Ursolic Acid, a Novel Liver X Receptor Î± (LXRÎ±) Antagonist Inhibiting

Article Cite This: J. Agric. Food Chem. 2018, 66, 11647−11662

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Ursolic Acid, a Novel Liver X Receptor α (LXRα) Antagonist Inhibiting Ligand-Induced Nonalcoholic Fatty Liver and Drug-Induced Lipogenesis Yen-Ning Lin,†,● Charles C. N. Wang,‡,● Hsiao-Yun Chang,§,● Fang-Yi Chu,† Yu-An Hsu,∥ Wai-Kok Cheng,† Wei-Chih Ma,† Chao-Jung Chen,⊥,# Lei Wan,∇ and Yun-Ping Lim*,†,∇,○ †

Department of Pharmacy, College of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan § Department of Biotechnology, Asia University, Taichung, Taiwan ∥ School of Chinese Medicine, China Medical University, Taichung, Taiwan ⊥ Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan # Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ∇ Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ○ Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan

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‡

S Supporting Information *

ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is a very common liver disease, and its incidence has significantly increased worldwide. The liver X receptor α (LXRα) is a multifunctional nuclear receptor that controls lipid homeostasis. Inhibition of LXRα transactivation may be beneficial for NAFLD and hyperlipidemia treatment. Ursolic acid (UA) is a plant triterpenoid with many beneficial effects; however, the mechanism of its action on LXRα remains elusive. We evaluated the effects of UA on T0901317 (T090)-induced LXRα activation and steatosis. UA significantly decreased the LXR response element and sterol regulatory element-binding protein-1c (SREBP-1c) gene promoter activities, mRNA, protein expression of LXRα target genes, and hepatic cellular lipid content in a T090-induced mouse model. A molecular docking study indicated that UA bound competitively with T090 at the LXRα ligand binding domain. UA stimulated AMP-activated protein kinase (AMPK) phosphorylation in hepatic cells and increased corepressor, small heterodimer partner-interacting leucine zipper protein (SMILE) but decreased coactivator, steroid receptor coactivator-1 (SRC-1) recruitment to the SREBP-1c promoter region. In contrast, UA induced SRC-1 binding but decreased SMILE binding to reverse cholesterol transport-related gene promoters in intestinal cells, increasing lipid excretion from intestinal cells. Additionally, UA reduced valproate-induced LXRα mediated and rifampin-induced pregnane X receptor mediated lipogenesis, offering potential treatments for drug-induced hepatic steatosis. Thus, UA displays liver specificity and can be selectively repressed while RCT stimulation by LXRα is preserved and enhanced. This is a novel therapeutic option to treat NAFLD and may be helpful in developing LXR agonists to prevent atherosclerosis. KEYWORDS: nonalcoholic fatty liver disease, liver X receptor α, pregnane X receptor, ursolic acid

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The liver X receptors α (LXRα; NR1H3) and β (LXRβ; NR1H2) are nuclear receptors (NRs) discovered in the mid1990s and have been found to be important regulators of cholesterol homeostasis.3 LXRα is highly expressed in lipidmetabolism-related organs, responsible for de novo synthesis and excretion of cholesterol and for glucose homeostasis, such as the liver, small intestine, kidney, spleen, and adipose tissue, whereas LXRβ is distributed ubiquitously.4 After they bind their ligands, LXRs heterodimerize with the retinoid X receptor (RXR; NR2B1) and then bind to the LXR response element (LXRE, consisting of two hexameric direct nucleotide repeats separated by four nucleotides; DR4) and induce the

INTRODUCTION

The liver plays an important role in the metabolism of lipids, participating in their transportation, production, and storage. A disruption in any of these processes, even without significant alcohol consumption, can lead to the development of nonalcoholic fatty liver disease (NAFLD).1 NAFLD is a growing public health problem, occurring in 11−46% of adults and affecting an increasing number of children in developed countries.1 The term NAFLD covers a spectrum of diseases, which result from dysregulation of lipid homeostasis and may lead to abnormal and excessive storage of triglycerides (TGs) in hepatocytes. TG accumulation has the potential to trigger an inflammatory response and to lead to nonalcoholic steatohepatitis (NASH). Although benign steatosis can be reversible, NASH can gradually progress to cirrhosis and result in hepatocellular carcinoma.2 © 2018 American Chemical Society

Received: Revised: Accepted: Published: 11647

August 1, 2018 October 14, 2018 October 15, 2018 October 25, 2018 DOI: 10.1021/acs.jafc.8b04116 J. Agric. Food Chem. 2018, 66, 11647−11662

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Journal of Agricultural and Food Chemistry transcription of target genes.4 The synthetic compound T0901317 (T090), an LXRα agonist developed through structure−activity relationship studies, stimulates the transcriptional activity of LXRs.5 Activation of LXRα can induce reverse cholesterol transport (RCT), which increases serum high-density lipoprotein (HDL) cholesterol, alleviates hypercholesterolemia, and prevents atherosclerosis. The induction of intestinal RCT gene expression upon LXRα activation is thought to reduce the efficiency of cholesterol absorption and to accelerate the fecal cholesterol disposal.6 However, activation of LXRα results in the development of hepatic steatosis, which is mediated via the hepatic lipogenic pathway, primarily through sterol regulatory element-binding protein-1c (SREBP-1c)4,7 and results in unwanted side effects, including hepatic steatosis and hyperlipidemia, through increased expression of genes critical for fatty acid (FA) synthesis, including SREBP-1c and a battery of its target genes, FA synthase (FAS), stearoyl-coenzyme A (CoA) desaturase-1 (SCD), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACLY), and FA elongase (FAE).8 This lipogenicity is a major limitation for the development of LXRα agonists as attractive antiatherosclerosis modulators. T090 was shown to be an effective antiatherogenic agent, able to induce RCT and enhance HDL cholesterol levels; however, the compound leads to undesirable side effects such as hypertriglyceridemia and hepatic steatosis.9 In addition, hepatic expression of LXRα, SREBP-1c, ACC, FAS, and SCD is significantly upregulated in liver biopsies from NAFLD patients.8 Thus, it is likely that antagonists of LXRs activation can be used as a treatment for NAFLD. The pregnane X receptor (PXR; NR1I2) is also a member of the NR superfamily. It is activated by a variety of ligands, including insecticides, pesticides, nutrients, and therapeutic compounds, and heterodimerized with RXR upon target gene activation.10 In addition to drug metabolism, PXR has been shown to be involved in endogenous functions of lipid and glucose metabolism. PXR activation induces FA uptake and lipogenesis as well as inhibits FA catabolism (β-oxidation) via several mechanisms.11,12 In addition, alterations in PXR function result in the induction and development of hepatic steatosis. Rifampin (RIF), a potent activator of PXR in humans, induces hepatic steatosis in patients with tuberculosis and causes steatotic lesions in rat livers.13−15 AMP-activated protein kinase (AMPK) is a serine/threonine heterotrimeric protein kinase complex consisting of a catalytic subunit (α) and two regulatory subunits (β and γ).16 When AMPK is activated by AMP and phosphorylated at a conserved threonine residue (Thr172 in the rat sequence) by an upstream kinase, it initiates the ATP-generating process, thus maintaining energy homeostasis by concomitant inhibition of energy-consuming biosynthetic pathways such as FA and sterol synthesis, suppression of SREBP-1c, and activation of ATPproducing catabolic pathways such as FA oxidation.17 Transcriptional activation and repression of NRs are regulated by NR coregulators, which are called coactivators and corepressors, respectively.18 Coactivators from the p160 family, including steroid receptor coactivator-1 (SRC-1), interact with the activation function 2 (AF2) domain of the ligand-binding domain (LBD) of an NR in the presence of a ligand. The small heterodimer partner-interacting leucine zipper protein (SMILE), belonging to the basic region leucine zipper family, was found to inhibit the T090-induced LXRα transcriptional activity by competing with the coactivator SRC-

1 and thus decreasing the SREBP-1c, FAS, and ACC gene expression.19 Expression of the SMILE gene may be induced through the liver AMPK pathway.19 Ursolic acid (UA; 3β-hydroxyurs-12-en-28-oic acid) is a naturally occurring pentacyclic triterpenoid carboxyl acid. It is found in several traditional Chinese medicinal herbs, such as Cornus officinalis,20 and it is well documented that UA exerts a wide range of biological activities, including anticancer, antiangiogenic,21 antioxidant, antiinflammatory, immunoregulatory,22 hepatoprotective,23 and hypolipidemic effects.24 UA has also been reported to reduce hepatic lipid accumulation.25 However, the effects of UA on the regulation of LXR activity, mediated by LXR ligands, have never been investigated in cultured cells and animal studies. In this study, we investigated whether UA could antagonize LXRα in vitro and in vivo and thus decrease hepatic lipid accumulation and improve the blood lipid profile.

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MATERIALS AND METHODS

Chemicals and Cell Culture. UA (purity ≥98.5%), T090, Oil Red O, valproate (VPA), RIF, compound C, and all chemicals with the highest-grade purity were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of chemicals were prepared in dimethyl sulfoxide (DMSO) or ultrapure water before use. The human hepatocellular carcinoma cell line, HepG2, and the intestinal adenocarcinoma cell line, LS174T, were available from the Food Industry Research and Development Institute (FIRDI, Taiwan, ROC) and cultivated in α-minimum essential medium (αMEM; Gibco BRL) containing 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and 1 × L-glutamine. Cell passages higher than 10 were not used for experiments. HepaRG cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Single used frozen cells were thawed and directly seeded and cultivated in William’s E medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FetalCloneIII serum (Hyclone, GE Healthcare, Chicago, IL, USA), 50 μM hydrocortisone hydrogen succinate, 5 μg/mL human insulin, and 1 × L-glutamine for 2 weeks. When cells reached confluence, the culture medium was replaced with a fresh medium plus 2% DMSO for 2 more weeks to induce differentiation of hepatocyte-like cells. The cells were cultured in a humidified atmosphere containing 5% CO2 in a 37 °C incubator. Cell viability was assessed using an acid phosphatase assay as described previously.26 Transient Transfection and Reporter Assay. Transient transfection was carried out as described previously.27 HepG2 cells were transfected, using the PolyJET transfection reagent (SignaGen Laboratories, Rockville, MD), with an LXRα27 or PXR26 expression vector and with a 3 × LXRE-luciferase (Luc),28 SREBP-1c-Luc,29 S14-Luc,30 or SCD-1-Luc12 reporter construct. A β-galactosidase reporter construct was transfected into HepG2 cells for normalization of efficiencies for 24 h. A LUC assay detection kit was used to determine LUC activities according to the manufacturer’s protocols (Promega, Madison, WI, USA). Drug treatments were performed at 6−7 h after the transfection. Cell lysates were produced after 20 h of drug treatment and analyzed for LUC activity, normalized to the corresponding β-galactosidase activity. Cellular Triglycerides, Total Cholesterol, and Oil Red O Staining. Cellular lipids were extracted as previously described.31 TGs and total cholesterol (TC) were quantified by a modified method using standard automatic clinical chemistry assays on a Cobas 8000 modular analyzer (Roche Diagnostics North America). For Oil Red O staining of cellular neutral lipids, differentiated HepaRG cells were treated with T090/VPA/RIF alone or in combination with 5 or 10 μM UA, as described in the graphical protocol provided as Figure S1. A stock solution of Oil Red O (0.5% in isopropyl alcohol) was freshly prepared before use. Treated cells were fixed with 10% formalin after washing with phosphate-buffered saline (PBS). After the cells were completely dried, they were stained with Oil Red O in 60% isopropyl alcohol for 20 min in the dark at 25 °C. After the cells 11648

DOI: 10.1021/acs.jafc.8b04116 J. Agric. Food Chem. 2018, 66, 11647−11662

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Journal of Agricultural and Food Chemistry

Animals. Male C57BL/6J mice (18−20 g, 4 weeks old) were purchased from the National Laboratory Animal Center (Taiwan, ROC). The experiments were carried out under the guidelines and approval from the Institutional Animal Care and Use Committee of China Medical University (approval numbers 2016-109 and 2018082). The mice were housed in a light- and temperature-controlled facility, with free access to food and water. All mice were adapted to the standard care and housing conditions for 1 week before the experiments. T0901317-Induced Fatty Liver Experiment. Mice were randomly assigned to four groups and treated by oral gavage once a day for 7 days as follows: (1) vehicle control (CON); (2) 50 mg/kg/ day T090 (dissolved in propylene glycol/Tween 80, 4:1) (T090); (3) 50 mg/kg/day T090 + 100 mg/kg/day UA (TU100); (4) 50 mg/kg/ day T090 + 250 mg/kg/day UA (TU250). At the end of the treatment period, the mice were sacrificed; the blood was immediately collected by cardiac puncture and centrifuged. The serum was aspirated and stored at −80 °C until analysis. The liver was immediately removed and divided into three parts as follows: one part was buffered in 10% neutral formalin for histopathology; the second part was carefully embedded in the optimal cutting temperature compound and frozen at −80 °C; the remaining part was collected into RNase-free tubes, snap-frozen in liquid nitrogen, and stored at −80 °C for RNA isolation and lipid analysis. Histological Analysis and Oil Red O Staining. Fixed livers were embedded in paraffin; 5 μm sections were prepared, stained with hematoxylin and eosin (H&E), and mounted by standard procedures. To further examine lipid droplet accumulation, frozen liver tissues were sliced and cryosectioning was carried out at −25 °C. The embedded liver was sectioned into 8−10 μm slices; these slices were stained with Oil Red O using a standard procedure, counterstained with Harris hematoxylin, and mounted in glycerin jelly as described.32 Serum Biochemistry and Hepatic Lipid Measurement. TGs and TC were extracted from the liver with 2 mL of isopropyl alcohol, and their content was determined as described previously.33 Serum/ liver TGs, serum/liver TC, serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) were analyzed by standard clinical chemistry assays on a Cobas 8000 modular analyzer (Roche Diagnostics North America). Nuclear Extract Preparation and DNA Affinity Precipitation Assay. Nuclear extracts were prepared from differentiated HepaRG and LS174T cells exposed to UA alone or in combination with T090 for 24 h, as described previously.34 The cellular nuclear extracts (500 μg) were mixed with 2 μg of a biotinylated SREBP-1c LXRE oligonucleotide (5′-biotin-CAG TGA CCG CCA GTA ACC CCA GC-3′, for HepaRG cells) or an ABCG1 LXRE oligonucleotide (5′biotin-GGC AAG AGG TAA CTG TCG GTC AAA TCC T-3′, for LS174T cells) and 20 μL of streptavidin-agarose beads (4%) (SigmaAldrich, St. Louis, MO, USA) with a 50% slurry. After 1 h of rotation at room temperature, the beads were pelleted and rinsed three times with ice-cold PBS. Bound protein complexes were eluted using SDSPAGE sample buffer and processed for Western blotting analysis with specific antibodies against SRC-1 and SMILE. Molecular Docking. Molecular docking of T090 (PubChem CID: 447912) and UA (PubChem CID: 64945) into LXRα (PDB id: 1UHL) was carried out using CDOCKER with Discovery Studio (DS) v. 4.5. Receptor−ligand interactions were further optimized by molecular dynamics using CHARMM and Clean Geometry of DS 4.5. The CDOCKER simulation parameters are shown in Table 2. To

were dried, cell images were taken under a phase-contrast microscope at a 400× magnification (Axio Observer A1, Zeiss Inc. Germany). Lipids were extracted with 100% isopropyl alcohol, transferred to microplates, and read at 510 nm using a multimode spectrophotometer. Quantitative Real-Time Polymerase Chain Reaction. For a quantitative real-time polymerase chain reaction (qRT-PCR), total RNA was extracted from cells and mouse livers using a Direct-zol RNA MiniPrep kit (ZYMO Research, Irvine, CA) according to the protocol provided. The resulting cDNA of each gene was analyzed by qRT-PCR using the Luminaris Color HiGreen qPCR master mix (ThermoFisher Scientific, Waltham, MA, USA) and a StepOnePlus real-time PCR system, as described previously.27 Each specific gene expression was normalized to the corresponding β-actin levels. The sequences of the gene-specific primers used in this study are given in Table 1.

Table 1. Sequences of PCR Primers gene

species

forward primer (5′-3′)

reverse primer (5′-3′)

hSREBP-1c

human

hFAS

human

hSCD

human

hACC

human

TGC AGA AAG CGA ATG TAG TCG AT GGA GCG AGA AGT CAA CAC GA GCG TAC TCC CCT TCT CTT TGA C TGA GTG CCG TGC TCT GGA T

hACLY

human

CGC TCC TCC ATC AAT GAC AA ACA TCA TCG CTG GTG GTC TG CCG ACG TGG CTT TTT CTT CT CTC TTG ACC CTG GCT GTG TAC TAG GTG TGG ACG TGG GTG ATG TG

hFAE

human

hABCA1

human

hABCG1

human

hS14

human

hActin

human

mSREBP-1c

mouse

mFAS

mouse

mSCD

mouse

mACC

mouse

mActin

mouse

TTC CGA GTC TCC CGG AAG T GAC ATC GTG GCG TTT TTG G CGG TGC TCT CAT CCC TTT CA TAT TTG CTC TGG CCC TTG CT CCT GGC ACC CAG CAC AAT CTG TCG TCT ACC ATA AGC TGC AC CCT GGA TAG CAT TCC GAA CCT CTA CAA GCC TGG CCT CCT GC ATG GGC GGA ATG GTC TCT TTC CTG TCC CTG TAT GCC TCT G

TTG ATG TCC TCA GGA TTC AGT TTC ACA GCC CAT CAG CAT CTG AGT CGA GAT ATG GTC CGG ATT GC GGT GTA CAG CCC GTC TTC CA GGT CGC CAA GTA AGA GGG TG GCC GAT CCA CAC GGA GTA CT ATA GCA TCT CCT GCA CAC TCA GC AGC ACA TCT CGA AGG CTA CAC A GGA CCC CAG GGA AAC CAG GA TGG GGA CCT TGT CTT CAT CAT ATG TCA CGC ACG ATT TCC

Western Blotting. Cells were harvested, and total proteins were extracted by lysing cells in RIPA buffer (containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM βglycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin) after 24 h of drug treatment. Total proteins (50 μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (BioTrace NT Nitrocellulose Transfer Membrane, PALL Corporation, Port Washington, NY, USA). Blots were hybridized with specific antibodies overnight at 4 °C, and signals were detected using the Immobilon western chemiluminescent horseradish peroxidase substrate (Merck Millipore, Billerica, MA, USA). Anti-SREBP-1c, FAE, ACC, ACLY, and S14 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-FAS, SCD, LXRα, SRC-1, and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SMILE and phospho-AMPK antibodies were purchased from GeneTex (Irvine, CA).

Table 2. Parameters for CDOCKER Simulation of Docking

11649

param name

param value

top hits random conformations orientation of refine forcefield use full potential

10 10 10 CHARMm False DOI: 10.1021/acs.jafc.8b04116 J. Agric. Food Chem. 2018, 66, 11647−11662

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Journal of Agricultural and Food Chemistry ascertain potential correlations between experimental activities and corresponding values of CDOCKER energy values, the best docked conformations of LXRα ligands were selected as preliminary binding conformations. Statistical Analysis. All data from separate measurements were represented as the mean ± standard error of the mean (SE). Multiple comparisons were carried out by one-way analysis of variance, followed by the least significant difference multiple comparison procedure, with P < 0.05 considered significant. Analyses were performed using the SPSS for Windows software (SPSS version 20.0, Armonk, NY).

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RESULTS Reduction of T0901317-Mediated LXRα Transactivation by Ursolic Acid. To rule out possible cytotoxic and antitumor effects of UA, we performed a cell viability test with two human hepatocarcinoma cell lines, HepG2 and HepaRG, and an intestinal cell line, LS174T. As shown in Figure S2, in comparison with the DMSO control, UA caused no cytotoxicity, either with or without T090 cotreatment, at 24 h. LXRα activation has been shown to promote hepatic lipogenesis and hyperlipidemia in vitro and in vivo through an important transcription factor, SREBP-1c. We assessed the effects of UA on LXRα transactivation via 3 × LXRE and the SREBP-1c promoter using LUC reporter gene assays. T090 significantly induced the transactivation of LXRα through both 3 × LXRE and SREBP-1c promoter activities (62.6- and 24.2fold, respectively; Figure 1). However, UA significantly reduced these transactivation effects in a dose-dependent manner (Figure 1A, 1B). Attenuation of T0901317-Induced Lipogenic Gene and Protein Expression in Vitro by Ursolic Acid. Previous studies have shown that de novo synthesis of FA- and TGrelated genes, LXRα, SREBP-1c, ACC, and FAS, was highly induced in NAFLD patients in comparison with that in patients with normal liver function.8 To further assess whether UA suppresses the downstream target genes of LXRα, the mRNA levels of SREBP-1c, FAS, SCD, ACC, ACLY, and FAE were analyzed in differentiated HepaRG cells (Figure 2A). The LXRα agonist T090 significantly induced these genes, to 15.4-, 8.5-, 14.8-, 5.4-, 9.5-, and 5.2-fold, respectively, of their levels of expression in the untreated group. However, cotreatment with UA significantly and dose-dependently decreased the mRNA levels of these genes (Figure 2A). We further assessed the effects of UA on protein expression of SREBP-1c, SCD, and FAS in differentiated HepaRG cells. T090 significantly induced the protein expression of SREBP-1c, SCD, and FAS (Figure 2B,C), while UA decreased the levels of these proteins in a dose-dependent manner, consistent with the transcriptional data (Figure 2A). These findings suggest that UA inhibits the expression of the LXRα−SREBP-1c signaling pathway-related battery of target genes both at the mRNA and protein levels. Inhibition of Ligand-Induced LXRα Activation and Lipogenesis in Differentiated HepaRG Cells by Ursolic Acid. We performed Oil Red O staining to detect intracytoplasmic lipid droplets in differentiated HepaRG cells after a repeated treatment procedure carried out for 14 days, as shown in Figure S1. Since we found that 20 μM UA showed cytotoxicity during this long-term treatment, we treated cells with 5 and 10 μM UA. Oil Red O staining of cells treated with UA showed significant reduction in cellular neutral lipid accumulation; however, T090-treated cells showed significant accumulation of lipid droplets after long-term treatment (Figure 3A,B). Furthermore, we treated these hepatocyte-like

Figure 1. Effects of ursolic acid (UA) and T0901317 (T090)activated LXRα. HepG2 cells were cotransfected with a LXRα expression plasmid along with (A) 3 × LXRE-Luc and (B) SREBP-1cLuc reporter genes. After 6 h, the cells were treated with 10 μM T090 alone or in combination with 10 or 20 μM UA and incubated for an additional 24 h. The luciferase assay was carried out as described in Materials and Methods. Data represent the mean ± SE: n = 4; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated).

cells for 72 h with T090 alone or in combination with 10 and 20 μM UA and found that the cellular TG concentrations were significantly decreased by cotreatment with UA, even though LXRα activation was induced (Figure 3C). In comparison with that in the T090-treated group, UA at 10 and 20 μM reduced the TG levels by 34% and 65%, respectively, whereas T090 increased cellular TGs up to 1.6-fold of their level in the untreated group (Figure 3C). However, T090 did not induce the accumulation of TC in these cells, while UA at 20 μM significantly decreased TC accumulation. These results show that UA treatment reduces the TG content in hepatocyte-like cells, likely through inhibition of expression of known LXRα target genes. Decrease in Lipid Accumulation and Reversal of T0901317-Induced Hepatic Steatosis in Mouse Liver by Ursolic Acid. We further explored whether inhibition of LXRα by UA could be observed in vivo. Male C57BL/6 mice were treated with 50 mg/(kg day) T090 for 7 days once daily by oral gavage. In the UA groups, 100 and 250 mg/(kg day) UA was coadministered with T090. Histopathologic observations (H&E staining; Figure 4A) and Oil Red O staining of frozen liver sections for neutral lipids (Figure 4B) revealed that 11650


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Figure 2. mRNA and protein expression of T0901317 (T090)-induced LXRα downstream genes affects by ursolic acid (UA). Well-differentiated HepaRG cells were treated with 10 μM T090 alone or along with 10 or 20 μM UA for 24 h. (A) Gene expression levels of SREBP-1c, FAS, SCD, ACC, ACLY, FAE, and β-actin (internal control) carried out by real-time PCR are shown. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (B, C) Total lysates collected from HepaRG cells treated with T090 or/and UA for 24 h. Protein expression of SREBP-1c, SCD, FAS, and β-actin (internal control) was analyzed by Western blotting. Quantitation of the indicated protein bands was corrected by each corresponding β-actin expression. The band density was quantified with ImageJ software, and a representative blot is shown. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated).

T090 caused hepatic lipid accumulation with microsteatosis; however, this phenomenon was significantly reduced by cotreatment with 100 and 250 mg/(kg day) UA. Hepatic TG levels were higher in the T090-treated mice than in the chow-fed mice and were significantly reduced by UA, especially in the TU250 group (Figure 4C). Liver TC was also decreased by UA cotreatment; however, the results did not reach statistical significance (Figure 4C). The serum TG and TC levels increased during treatment with T090 alone but were significantly reduced in the UA-cotreated groups (TU100 and TU250; Figure 4D). Several serum parameters, including ALT, BUN, and CREA, were not significantly affected by T090

and UA treatment, suggesting that these treatments did not alter basic liver and kidney functions (Figure 4E). To test whether UA inhibits lipid production through inhibition of the expression of lipogenic genes, the SREBP-1c gene and its downstream genes, FAS, SCD, and ACC, were analyzed in the mouse liver. The liver expression levels of several lipogenic genes in the mice treated with T090 alone or in combination with 100 and 250 mg/kg UA are shown in Figure 4F. The mRNA expression of SREBP-1c, FAS, SCD, and ACC was significantly upregulated in the liver of T090-fed mice. However, these genes were significantly downregulated by cotreatment with 100 and 250 mg/(kg day) UA (Figure 4F). 11651


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Figure 3. Determination of lipid accumulation in differentiated HepaRG cells with T0901317 (T090) alone or in combination with ursolic acid (UA). (A) Well-differentiated HepaRG cells were repeatedly exposed to (a) solvent, (b) 10 μM T090, (c) 10 μM T090 + 5 μM UA, or (d) 10 μM T090 + 10 μM UA for 14 days as described in Figure S1. Lipid accumulation was then visualized by Oil Red O staining, to detect the presence of triglycerides and cholesterol esters, and then cells were observed and photographed under a phase-contrast microscope (original magnification ×400). (B) Oil red O dye was then extracted by isopropyl alcohol and quantified with a microplate reader at 510 nm. Values represent the mean ± SE: n = 3; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (C) Differentiated HepaRG cells were treated with 10 μM T090 alone or in combination with 10 or 20 μM UA for 72 h. Cellular triglycerides and total cholesterol were measured. Values represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated).

ligand−target interactions, T090 and UA were docked into LXRα. T090 could dock stably within the LXRα endogenous ligand-binding site. In this structural domain, six amino acid residues (Arg305, Thr302, His421, Met298, Trp443, and Phe257) are involved in the interaction between T090 and LXRα, as shown in Figure 5A. The docking binding energy results showed an active CDOCKER energy score of 45.7965 kcal/mol. The docking results showed that the ligand with the best binding energies, UA, occupied the active site of LXRα. CDOCKER calculates the interaction energy profiles of known inhibitor molecules cocrystallized with target proteins. The energy of docking UA into LXRα with CDOCKER was −37.5211 kcal/mol. Figure 5B shows the three-dimensional structure of LXRα with UA bound, which was generated by DS

The elevated levels of expression of lipogenic genes in T090treated mice were decreased on cotreatment with UA, indicating that UA exerts potent antisteatotic effects against the LXRα signaling pathway. Molecular Interactions between LXRα and UA. Molecular docking using the CDOCKER module in DS 4.5 was performed on the test compounds to investigate their binding modes and reveal essential residues involved in binding interactions. The protein crystal structure of LXRα and of its binding site was retrieved from the PDB (PDB ID: 1UHL). The structure of 1UHL has four chains and consists of 488 amino acid residues. These are represented by three unique entities. In addition to the endogenous ligand-binding site, automatic searching using DS 4.5 revealed two active sites. To determine the putative binding model and potential 11652


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Figure 4. Effects of ursolic acid (UA) on T0901317 (T090)-induced hepatic steatosis in vivo. Male C57BL/6J mice were administered through oral gavage daily for 7 days solvent control (CON), 50 mg/(kg day) T090 (T090), 50 mg/(kg day) T090 + 100 mg/(kg day) UA (TU100), or 50 mg/(kg day) T090 + 250 mg/(kg day) UA (TU250). After this administration schedule, the mice were fasted for 10 h and sacrificed. Livers were immediately removed, and blood was immediately collected by cardiac puncture and centrifuged. (A) H & E stained mouse liver sections of (a) CON, (b) T090, (c) TU100, and (d) TU250 are shown (original magnification ×400). (B) Oil Red O staining for neutral lipid content in liver sections of (a) CON, (b) T090, (c) TU100, and (d) TU250. A representative slice from each group is shown (original magnification ×400). (C) Liver contents of triglycerides and total cholesterol. (D) Serum triglycerides and total cholesterol concentration. (E) Serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) were measured as described in Materials and Methods. Values represent the mean ± SE: n = 6; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (F) Liver total RNA was isolated from 100 mg of liver tissue from male C57BL/6J mice (control; 50 mg/(kg day) T090-treated (T090); 50 mg/(kg day) T090 + 100 mg/ (kg day) UA (TU100); 50 mg/(kg day) T090 + 250 mg/(kg day) UA (TU250)) and converted to cDNA. Levels of cDNA were measured by realtime PCR as described inMaterials and Methods. Results are normalized to β-actin mRNA levels, and levels in the control mice group were set at 1. SREBP-1c, FAS, SCD, and ACC levels are shown. Values represent the mean ± SE: n = 6; ***, p < 0.001.

His421, Trp443, Leu439, Phe254, Ala261, and Met298. Some of these amino acid residues were also found to be involved in binding of ligand molecules to LXRα in other docking studies.

4.5 and used to study the ligand and receptor interactions. The binding mode clearly indicates UA interactions with residues such as Arg305, Glu301, Thr302, Phe326, Phe257, Leu331, 11653


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Figure 5. Binding of T0901317 (T090) and ursolic acid (UA) with LXRα. (A) Binding mode of the interactions of T090 ligand with the residues of LXRα. (B) Binding mode of the interactions of UA ligand with the residues of LXRα.

Effects of Ursolic Acid on Reverse Cholesterol Transport in Intestinal Cells. UA inhibits hepatic lipogenesis through inhibition of LXRα activation. The activation of LXRα reduces the body load of cholesterol by stimulating RCT via induction of key target genes, ATP-binding cassette subfamily A member 1 (ABCA1) and ABCG1, in macrophages and the intestine and may block the development of atherosclerosis.35,36 Thus, we assessed whether UA inhibits the expression of ABCA1 and ABCG1 in human intestinal cells, LS174T.37 Interestingly, UA did not inhibit the T090-induced ABCA1 and ABCG1 gene expression in these cells; instead, UA significantly increased the expression of both genes in combination with T090 treatment (Figure 6A), while the SREBP-1c gene remained transcriptionally silent in these cells.38 However, we found that the ABCG1 gene expression decreased upon cotreatment with UA and T090 in HepaRG cells (Figure S3). We next determined the effects of T090 and UA on the cellular contents of TGs and TC in LS174T cells. There was not much difference in the TG content between T090- and UA-treated and control cells; however, TC in the

cells treated with T090 alone or in combination with UA significantly decreased (Figure 6B). These results may indicate that the activation of RCT by T090 and UA increases the efflux of cholesterol from LS174T cells, which may be due to the increase in RCT-related gene expression. Previous studies have demonstrated that AMPK activation leads to the inhibition of the production of endogenous LXR ligands via inhibiting the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) activity and thus decreasing the SREBP-1c gene expression.39 Activation of AMPK by 5aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin decreased the T090-induced SREBP-1c promoter activity.40 The authors concluded that activation of AMPK directly affects the ligand-induced LXR activity. We used compound C, a cell-permeable AMPK inhibitor,41 to inhibit AMPK activation and found that, in the presence of compound C, SREBP-1c, FAS, and SCD gene expression significantly increased, by 58%, 50%, and 84%, respectively, compared with that in T090-treated cells, suggesting AMPK is involved in the expression of these genes in HepaRG cells (Figure 6C). The 11654


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Figure 6. Effects of ursolic acid (UA) on reverse cholesterol transport (RCT)-related genes in LS174T cells, cholesterol excretion, involvement of AMPK phosphorylation, and recruitment of differential coregulators to the lipogenic and RCT genes. (A) LS174T cells were treated with T090 or/ and UA for 24 h. The expression of RCT-related genes, ABCA1 and ABCG1, and lipogenesis gene, SREBP-1c, was analyzed by real-time PCR. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (B) LS174T cells were treated with 10 μM T090 or/and 10 or 20 μM UA for 72 h. Cellular triglycerides and total cholesterol were measured. Values represent the mean ± SE: n = 3; ***, p < 0.001 (compared with control as indicated). (C) Differentiated HepaRG and LS174T cells were treated with 10 μM T090 or/ and 20 μM UA and/or 3 μM compound C as indicated in the figure for 24 h. Gene expression levels of SREBP-1c, FAS, SCD (for HepaRG cells) and ABCA1, ABCG1 (for LS174T cells) by real-time PCR are shown. β-Actin was used as an internal control. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (D) Total lysates from differentiated HepaRG cells or LS174T cells treated with T090 or/and UA for 24 h were collected. Protein expression of phospho-AMPK and β-actin (internal control) was analyzed by Western blotting. Quantitation of the indicated protein bands with ImageJ software and was corrected by β-actin expression. A representative blot is shown. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control group as indicated). (E) Nuclear extracts after T090 or/and UA treatment were collected and analyzed by DAPA as mentioned in Materials and Methods of SREBP-1c (upper panel) and ABCG1 (lower panel) response element from HepaRG and LS1714T cells, respectively. A representative blot is presented, and the protein expression in the mock control was set at 1. 11655


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Figure 7. Effects of ursolic acid (UA) on valproate (VPA)-induced transient transcriptional activation of 3 × LXRE and SREBP-1c reporters, mRNA and protein expression of LXRα target genes, and lipid contents. HepG2 cells were cotransfected with an LXRα expression plasmid and (A) 3 × LXRE-Luc and (B) SREBP-1c-Luc reporter genes and treated with 693.4 μM VPA or/and UA, and luciferase activities were measured after 24 h treatment. Data represent the mean ± SE: n = 4; *, p < 0.05; ***, p < 0.001 (compared with control or T090-treated groups as indicated). (C) Differentiated HepaRG cells were treated with 693.4 μM VPA or/and UA for 24 h. Gene expression levels of SREBP-1c, FAS, SCD, ACC, ACLY, FAE, and β-actin (internal control) by real-time PCR are shown. Data represent the mean ± SE: n = 3; ***, p < 0.001 (compared with control or VPA-treated groups as indicated). (D) Differentiated HepaRG cells were treated with VPA or/and UA, and total lysates were collected after 24 h treatment. Protein expression of LXRα target genes and β-actin was analyzed by Western blotting. Quantitation of the indicated protein bands with 11656


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Journal of Agricultural and Food Chemistry Figure 7. continued

ImageJ software was corrected by β-actin expression. A representative blot shown is shown. (E) Differentiated HepaRG cells were repeatedly exposed to (a) solvent, (b) 693.4 μM VPA, (c) 693.4 μM VPA + 5 μM UA, or (d) 693.4 μM VPA + 10 μM UA for 14 days. Lipid accumulation was then visualized by Oil Red O staining, to detect the present of triglycerides and cholesterol esters, and then cells were observed and photographed under a phase-contrast microscope (original magnification ×400). Oil Red O dye was then extracted by isopropyl alcohol and quantified with a microplate reader at 510 nm. Values represent the mean ± SE: n = 3; ***, p < 0.001 (compared with control or T090-treated groups as indicated).

of differentiated HepaRG cells with UA as described in Figure S1, VPA induced lipid accumulation in these cells, which disappeared in the presence of UA based on Oil Red O staining (Figure 7E). Reduction of Rifampin-Mediated PXR Transactivation, Expression of Lipogenic Genes, and Lipid Contents in Hepatic Cells by Ursolic Acid. RIF, a potent activator of PXR in humans, induces hepatic steatosis in patients with tuberculosis.44 In our previous work, we have found that UA may inhibit cytochrome P450 3A4 (CYP3A4) expression through a PXR-mediated pathway.23 Thus, we analyzed the effects of UA on PXR signaling using its lipogenesis-related target genes, including S14, SCD, FAS, and FAE.12,30 On the basis of the reporter assay, RIF induced the S14 and SCD promoter activity, which was reduced by cotreatment with UA (Figure 8A). This inhibition was confirmed in mRNA (Figure 8B) and protein expression (Figure 8C,D) experiments and resulted in a decreased accumulation of RIF-induced lipid droplets in HepaRG cells (Figure 8E). The results showed that UA changes the transactivation of these NRs, induced by their agonists, suggesting that UA may be an antagonist of both LXRα and PXR.

increase was attenuated by cotreatment with UA (Figure 6C). However, we did not observe inhibitory effects of compound C on the increase of ABCA1 and ABCG1 gene expression in LS174T cells, while cotreatment with UA increased the expression of these RCT genes (Figure 6C), suggesting a mechanism involving another molecule in the regulation of these genes. To explore AMPK activation in both cell lines, cells were exposed to T090 and UA alone or in combination. Our results showed that the phosphorylation of AMPK significantly increased in HepaRG cells and decreased in LS174T cells in the presence of UA (Figure 6D). These observations were consistent with our previous results showing that UA inhibited the LXRα activation in HepaRG cells but not in LS174T cells, suggesting that UA controls LXRα signaling in a cell- and tissue-specific manner. LXR activity is regulated by agonists, antagonists, coactivators (such as SRC-1), and corepressors (such as SMILE).18,19 We performed a DAPA to confirm the recruitment of SRC-1 and SMILE to the SREBP-1c and ABCG1 LXR-binding motifs in HepaRG and LS174T nuclear extracts obtained after T090 and UA treatment. As shown in upper panel of Figure 6E, SRC-1 occupied the SREBP-1c promoter in the T090-treated HepaRG nuclear extract. However, the recruitment of this coactivator decreased in the presence of UA. In contrast, the interaction of SMILE with the SREBP-1c promoter decreased upon T090 activation of LXR but gradually increased with the addition of UA. Interestingly, in LS174T cells, the opposite effects were observed: that is, UA did not promote the recruitment of SMILE to the ABCG1 promoter but increased the binding of SRC-1 to this site (Figure 6E, lower panel). These results suggest that UA differently affects LXRα signaling in hepatocytes and intestinal cells, which may account for its differential effects on the recruitment of coregulators to lipogenic and RCT gene promoters. Reduction of Valproate-Mediated LXRα Transactivation, Expression of Lipogenic Genes, and Lipid Contents in Hepatic Cells by Ursolic Acid. VPA is a widely used medication in the management of epilepsy.42 In spite of its therapeutic benefits, VPA has been reported to cause drug-induced adverse reactions,42 including the development of microvesicular steatosis, in 80% of treated patients.43 In our previous work, we have found that VPA may activate the LXRα transactivation, leading to lipogenesis in hepatocytelike cells.27 Thus, we investigated whether UA may attenuate the effects from VPA-mediated LXRα activation, as well as mRNA and protein expression of lipogenic genes. We found that UA decreased the VPA-induced LXRα-mediated 3 × LXRE and SREBP-1c promoter activity (Figure 7A,B). The VPA-induced expression of a series of LXRα target genes related to the lipogenic process, including SREBP-1c, FAS, SCD, ACC, ACLY, and FAE, was also dose-dependently attenuated by UA (Figure 7C), which was consistent with the protein expression data (Figure 7D). Using repeated treatment

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DISCUSSION In this study, we have provided strong evidence demonstrating that UA is a naturally occurring LXRα and PXR antagonist for inhibition of hepatic steatosis. First, evaluation of promoter activities of several target genes showed that UA inhibited both LXRα and PXR transcriptional activities, induced by T090 and VPA (for LXRα) and RIF (for PXR). UA also inhibited the expression of LXRα and PXR downstream genes in human hepatic HepaRG cells, which was confirmed for LXRα in a mouse model. Second, in silico modeling indicated that UA may compete with the LXRα agonist T090 in binding to the LXRα LBD. In addition, UA enhanced the RCT effects in an intestinal cell line by triggering the recruitment of the SRC-1 coactivator and dissociation of the SMILE corepressor from the ABCG1 LXRE region, along with the inhibition of AMPK phosphorylation. The effects of UA were found to be cell type specific, and the mechanisms involved were different between hepatic and intestinal cells. Thus, we concluded that UA was a novel and potent antagonist of LXRα- and PXR-mediated lipogenesis. UA is a natural pentacyclic triterpenoid carboxylic acid, which has been used in Chinese medicine to treat liver disorders for more than 20 years.45 Several herbs and fruits, such as apple, cranberry, and olive, contain high amounts of UA and provide a number of biological benefits. It has been reported that UA ameliorated high-fat-diet-induced hepatic steatosis and liver injury in combination with rosiglitazone.46 In the LXRE- and SREBP-1c reporter gene assay, UA suppressed the transcriptional activity of LXRα, induced by T090 and VPA. We found that there is no significant change in 11657


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Figure 8. Effects of ursolic acid (UA) on rifampin (RIF)-induced transient transcriptional activation of S14 and SCD reporter activity, mRNA and protein expression of PXR target genes, and lipid contents. (A) HepG2 cells were cotransfected with a PXR expression plasmid and S14-Luc and SCD-Luc reporter genes and treated with 20 μM RIF or/and UA; luciferase activities were measured after 24 h treatment. Data represent the mean ± SE: n = 4; *, p < 0.05; ***, p < 0.001 (compared with control or RIF-treated groups as indicated). (B) Differentiated HepaRG cells were treated with 20 μM RIF or/and UA for 24 h. Real-time PCR results of gene expression levels of S14, FAS, SCD, β-actin (internal control) are given. Data represent the mean ± SE: n = 3; **, p < 0.01; ***, p < 0.001 (compared with control or RIF-treated groups as indicated). (C) Differentiated HepaRG cells treated with RIF or/and UA for 24 h were harvested, and total lysates were collected. Protein expression of PXR target genes and βactin (internal control) was analyzed by Western blotting. (D) Quantitation of the indicated protein bands was corrected by β-actin expression. 11658


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Journal of Agricultural and Food Chemistry Figure 8. continued

The representative blot shown was quantified with ImageJ software. Data represent the mean ± SE: n = 3; *, p < 0.05; ***, p < 0.001 (compared with control or RIF-treated groups as indicated). (E) Differentiated HepaRG cells were repeatedly exposed to (a) solvent, (b) 20 μM RIF, (c) 20 μM RIF + 5 μM UA, or (d) 20 μM RIF + 10 μM UA for 14 days. Lipid accumulation was then visualized by Oil Red O staining to detect the presence of triglycerides and cholesterol esters, and then cells were observed and photographed under a phase-contrast microscope (original magnification ×400). Oil Red O dye was then extracted by isopropyl alcohol and quantified with a microplate reader at 510 nm. Values represent the mean ± SE: n = 3; ***, p < 0.001 (compared with control or RIF-treated groups as indicated).

upregulation of RCT. None of the treatment groups showed significant hepatocellular or kidney damage induced by T090 or UA in combination, on the basis of the serum ALT, BUN, and CREA levels. Consistent with the data of the in vivo study, treatment of differentiated HepaRG cells with T090 induced TG accumulation and the expression of hepatic lipogenesis genes; however, these effects were significantly inhibited by cotreatment with UA. Since LXRα promotes hepatic lipogenesis, efforts to investigate LXRα ligands that antagonize these effects and enhance RCT (thus providing benefits for the prevention of atherosclerosis) are needed. Although we did not confirm the involvement of intestinal and peritoneal macrophages in RCT expression in mice, we believe that UA may increase RCT in mice and decrease the intestinal cholesterol content, on the basis of the serum TC content after stimulation with T090 and cotreatment with UA. Our results were consistent with those of a previous report, which showed that the ABCA1 and ABCG1 mRNA expression increased after T090 stimulation in Caco2 intestinal cells.52 Activation of AMPK inhibited hepatic FA synthesis by reducing the SREBP-1c expression through inhibition of production endogenous LXRα ligands in a rat hepatoma cell model.40 In the presence of AMPK activators, AICAR and metformin, a 25% decrease in the SREBP-1c promoter activity, in comparison with that in the control, was found in this cell model. In the presence of compound C and T090, SREBP-1c and the expression of its downstream genes highly increased in HepaRG cells in comparison with those in the T090-treated group, suggesting that AMPK signaling was involved in these gene induction pathways. However, cotreatment with UA reduced the expression of these genes in comparison with that in the T090 + compound C group. These results indicated that UA might be involved in AMPK signaling, and we confirmed that UA enhanced the AMPK phosphorylation in HepaRG cells. Additionally, we found that the phosphorylation of ACC was also slightly increased by UA treatment; therefore, AMPK signaling was more active in the presence of UA (Figure S6). This finding suggests another mechanism of inhibition of SREBP-1c activation, different from that through LXRα inhibition. SMILE, as a corepressor, represses transactivation of several NRs, including LXRs, through direct interaction, competition with coactivators, or active repression through recruitment of histone deacetylases.18,19 We showed that UA inhibited the T090-induced SRC-1 recruitment to the SREBP-1c promoter but enhanced the binding of SMILE and thus led to the repression of the SREBP-1c gene expression in a hepatic cell model. In contrast, in an intestinal cell model, UA reduced the AMPK phosphorylation and thus decreased the recruitment of SMILE to the ABCG1 promoter region but enhanced the binding of SRC-1. As a consequence, expression of RCT genes and excretion of cellular cholesterol increased. Thus, the increase in SMILE binding may reduce hepatic steatosis caused by LXRα-induced lipid accumulation. However, we may not

mRNA and protein expression of these NRs during our drug treatment (Figures S4 and S5), suggesting that UA inhibits LXR transactivation but not through these upstream control elements. UA also inhibited ligand-induced lipid accumulation, as well as the mRNA and protein expression of lipogenic genes, both in vitro and in vivo. As shown in our previous study, UA is also an antagonist of PXR. We found that UA also inhibited the RIF-induced expression of lipogenic genes and thus decreased the lipid accumulation in a hepatic cell line. Several naturally occurring LXR antagonists have been explored in recent studies, which showed that these compounds might have some potential utility as therapeutic agents against hypertriglyceridemia and reduce the incidence of hepatic steatosis; these were reviewed by Komati et al.47 Here, we report UA as a new LXRα antagonist and lipogenesis inhibitor in hepatic cells and the liver. NRs are involved in a wide range of pathophysiological conditions and are thus always considered potential drug targets. Previous reports have shown that patients with NAFLD, in comparison with the general population, had a greater incidence of hypertriglyceridemia (44% vs 27%), hypercholesterolemia (54% vs 44%), and hyperglycemia (7% vs 4%) and decreased HDL cholesterol (15% vs 8%).48 In Taiwan, NAFLD shows a prevalence of approximately 25−37% on the basis of medical checkup reports. Furthermore, it was estimated that there are 20 million NAFLD patients worldwide.8,49 The level of serum TGs is positively correlated with the severity of fatty liver disease.50 NAFLD is highly associated with a high body mass index, diabetes, hyperlipidemia, and hypertension, representing hepatic demonstration of metabolic syndrome, considered a harmful that needs to be given an appropriate management. Activation of LXRs by agonists have been reported to induce NAFLD and hypertriglyceridemia.28 In comparison to LXR+/+ mice, LXR−/− mice have lower levels of TGs and increased levels of HDL cholesterol.38 Thus, suppression of the LXR activity may be beneficial for the cure of NAFLD, as we confirmed in an in vivo model. Since LXRα is a main regulator of lipid homeostasis, accumulation of intracellular oxysterols induces LXRα transactivation and results in hepatic lipogenesis and RCT via induction of the expression of related genes.36 T090 stimulates the hepatic free FA uptake and hepatic lipogenesis by induction of cluster of dif ferentiation 36 (CD36) and SREBP1c, respectively, thus causing hepatic steatosis and hypertriglyceridemia.7 Cholesterol may be metabolized to bile acids in the liver or directly excreted into the bile, and LXRα promotes intestinal cholesterol excretion and decreases cholesterol absorption.51 We found an increase in hepatic and serum TGs after T090 administration for 7 days, which was associated with the stimulation of hepatic lipogenic genes. However, these effects were ameliorated by coadministration of UA, resulting in a significant reduction of lipogenesis and hepatic steatosis. Serum cholesterol was higher in the TU100 and TU250 groups than in the control, likely due to the 11659


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also be helpful in the development of LXRα agonists that effectively prevent atherosclerosis.

rule out that UA increases the phosphorylation of AMPK and recruitment of SMILE and also disrupts the interaction between LXRα and RXR. T090 has been commonly used in experimental studies for the activation of both LXRs, especially LXRα.47 However, it is not a specific LXR ligand since it also induces the transactivation of PXR.53 In our study, we transfected into cells an expression vector with a full-length human LXRα and found that T090 significantly induced the promoter activity of both 3 × LXRE and SREBP-1c, but these effects were attenuated by the addition of UA, suggesting that UA is an LXRα antagonist. We also performed RIF-induced PXRmediated S14 and SCD promoter assays and found that UA also represented an effective PXR antagonist, which was consistent with our previous findings.23 The ligand-binding pocket of LXRα can accommodate a wide range of compounds with diverse shapes, structures, and volumes.54 Through virtual docking, we identified several amino acid residues that could interact with UA but were different from those in the T090binding site of LXRα. T090 binds to six amino acid residues of LXRα, while UA forms 12 hydrogen bonds with amino acid residues, among which six residues are the same as those in the T090−LXRα complex. The results show that UA may compete with T090 binding to LXRα. Since the liver is the main drug/xenobiotic metabolism factory of the body, it is highly susceptible to drug toxicity. Drug-induced steatosis is a significant issue for the pharmaceutical industry and can lead to discontinuation of the development of a new drug or even its withdrawal from the market.55 Antiepileptic drugs show many serious adverse reactions, due to their hepatotoxic effects.42 As reported in our previous study, VPA, which is used in the treatment of epilepsy, induces lipid accumulation in hepatic cells, partly via the activation of the LXRα signaling pathway.27 Although the typical target gene of PXR is CYP3A4, the spectrum of PXR targets has expanded to genes involved in energy homeostasis, such as S14, SCD, FAE, and ACLY.11,56 PXR activation by RIF was shown to worsen steatosis, obesity, and insulin resistance because of increased hepatic FA uptake and lipid synthesis and decreased β-oxidation.15 This study reports a prominent protection by UA against drug-induced hepatic steatosis by inhibiting VPA-induced LXRα and RIF-induced PXR activation. In summary, we demonstrated that UA is a novel LXRα antagonist, displaying efficacy in the reduction of hepatic steatosis pathology in vitro and in a mouse model of T090induced steatosis through modulation of the hepatic LXRαmediated pathway, including SREBP-1c and a battery of downstream target genes. UA reduces cellular and hepatic lipid contents and decreases blood lipids, partially via AMPK activation. Moreover, UA induces RCT in the presence of T090 in intestinal cells through downregulation of AMPK phosphorylation and induces the recruitment of the coactivator SRC-1 but decreases that of the corepressor SMILE to the ABCG1 promoter region, resulting in a decrease in the cellular lipid content. Furthermore, UA reduces the VPA-induced LXRα-mediated and RIF-induced PXR-mediated lipogenesis and is thus potentially beneficial for drug-induced hepatic steatosis. Finally, UA displays liver specificity on lipogenesis repression while preserving and enhancing the RCT stimulation by LXRα. Thus, UA may not only offer a novel and promising therapeutic option to treat NAFLD but may

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04116. Figures S1−S6 as described in the text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Y.-P.L: tel, +886-4-2205-3366 ext. 5802; fax, +886-4-22078083; e-mail, [email protected], [email protected]. tw. ORCID

Yun-Ping Lim: 0000-0001-9312-048X Author Contributions ●

Y.-N.L., C.C.N.W., and H.Y.-C. contributed equally to this work. Funding

This study was supported by the Ministry of Science and Technology, Taiwan, ROC (MOST107-2320-B-039-042MY3), China Medical University, Taichung, Taiwan (CMU106-ASIA-22), partially supported by the Taiwan Ministry of Health and Welfare, Taiwan (MOHW107-TDUB-21-−123004), China Medical University Hospital, Academia Sinica Stroke Biosignature Project (BM10701010021), and MOST Clinical Trial Consortium for Stroke (MOST1062321-B-039-005). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor David J. Mangelsdorf (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA), Professor Marta Casado (Instituto de Biomedicina de Valencia, IBV-CSIC, Jaime Roig 11, 46010 Valencia, Spain), Professor Yonggong Zhai (College of Life Sciences, Beijing Normal University, Beijing, China), and Professor Youfei Guan (Department of Physiology, Dalian Medical University, China) for providing the LXRα and the reporter constructs.

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ABBREVIATIONS ABC,ATP-binding cassette; ACC,acetyl-CoA carboxylase; ACLY,ATP-citrate lyase; AEDs,antiepileptic drugs; AF-2,activation function 2; AICAR,5-aminoimidazole-4-carboxamide ribonucleotide; AMPK,AMP-activated protein kinase; DAPA,DNA affinity precipitation assay; DMSO,dimethyl sulfoxide; DR4,direct repeat 4; DS,Discovery Studio; FA,fatty acid; FAE,fatty acid elongase; FAS,fatty acid synthase; HDLC,high-density lipoprotein cholesterol; HMGCR,3-hydroxy-3methyl-glutaryl-CoA reductase; LBD,ligand-binding domain; LXRα,liver X receptor alpha; LXRE,liver X receptor response element; NAFLD,nonalcoholic fatty liver disease; NASH,nonalcoholic steatohepatitis; NR,nuclear receptor; OA,oleanolic acid; PDB,Protein Data Bank; PXR,pregnane X receptor; RCT,reverse cholesterol transport; RIF,rifampin; RXR,retinoid X receptor; S14,thyroid hormone responsive spot 14; SCD,stearoyl-CoA desaturase-1; SMILE,small heterodimer partner-interacting leucine zipper protein; SRC-1,steroid 11660


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receptor coactivator-1; SREBP-1c,sterol regulatory element binding protein-1c; T090,T0901317; TC,total cholesterol; TGs,triglycerides; VPA,valproate

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Ursolic Acid, a Novel Liver X Receptor Î± (LXRÎ±) Antagonist Inhibiting

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