protein in

The pattern of ERRγ and Srebp-1c gene expression in the liver of mice exposed to alcohol or 2-AG suggest that ERRγ might regulate Srebp-1c gene transcription. To examine this idea, we infected hepatocytes with adenovirus-mediated expression of GFP (Ad-GFP) or ERRγ (Ad-ERRγ) and measured mRNA and protein expression of SREBP-1c. Gene expression of Srebp-1c and Fasn was significantly increased in AdERRγ-infected primary mouse hepatocytes compared with control (Fig. 2A). Gene expression of Pdk4 (encoding pyruvate dehydrogenase kinase 4) and Pck1 (encoding phosphoenolpyruvate carboxylase), both known ERRγ target genes, was also higher in Ad-ERRγ-infected hepatocytes. In addition, overexpression of ERRγ in mouse primary 4

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Fig. 2. ERRγ increases SREBP-1c expression and hepatic lipogenesis in vivo. (A) Q-PCR analysis showing mRNA levels of lipogenic genes in mouse primary hepatocytes infected with Ad-GFP or Ad-flag-ERRγ for 48 h. (B) Western blot analyses showing expression of precursor SREBP-1 (pSREBP-1) and nuclear SREBP-1 (nSREBP-1) in mouse primary hepatocytes infected with Ad-GFP or Ad-flag-ERRγ for 48 h. (C) Top, ERRE-dependent activation of SREBP-1c promoter in HepG2 cells transfected with the indicated plasmids for 48 h. Bottom, sequence alignments of ERRE on human and mouse SREBP-1c promoters. The ERRE is underlined. The ERREmut is underlined and italicized. Numbers at the right are the distance (bp, base pairs) from the transcription start site. (D) ChIP assay showing the binding of ERRγ to SREBP-1c promoter in HepG2 cells and mouse primary hepatocytes treated with 2-AG ether (10 μM) and AM251 (10 μM) for 18 h. (E-G) Either Ad-GFP or Adflag-ERRγ was injected via tail-vein into mice (n = 3–5 per group) and the mice were sacrificed at day 8 after the injection. H&E (scale bar, 200 μm) and Oil-Red O (scale bar, 50 μm) staining of liver (E). TG levels in liver (F). Lipogenic genes expression in liver (G). Equal amounts of RNA from each mouse in the same group was pooled for analysis. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 by two-tailed Student t-test.

transactivation of Srebp-1c promoter was significantly decreased by GSK5182 treatment in 293 T cells (Fig. 5A). In addition, ERRγ-induced SREBP-1c activation is strongly diminished by GSK5182 treatment compared to control in primary mouse hepatocytes, as shown by western blot analysis (Fig. 5B). The results suggest that the inverse agonist decreases SREBP-1c-mediated hepatic fat accumulation in cultured hepatocytes through downregulation of ERRγ transcriptional activity. Finally, to examine an ameliorative effect of GSK5182 on alcoholic fatty liver in vivo, C57BL/6 J mice were pair-fed an alcohol or control liquid diet for 4 weeks and GSK5182 was given by oral gavage administration once-daily for the last 2 weeks. Oil red O and H&E staining analysis revealed that fat accumulation and lipid droplet formation were increased in liver of mice chronically fed with alcohol when compared to control, which were significantly decreased by GSK5182 treatment (Fig. 5C, D). In addition, increased liver TG levels in alcoholfed mice was almost entirely blunted in mice fed with alcohol plus GSK5182 (Fig. 5E). These findings are consistent with results showing that GSK5182 significantly decreases alcohol-induced Srebp-1c, Acaca and Fasn mRNA and protein expression (Fig. 5F, G). Importantly, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, markers of liver injury, were reduced upon GSK5182 treatment, indicating improvement in liver damage from chronic alcohol feeding (Fig. 5H). Taken together, our findings suggest that an ERRγ inverse agonist has therapeutic potential to ameliorate alcoholic fatty liver disease through inhibition of SREBP-1c-mediated de novo lipogenesis.

liver of mice (Fig. 3A, B). Furthermore, 2-AG-mediated Srebp-1c mRNA and protein expression was as well almost completely blunted by AdshERRγ in AML12 cells (Fig. 3C, D). Therefore, we conclude that ERRγ contributes to hepatic SREBP-1c expression and de novo lipogenesis in liver of mice fed with alcohol. 3.4. SREBP-1c is required for ERRγ-mediated de novo lipogenesis To rule out an off-target effect of ERRγ on the regulation of hepatic lipogenesis, we employed primary hepatocytes isolated from WT and Srebp-1c KO mice. Expression of nuclear form of SREBP-1c was significantly increased in WT hepatocytes infected with Ad-ERRγ compared to Ad-GFP. However, these induction was attenuated in Srebp-1c KO hepatocytes infected with Ad-ERRγ (Fig. 4A). In addition, Ad-ERRγmediated upregulation of FAS expression in WT hepatocytes was blunted in Srebp-1c KO hepatocytes. An increase of lipid accumulation in WT hepatocytes infected with Ad-ERRγ did not occur in Srebp-1c KO hepatocytes infected with Ad-ERRγ (Fig. 4B, C). Taken together, these findings indicate that ERRγ regulates hepatic de novo lipogenesis mainly through induction of SREBP-1c. 3.5. Inverse agonist of ERRγ ameliorates alcoholic fatty liver through inhibition of SREBP-1c Having delineated the role of ERRγ in regulation of SREBP-1c expression and hepatic de novo lipogenesis, we next examined whether interfering with ERRγ transcriptional activity by its inverse agonist, GSK5182, can ameliorate development of alcoholic fatty liver. First of all, we investigated if GSK5182 inhibits ERRγ-induced Srebp-1c promoter activity in cultured cell lines. We found that ERRγ-mediated

4. Discussion In the current study, we identified a previously unrecognized role of 5

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Fig. 3. ERRγ deficiency attenuates alcohol-induced lipogenic gene expression. (A-B) Male C57BL/6 J mice (n = 4 per group) were injected with an adenovirus expressing an unspecific short hairpin (sh) RNA (Ad-US) or Ad-shERRγ and then administered vehicle or alcohol (6 g/kg) for 24 h at day 5 after the injection. Q-PCR analysis showing mRNA levels of ERRγ and lipogenic genes in liver of mice (A). Western blot analysis showing hepatic ERRγ, nSREBP-1 and FAS expression (B). (C-D) AML12 cells were infected with either Ad-US or Ad-shERRγ and treated with vehicle or 2-AG ether (10 μM) for 18 h. Q-PCR analysis showing mRNA levels of ERRγ and Srebp-1c (C). Western blot analysis showing ERRγ, nSREBP-1 and FAS expression (D). Equal amounts of RNA from each mouse in the same group was pooled for analysis. ⁎P < 0.05, ⁎⁎⁎P < 0.001 by two-tailed Student t-test.

damage by alcohol-mediated oxidative stress is an important contributor to the progress from alcoholic fatty liver to steatohepatitis or fibrosis [27,28]. Recently, we found that ERRγ expression is induced by 2-AG in liver of chronic alcohol-fed mice, and that this induction causes oxidative stress and liver injury through upregulation of CYP2E1, a major pathway of ROS generation [22]. In addition, activation of hepatic CB1 receptor signaling by 2-AG increases Srepb-1c gene expression resulting in alcoholic fatty liver [10]. These findings suggest a mechanistic link between ERRγ expression and hepatic steatosis in the pathogenesis of alcoholic fatty liver disease. Interestingly, we found that hepatic ERRγ expression is highly inducible at the transcriptional

ERRγ in the pathogenesis of alcoholic fatty liver. Hepatic ERRγ expression is induced through activation hepatic CB1 receptor signaling in chronic alcohol-fed mice and promotes hepatic de novo lipogenesis through induction of SREBP-1c expression, while ablation of hepatic ERRγ inhibits alcohol-mediated SREBP-1 expression (Fig. 6). In addition, we also demonstrate that SREBP-1c is required for ERRγ-mediated hepatic de novo lipogenesis. Finally, we show an ameliorative effect of inverse agonist of ERRγ on alcoholic fatty liver in a mouse model. Alcoholic fatty liver, an initial response of the liver to binge or chronic alcohol exposure, enhances susceptibility of the liver to more advanced stages of alcoholic liver disease [1]. It is reported that liver

Fig. 4. SREBP-1c is required for ERRγ-mediated lipogenesis. (A-C) Primary hepatocytes isolated from WT and Srebp-1c KO mice were infected with either Ad-GFP or Ad-flag-ERRγ for 48 h. Western blot analysis showing nSREBP-1, FAS and ERRγ expression (A). Oil-Red O staining of the primary hepatocytes (B). Graphical representation showing Oil-red O staining (C). ⁎P < 0.05 by two-tailed Student t-test. 6

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Fig. 5. ERRγ inverse agonist improves alcoholic fatty liver through inhibition of SREBP-1c. (A) Effect of GSK5182 on ERRγ-induced Srebp-1c promoter activity. 293 T cells were transfected with the indicated plasmids for 24 h and then treated with vehicle or GSK5182 (10 μM) for 24 h. (B) Western blot analysis showing ERRγ and nSREBP-1 expression. Mouse primary hepatocytes were infected with Ad-GFP or Ad-flag-ERRγ for 24 h and then treated with vehicle or GSK5182 (10 μM) for 24 h. Insulin (100 nM) was treated for 24 h. (CeH) Male C57BL/6 J mice (n = 4–5 per group) were fed an alcohol-containing diet for 4 weeks and GSK5182 (40 mg/kg once daily) was given by oral gavage for the final 2 weeks of alcohol feeding. Oil-Red O (scale bar, 100 μm) staining of liver (C). H&E (scale bar, 50 μm) staining of liver (D). TG levels in liver (E). Q-PCR analysis showing mRNA levels of hepatic ERRγ, Srebp-1c and Fasn in mice (F). Western blot analysis showing hepatic ERRγ, nSREBP-1 and FAS expression (G). Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (H). Equal amounts of RNA from each mouse in the same group was pooled for analysis. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 by two-tailed Student t-test.

basal SREBP-1c expression seems not to be under control of ERRγ. In contrast, overexpression of ERRγ is able to significantly increase SREBP-1c expression in the liver and in cultured hepatocytes. Moreover, the ChIP assays performed in HepG2 cells and mouse primary hepatocytes show that ERRγ strongly binds to the ERRE of the Srebp-1c gene promoter only in the presence of 2-AG and upregulated ERRγ expression. Therefore, we conclude that alcohol-inducible ERRγ expression is required for transcriptional regulation of the Srebp-1c gene. It is reported that Mg2+-dependent PA phosphatase (PAP) activity of Lipin-1 catalyzes PA conversion to DAG, which in turn promotes activation of hepatic protein kinase C ε resulting in impaired insulin signaling [29]. In addition, alcohol exposure significantly increased Lipin-1 expression and promoted its cytoplasmic localization through inhibition of adenosine monophosphate-dependent protein kinase and activation of SREBP-1, which significantly contributes to alcoholic fatty liver [30]. We have previously found that ERRγ is involved in the direct regulation of Lipin-1 expression and DAG production in liver of mice, and that its inverse agonist significantly could reduce ERRγ-mediated DAG production [21]. These findings together with our findings imply that ERRγ is associated with alcohol-induced hepatic lipogenesis at multiple levels. The hypothesis is confirmed by the current study: hepatic ERRγ expression is induced by alcohol consumption, and subsequently increases SREBP-1c expression by direct binding to the Srebp-1c gene promoter. The ability of ERRγ to regulate multiple lipogenic and ROS producing genes, such as Lipin-1, SREBP-1c and CYP2E1, suggests that this receptor is a major contributor to the pathogenesis of alcoholic fatty liver, and can also accelerate disease progression to more advanced stages of alcoholic liver disease. Two major transcription factors, peroxisome proliferator-activated receptor alpha (PPARα) and SREBP-1c, act downstream of alcohol to regulate hepatic lipid metabolism [2]. PPARα, a member of the NR superfamily, regulate transcription of a set of enzyme involved mainly

Fig. 6. Proposed role of ERRγ in the pathogenesis of alcoholic fatty liver. Hepatic ERRγ expression is increased by alcohol-mediated activation of CB1 receptor signaling and subsequently contributes to induction of SREBP-1c expression and de novo lipogenesis leading to alcoholic fatty liver. GSK5182, an ERRγ inverse agonist, suppresses alcohol-mediated hepatic de novo lipogenesis through inhibition of SREBP-1c expression.

level in the liver downstream of 2-AG-mediated activation of CB1 receptor signaling. This suggests that alcohol induced ERRγ binds to the Srebp-1c gene promoter, leading to induction of SREBP-1c gene expression. Knockdown of basal ERRγ expression in cultured cells had no effect on SREBP-1c expression, suggesting that the low amounts of ERRγ are not directed towards the Srebp-1c promoter, and vice versa, 7

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Acknowledgements

in transport and oxidation of free fatty acids, while SREBP-1c increases gene expression of lipogenic enzyme involved in hepatic de novo lipogenesis [6,31]. Alcohol induces hepatic SREBP-1c expression, while decreasing expression of PPARα, which results in alcoholic fatty liver [2]. Downstream of alcohol consumption, SREBP-1c expression is regulated directly by alcohol metabolites, such as acetaldehyde [3], or indirectly by activating processes and factors, such as endoplasmic reticulum stress and endocannabinoid [10,32]. Parameters, linking alcohol and SREBP-1c expression are still incompletely described. Liver X receptors (LXRs), other members of the NR superfamily, are able to directly regulate Srebp-1c gene expression by dietary cholesterol [33]. However, it remains unclear whether this receptor is involved in alcohol-mediated SREBP-1c expression in liver. In this study, we demonstrate that ERRγ is a major downstream mediator of alcohol on regulation of SREBP-1c expression. Interestingly, in a distal region upstream of the Srebp-1c gene promoter, and different from the previously described LXRE [33], we found a half site located ERRE, which is conserved in human and mouse Srebp-1c gene promoters, and using site-specific mutagenesis, we demonstrated its functional significance in ERRγ-mediated regulation of Srebp-1c gene expression. Our findings are supported by previous results showing that alcohol-mediated production of 2-AG from hepatic stellate cells activates CB1 receptor signaling in hepatocytes, which increases ERRγ gene expression in this cell type [22]. In addition, we show that ERRγ-induced SREBP-1, FAS and ACCα expression, major hepatic lipogenic genes, is significantly decreased in Srebp-1c KO hepatocytes, but interestingly is not completely abolished. This suggests a possibility that ERRγ may also regulate hepatic lipogenic enzyme gene expression in a SREBP-1c-independent manner. This idea is further supported by studies on chronic alcohol consumption, where treatment with ERRγ inverse agonist almost completely reversed hepatic steatosis. Most interestingly, the inverse agonist of ERRγ, GSK5182, was able to normalize liver toxicity and fatty liver caused by progression of diabetic phenotypes or hyperglycemia in mouse models of type 2 diabetes mellitus, such as db/db and diet-induced obesity mice, without toxic effects on other tissues such as liver, kidney, muscle or heart [17,23]. These findings are suggestive to use the ERRγ inverse agonist alcoholic fatty liver disease for disease amelioration without high risk for adverse effects. However, since in vivo effects of the inverse agonist on alcoholic fatty liver disease have not been fully elucidated, its therapeutic efficacy and possible adverse effects need to be further characterized.

We would like to thank Robert R. Harris (Kansas University Medical center, USA) and Honglei Weng (Heidelberg University, Germany) for critical reading and helpful discussions. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions D.K.K., Y.H.K., C.H.L., and H.S.C. designed the study, researched data and wrote the manuscript. Y.S.J., R.F., T.I.J., and S.S.I. developed methodologies for analyzing alcoholic fatty liver. I.K.L., J.K. and S.J.C. synthesized and provided GSK5182. R.F., T.I.J., S.S.I., S.D., and T.F.O. reviewed and edited manuscript. S.S.I. and J.H.L. performed Srebp-1c KO study. All authors have read and approved the manuscript. Funding This work was supported by National Creative Research Initiatives Grant (20110018305 to H.S.C., NRF-2018R1D1A1B07043953 to D.K.K., NRF-2019R1C1C1005319 to Y.H.K.) through the National Research Foundation (NRF) funded by the Korean government (MSIP and Ministry of Education); a grant of the Cooperative Research Program for Agriculture Science and Technology Development (No. PJ01280701 to D.K.K.) funded by Rural Development Administration, Republic of Korea; a grant of the Korea Health technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI16C1501) and a grand of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Program of the Republic of Korea. References [1] B. Gao, R. Bataller, Alcoholic liver disease: pathogenesis and new therapeutic targets, Gastroenterology 141 (2011) 1572–1585. [2] V. Purohit, B. Gao, B.J. Song, Molecular mechanisms of alcoholic fatty liver, Alcohol. Clin. Exp. Res. 33 (2009) 191–205. [3] M. You, M. Fischer, M.A. Deeg, D.W. Crabb, Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP), J. Biol. Chem. 277 (2002) 29342–29347. [4] J.M. Lluis, A. Colell, C. Garcia-Ruiz, N. Kaplowitz, J.C. Fernandez-Checa, Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress, Gastroenterology 124 (2003) 708–724. [5] M. You, D.W. Crabb, Molecular mechanisms of alcoholic fatty liver: role of sterol regulatory element-binding proteins, Alcohol (Fayetteville, NY) 34 (2004) 39–43. [6] T.I. Jeon, T.F. Osborne, SREBPs: metabolic integrators in physiology and metabolism, Trends Endocrinol Metab 23 (2012) 65–72. [7] S.S. Im, L. Yousef, C. Blaschitz, J.Z. Liu, R.A. Edwards, S.G. Young, M. Raffatellu, T.F. Osborne, Linking lipid metabolism to the innate immune response in macrophages through sterol regulatory element binding protein-1a, Cell Metab. 13 (2011) 540–549. [8] I. Shimomura, H. Shimano, J.D. Horton, J.L. Goldstein, M.S. Brown, Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells, J. Clin. Invest. 99 (1997) 838–845. [9] C. Ji, C. Chan, N. Kaplowitz, Predominant role of sterol response element binding proteins (SREBP) lipogenic pathways in hepatic steatosis in the murine intragastric ethanol feeding model, J. Hepatol. 45 (2006) 717–724. [10] W.I. Jeong, D. Osei-Hyiaman, O. Park, J. Liu, S. Batkai, P. Mukhopadhyay, N. Horiguchi, J. Harvey-White, G. Marsicano, B. Lutz, B. Gao, G. Kunos, Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver, Cell Metab. 7 (2008) 227–235. [11] J. Misra, D.K. Kim, H.S. Choi, ERRgamma: a junior orphan with a senior role in metabolism, Trends Endocrinol Metab 28 (2017) 261–272. [12] J.M. Huss, R.P. Kopp, D.P. Kelly, Peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) coactivates the cardiac-enriched nuclear receptors estrogen-related receptor-alpha and -gamma. Identification of novel leucine-rich interaction motif within PGC-1alpha, J. Biol. Chem. 277 (2002) 40265–40274. [13] S. Sanyal, J.Y. Kim, H.J. Kim, J. Takeda, Y.K. Lee, D.D. Moore, H.S. Choi,

5. Conclusions NRs have diverse biological roles in the maintenance of cellular homeostasis because they act as a major transcriptional mediator of hormonal, nutrient and metabolite signals into specific gene expression networks in response to distinct physiological and pathophysiological stimuli [34]. Therefore, their modulators are able to provide therapeutic potential for a variety of metabolic disorders. Here, we identified ERRγ as a major downstream mediator of adverse alcohol effects, contributing to the development of alcoholic fatty liver through regulation of hepatic SREBP-1c expression. Furthermore, we demonstrated an ameliorative effect of ERRγ inverse agonist on alcoholic fatty liver. Our results indicated that the ability of ERRγ inverse agonist to suppress alcohol-induced hepatic lipogenesis could be promising therapeutic approach for the treatment of alcoholic fatty liver disease.

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