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Journal of Pharmacological Sciences 128 (2015) 71e77

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20(S)-protopanaxatriol inhibits liver X receptor a-mediatedexpression of lipogenic genes in hepatocytes

Gyun-Sik Oh a, b, Jin Yoon a, b, Gang Gu Lee a, b, Won Keun Oh c, Seung-Whan Kim a, b, *

a Department of Pharmacology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Koreab Bio-medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Koreac Korea Bioactive Natural Material Bank, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

a r t i c l e i n f o

Article history:Received 2 February 2015Received in revised form23 April 2015Accepted 20 May 2015Available online 29 May 2015

Keywords:20(S)-protopanaxatriolLiver X receptor aSterol-regulatory element binding protein-1cLipogenesisSteatosis

* Corresponding author. Department of PharmacUniversity of Ulsan College of Medicine, Seoul 138-7362 3010 4297; fax: þ82 2 3010 2941.

E-mail address: swkim7@amc.seoul.kr (S.-W. Kim)Peer review under responsibility of Japanese Pha

http://dx.doi.org/10.1016/j.jphs.2015.05.0071347-8613/© 2015 The Authors. Production and hostinlicense (http://creativecommons.org/licenses/by-nc-n

a b s t r a c t

20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has severalinteresting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT wasidentified as an inhibitor against the ligand-dependent transactivation of liver X receptor a (LXRa) usinga Gal4-TK-luciferase reporter system. LXRa is a transcription factor of nuclear hormone receptor familyand stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesteroltransport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRa-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part,a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) ofthe SREBP-1c promoter. Furthermore, LXRa-dependent triglyceride accumulation in primary mousehepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRa-dependenttranscription of ABCA1, a crucial LXRa target gene involved in RCT. Chromatin immunoprecipitationassays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1cLXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activityagainst LXRa-mediated lipogenesis, but not LXRa-stimulated RCT.

© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

1. Introduction

Liver X receptor a (LXRa) is a transcription factor of nuclearreceptor superfamily and regulates the expression of many lipo-genic genes, including sterol regulatory element binding protein-1c(SREBP-1c), fatty acid synthase (FAS), and stearoyl CoA desaturase 1(SCD1) (1e5). Because of the increased expression of these genes,treatment of mice with LXR agonists, such as T0901317 or GW3965,can induce a fatty liver. Moreover, SREBP-1c, one of the LXRa targetgenes, is a transcription factor of the basic-helix-loop-helix-leucinezipper family and is known to be a master regulator of the lipogenicpathway (6). Therefore, LXRa is considered to be a crucial target forthe development of anti-lipogenic agents. On the other hand, LXRaalso stimulates the expression of reverse cholesterol transport

ology, Asan Medical Center,, Republic of Korea. Tel.: þ82

.rmacological Society.

g by Elsevier B.V. on behalf of Japad/4.0/).

(RCT)-related genes, such as ATP-binding cassette transporter A1(ABCA1) and G1 (ABCG1), which contribute to the increase ofHDL-cholesterol levels and are beneficial for the treatment ofatherosclerosis (7, 8).

For more than 2000 years, the root of Panax ginseng C. A. Meyer(Family Araliaceae) has been used as a panacea because of its tonicfunctions. For metabolic diseases, ginseng and its constituent gin-senosides have been shown to exert therapeutic effects in severaldiseasemodels of diabetesmellitus and obesity (9e16), as well as indiabetic patients (17). However, the specific active constituents andtheir underlying action mechanisms remain largely unknown.

Ginsenosides are a special group of triterpenoid saponins foundexclusively in ginseng and can be classified based on their agly-cones (nonsugar components) into two groups: 20(S)-proto-panaxadiol (PPD)-type and 20(S)-protopanaxatriol (PPT)-typeginsenosides. The ginsenosides Rb1, Rb2, Rc, Rd, and Rh2 are thePPD-type ginsenosides, while the PPT-type ginsenosides includeRe, Rf, Rg1, Rg2, and Rh1. Both the PPD- and PPT-type ginsenosidescan be deglycosylated by gastric acid and/or intestinal bacteria,

nese Pharmacological Society. This is an open access article under the CC BY-NC-ND

G.-S. Oh et al. / Journal of Pharmacological Sciences 128 (2015) 71e7772

thereby leading to the formation of secondary metabolites andaglycones.

In our current study, the aglycone PPT was identified as aninhibitor of autonomous LXRa transactivation using a Gal4-UASscreening system. Previous studies by other researchers haverevealed several interesting activities of PPT. Oh et al. reported thatPPT reduces the expression levels of inducible nitric oxide synthaseand cyclooxygenase-2 through the inactivation of NFkB by pre-venting IkBa phosphorylation in RAW 264.7 macrophages stimu-latedwith lipopolysaccharide (18). Kwok et al. have shown that PPTprotects endothelial cells from hydrogen peroxide-induced celldeath (19). Han et al. reported that PPT increases the trans-activation activity of peroxisome proliferator-activated receptor g(PPARg) in a dose-dependent manner and enhances adipogenesisby increasing the expression of PPARg target genes, such as fattyacid binding protein 4, lipoprotein lipase, and phosphoenolpyr-uvate carboxykinase (20). However, no study has yet examined theeffect of PPT on LXRa-mediated lipogenesis.

Our present study aimed to identify an anti-lipogenic compoundbased on the inhibition of LXRa transactivation and to furtherinvestigate the mechanism of action of such a compound at thetranscriptional level. Our data show that PPT is a novel LXRainhibitor with selective effects on lipogenic process. Interestingly,PPT did not inhibit the transcription of LXRa target gene related toRCT process. We suggest a possible mechanism for the differentialinhibition of the lipogenesis and RCT pathways by PPT.

2. Materials and methods

2.1. Reagents and antibodies

Phytochemicals, including PPT, were provided by Dr. Won KeunOh (Korea Bioactive Natural Material Bank, Seoul National Univer-sity, Seoul, Korea). T0901317 and dimethyl sulfoxide (DMSO) werepurchased from SigmaeAldrich (St. Louis, MO, USA). Percoll wasobtained from GE Healthcare (Pittsburgh, PA, USA). M199 mediumwas from Gibco (Grand Island, NY, USA). Dulbecco's Modified Ea-gle's Medium (DMEM) and fetal bovine serum (FBS) were obtainedfrom Hyclone (Logan, UT, USA). The primers used for PCR weresynthesized by Genotech (Daejeon, Korea). Rabbit anti-RNA poly-merase II (sc-900X) and goat anti-TRAP80 (sc-12453) antibodieswere obtained from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Rabbit anti-IgG control was purchased from Abcam(Cambridge, UK).

2.2. Isolation of mouse primary hepatocytes and cell culture

Mouse primary hepatocytes were isolated from 8 to 10-week-old male C57BL/6 mice (Orient Bio, Seongnam, Korea) using apreviously described collagenase perfusion method with minormodifications (21). Briefly, livers were perfused with Mg2þ/Ca2þ-free Hanks' balanced salt solution (SigmaeAldrich) containing100 U/mL collagenase type II (Gibco) and 48 mg/mL trypsin inhibitor(SigmaeAldrich). Next, viable hepatocytes were collected usingPercoll density gradient centrifugation after filtering liver cellsthrough a 100-mm nylon mesh strainer (BD Biosciences, San Jose,CA, USA). Isolated primary hepatocytes were plated onto collagentype I-coated culture dishes and maintained in M199 media. Hu-man hepatoma HepG2 cells were maintained in DMEM containing10% FBS at 37 �C in a humidified 5% CO2 incubator. All animalprotocols were approved by the Institutional Animal Care and UseCommittee (IACUC) of the Asan Institute for Life Sciences, AsanMedical Center (Approval No. 2013e12-168). Mice were main-tained in a temperature-controlled facility with a 12 h light/12 hdark cycle and were provided ad libitum access towater and regular

rodent chow diet. Following acclimatization for 1 week, mice werehumanely euthanized for the preparation of primary hepatocytes.

2.3. Transient transfection and luciferase reporter assays

HepG2 cells were seeded in 24-well plates 24 h prior to trans-fection and were grown in DMEM supplemented with 10%charcoal-stripped FBS. Transfections were performed with Gal4-TK-luciferase, Gal4-hLXRa ligand binding domain (LBD), SREBP-1cLXRE (LXR response element)-luciferase, or ABCA1 LXRE-luciferase constructs using Lipofectamine 2000 reagent (Invi-trogen, Grand Island, NY, USA) according to the manufacturer'sprotocol. After 24 h, cells were treated with 1 mM T0901317 and10 mg/mL phytochemicals for 18 h. Luciferase activity was thenmeasured using a Centro LB 960 luminometer (Berthold Technol-ogy, Bad Wildbad, Germany), and enzyme activity values werenormalized to those of b-galactosidase. For normalization, pActin-bgal plasmids were co-transfected into HepG2 cells along with theplasmids containing promoter-luciferase reporter genes.

2.4. Analysis of transcript levels by conventional or quantitativeRT-PCR

Total RNAwas extracted from primary hepatocytes treated with1 mM T0901317 and 10 mg/mL PPT using TRIzol-RNA Lysis Reagent(Invitrogen) according to the manufacturer's instructions. PurifiedRNA was reverse transcribed by M-MLV reverse transcriptase(Promega, Madison, WI, USA). Next, specific mRNA levels weremeasured by conventional or quantitative RT-PCR. The oligonu-cleotide primers used for conventional RT-PCR were as follows:SREBP-1c, 5ʹ-GGC GCA TGG ATT GCA CAT TT-3ʹ and 5ʹ-GCA GGC TGTAGG ATG GTG A-3ʹ; and 18S rRNA, 5ʹ-CGT CCC CCA ACT TCT TAGAG-3ʹ and 5ʹ-CAC CTA CGG AAA CCT TGT TAC-3ʹ. For conventionalRT-PCR, 18S rRNA was used as an internal control. Quantitative RT-PCR analyses were performed on a LightCycler 480 system (Roche,Basel, Switzerland). The following oligonucleotide primers wereused for quantitative RT-PCR: SREBP-1c, 5ʹ-CAG CCA TGG ATT GCACAT TTG-3ʹ and 5ʹ-GTC TTG GTT GTT GAT GAG CTG G-3ʹ; SCD1, 5ʹ-AGC TCAGTC TCA CTC CTT CCC TTA-3ʹ and 5ʹ-CAG CCAGCC TCT TGACTA TTC C-3ʹ; FAS, 5ʹ-CTG CAG CTG TCA GTG TGA AGA AG-3ʹ and 5ʹ-GCA GCA TTT TTA CCA GGT TGG T-3ʹ; Glycerol-3-phosphate acyl-transferase (GPAT), 5ʹ-TCA TCC AGT ATG GCA TTC TCA CA-3ʹ and5ʹ-GCA AGG CCA GGA CTG ACA TC-3ʹ; ABCA1, 5ʹ-GAC CCG TAC TCTCGC AGG G-3ʹ and 5ʹ-CCT TGC CGG TAT TTT AGC AGG-3ʹ; andribosomal protein S29 (RPS29), 5ʹ-CGC AAA TAC GGG CTG AAC A-3ʹand 5ʹ-GCC TAT GTC CTT CGC GTA CTG-3ʹ. RPS29 was used as aninternal control for quantitative RT-PCR.

2.5. Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed in primary hepatocytes treatedwith 1 mM T0901317 and 10 mg/mL PPT for 30 min, as describedpreviously, with minor modifications (22). Briefly, cells were fixedwith 1% formalin (SigmaeAldrich) for 20 min and then quenchedwith 0.125 M glycine for 5 min at room temperature. Cells werewashed twice with cold PBS and lysed in SDS lysis buffer con-taining 50 mM TriseHCl (pH 8.0), 10 mM EDTA, and 1% SDS.Soluble chromatin was prepared by sonication (VCX-600Sonicator, Sonics & Materials, Newton, CT, USA) and then was pre-cleared using protein G-agarose beads (Santa Cruz Biotechnology).Pre-cleared supernatants were then immunoprecipitated withanti-RNA polymerase II (Santa Cruz Biotechnology), anti-TRAP80(Santa Cruz Biotechnology), or IgG antibodies (Abcam) for12 h at 4 �C. The remaining DNA was extracted from the immu-noprecipitate and analyzed by quantitative RT-PCR using the

Fig. 1. Effects of ginsenosides and their aglycones on (A) the autonomous trans-activation of Gal4-LXRa LBD and (B) LXRa agonist-stimulated increases of SREBP-1ctranscript levels. (A) Gal4-LXRa LBD transactivity was measured in the presence of1 mM T0901317 and 10 mg/mL ginsenosides or their aglycones using a Gal4-TK-lucif-erase assay (n ¼ 6). (B) The effects of ginsenosides and their aglycones on T0901317-stimulated SREBP-1c mRNA levels were determined in primary hepatocytes byconventional RT-PCR (n ¼ 3). Band intensities were quantified with the ImageJ pro-gram. Data are presented as means ± SD. T1317, T0901317; PPT, 20(S)-proto-panaxatriol; Re, ginsenoside Re; Rg2, 20(S)-ginsenoside Rg2; Rh1, 20(R)-ginsenosideRh1; Rd, ginsenoside Rd; PPD, 20(S)-protopanaxadiol. *, P < 0.05 vs. without T1317; **,P < 0.01 vs. without T1317; ***, P < 0.001 vs. without T1317; #, P < 0.05 vs. T1317 alone;##, P < 0.01 vs. T1317 alone; ###, P < 0.001 vs. T1317 alone.

G.-S. Oh et al. / Journal of Pharmacological Sciences 128 (2015) 71e77 73

LightCycler 480 system (Roche). The oligonucleotide primers usedfor the ChIP assay were as follows: SREBP-1c LXRE region, 5ʹ-AGGCTC TTT TCG GGG ATG G-3ʹ and 5ʹ-TGG GGT TAC TGG CGG TCAC-3ʹ; and ABCA1 LXRE region, 5ʹ-GGG GAA AGA GGG AGA GAACAG-3ʹ and 5ʹ-GAA TTA CTG GTT TTT GCC GC-3ʹ. Immunoprecipi-tated DNA levels were presented as fold enrichments normalizedto 10% input DNA levels.

2.6. Cellular triglyceride (TG) accumulation

Primary hepatocytes were seeded onto 60 mm collagen-coatedculture dishes and incubated overnight in M199 medium. Aftertreatment with 1 mM T0901317 and 10 mg/mL PPT for 24 h, cellswere washed twice with cold phosphate buffered saline (PBS) andlysed in PBS containing 1% Triton X-100 for 30 min at 4 �C. Lysateswere centrifuged at 10,000 � g for 10 min at 4 �C. Cellular lipidswere then extracted from the supernatant using the Bligh and Dyermethod (23). TG levels were measured using a TG assay kit (AsanPharmaceutical, Gyeonggi-do, Korea) and normalized to total pro-tein levels.

2.7. Statistical analyses

Results are presented as means ± SD. Data were analyzed usingSPSS version 19 (SPSS/IBM, Chicago, IL, USA). Statistical analyseswere performed using one-way analysis of variance (ANOVA).Bonferroni multiple comparison tests were conducted for post-hocanalysis. Differences with P values <0.05 were considered to bestatistically significant. All experiments were repeated at leastthree times.

3. Results

3.1. PPT inhibits the autonomous transactivation of Gal4-LXRa LBDand the T0901317-dependent transcription of SREBP-1c

A Gal4/UAS reporter assay was used to determine the effectsof ginsenosides and their aglycones (PPT and PPD) on thetransactivation of Gal4-LXRa LBD. Among many phytochemicalsof ginseng, PPT and 20(R)-ginsenoside Rh1 exerted marked in-hibition of the T0901317-dependent transactivation of Gal4-LXRaLBD (Fig. 1A). Because SREBP-1c is a master regulator of lipo-genesis and is also a major LXRa target gene, we investigatedthe effects of PPT and other ginsenosides on the T0901317-dependent stimulation of SREBP-1c transcription to estimatethe anti-lipogenic activities of these compounds. ConventionalRT-PCR analysis showed that PPT markedly suppressed theLXRa-dependent transcription of SREBP-1c (Fig. 1B). The inhibi-tory effect of 20(R)-ginsenoside Rh1 on T0901317-dependenttranscription of SREBP-1c was not statistically significant(Fig. 1B).

3.2. PPT inhibits the T0901317-dependent transactivation of theSREBP-1c promoter and the recruitment of RNA polymerase II to theLXRE region of SREBP-1c

To elucidate the mechanism of transcriptional inhibition ofSREBP-1c by PPT, we examined the effect of PPT on the activity ofSREBP-1c LXRE luciferase (a luciferase reporter containing SREBP-1c LXRE promoter region). Importantly, PPT suppressed theT0901317-dependent stimulation of SREBP-1c LXRE luciferaseactivity (Fig. 2A). The recruitment of RNA polymerase II to theendogenous LXRE of the SREBP-1c gene was then assessed using aChIP assay. Interestingly, the T0901317-dependent recruitment ofRNA polymerase II to the LXRE region of the SREBP-1c promoter

was abrogated by PPT treatment (Fig. 2B). These results suggest thatPPT directly regulates the transcription of SREBP-1c by modulatingthe recruitment of RNA polymerase II.

3.3. PPT inhibits T0901317-dependent transcription of LXRa targetgenes related to lipogenesis, and reduces T0901317-induced TGaccumulation in primary hepatocytes

Because both LXRa and SREBP-1c are transcription factors thatcan stimulate the transcription of many lipogenic enzymes, wetested whether PPT could influence the expression of other lipo-genic genes, such as SCD1, FAS, and GPAT. SREBP-1c, SCD1, and FASare LXRa target genes, while SCD1, FAS, and GPAT are SREBP-1ctarget genes. Quantitative RT-PCR analysis revealed that PPT sup-pressed the T0901317-dependent transcription of SREBP-1c, SCD1,FAS, and GPAT in primary mouse hepatocytes (Fig. 3A). TheT0901317-dependent inductions of lipogenic SREBP-1c and FASproteins were also decreased by PPT in primary mouse hepatocytes(Fig. 3B). Subsequently, we investigated the effect of PPT on cellularTG accumulation induced by T0901317 in primary hepatocytes.Importantly, T0901317-induced TG accumulation in hepatocyteswas significantly reduced by PPT treatment (Fig. 3C). Together,these data indicate that the inhibitory activity of PPT against LXRatransactivation can result in reduced expression of lipogenic genesand ultimately the suppression of TG accumulation in hepatocytes.

Fig. 3. Effects of PPT on (A, B) T0901317-dependent increases of lipogenic gene transcript and protein levels and (C) cellular TG accumulation in mouse primary hepatocytes.(A) T0901317-induced transcription of SREBP-1c, SCD1, FAS, and GPAT was determined in the presence or absence of PPT using qRT-PCR (n ¼ 4). (B) The effects of PPT on T0901317-stimulated expression of lipogenic proteins were determined by immunoblot analysis. (C) TG accumulation in response to co-treatment with T0901317 and PPT. Intracellular TGlevels were measured enzymatically after treatment of primary hepatocytes with 1 mM T0901317 and 10 mg/mL PPT and normalized to total protein levels (n ¼ 4). Data are presentedas means ± SD. DMSO, dimethyl sulfoxide; T1317, T0901317. *, P < 0.05 vs. DMSO; **, P < 0.01 vs. DMSO; ***, P < 0.001 vs. DMSO; #, P < 0.05 vs. T1317; ##, P < 0.01 vs. T1317; ###,P < 0.001 vs. T1317.

Fig. 2. Effects of PPT on (A) the transcriptional activity of the SREBP-1c promoter and (B) the T0901317-dependent recruitment of RNA polymerase II to the LXRE region of theSREBP-1c gene. (A) LXRa-mediated transcriptional activity was determined using SREBP-1c LXRE-luciferase reporters in the presence of 1 mM T0901317 and 10 mg/mL PPT inHepG2 cells (n ¼ 8). Luciferase activity was normalized to cotransfected b-galactosidase activity. (B) After treatment of primary hepatocytes with 1 mM T0901317 and 10 mg/mLPPT, ChIP assays were performed using anti-RNA polymerase II or IgG antibodies (n ¼ 4). Immunoprecipitated LXRE-containing DNA levels were determined by qRT-PCR andnormalized to input DNA. Data are presented as means ± SD. DMSO, dimethyl sulfoxide; T1317, T0901317. **, P < 0.01 vs. DMSO; ***, P < 0.001 vs. DMSO; #, P < 0.05 vs. T1317;###, P < 0.001 vs. T1317.

G.-S. Oh et al. / Journal of Pharmacological Sciences 128 (2015) 71e7774

3.4. PPT does not affect the transcription of ABCA1, an LXRatarget gene

LXRa regulates not only lipogenic genes, but also RCT-relatedgenes, such as ABCA1 (7, 24). Moreover, RCT is considered to be abeneficial process for maintaining high HDL-cholesterol levels,while lipogenesis is thought to be potentially harmful. Therefore,

we investigated the effect of PPT on ABCA1 transcription. Inter-estingly, PPT did not inhibit the promoter activity of ABCA1 in aluciferase reporter system (Fig. 4A). T0901317-dependent increasesin ABCA1 transcript and protein levels were not repressed by PPT(Fig. 4: B and C). Furthermore, PPT did not affect the T0901317-stimulated recruitment of RNA polymerase II to the ABCA1 LXREregion (Fig. 4D). These data indicate that PPT differentially affects

Fig. 4. Effects of PPT on (A) the LXRa-mediated promoter activity of ABCA1-Luc, (B) the mRNA transcript levels of ABCA1, (C) the protein levels of ABCA1 and (D) the recruitment ofRNA polymerase II to the LXRE region of the ABCA1 gene. (A) The activity of ABCA1 LXRE-luciferase was determined, following treatment with 1 mM T0901317 and 10 mg/mL PPT, inHepG2 cells and normalized to cotransfected b-galactosidase activity (n ¼ 8). (B) The mRNA levels of ABCA1 were measured in primary hepatocytes treated with 1 mM T0901317 and10 mg/mL PPT using qRT-PCR (n ¼ 4). (C) The protein levels of ABCA1 were determined in the presence or absence of T0901317 and/or PPT using immunoblot analysis. (D) ChIPassays were performed using anti-RNA polymerase II or IgG antibodies after the treatment of primary hepatocytes with 1 mM T0901317 and 10 mg/mL PPT (n ¼ 4). Levels ofimmunoprecipitated LXRE-containing DNA were determined by qRT-PCR and normalized to input DNA. Data are presented as means ± SD. DMSO, dimethyl sulfoxide; T1317,T0901317. **, P < 0.01 vs. DMSO; ***, P < 0.001 vs. DMSO.

G.-S. Oh et al. / Journal of Pharmacological Sciences 128 (2015) 71e77 75

the expression levels of SREBP-1c and ABCA1, even though bothSREBP-1c and ABCA1 have LXREs in their promoters and are well-known LXRa target genes.

3.5. Recruitment of the coactivator TRAP80 to the SREBP-1cpromoter can be inhibited by PPT

Recently, TRAP80 was reported to be a lipogenic coactivator inhepatocytes (25). TRAP80 is selectively recruited to the SREBP-1cpromoter, but not the ABCA1 promoter. Herein, we investigatedwhether PPT has any effect on the recruitment of TRAP80 to the

Fig. 5. Recruitment of the lipogenic coactivator TRAP80 to the LXRE regions of SREBP-1c (A)mL PPT, ChIP assays were performed using anti-TRAP80 or IgG antibodies (n ¼ 4). Levels of iby qRT-PCR and normalized to input DNA. Data are presented as means ± SD. DMSO, dime

SREBP-1c promoter using ChIP assay. Surprisingly, PPT inhibitedthe T0901317-dependent recruitment of TRAP80 to the LXREregion of the endogenous SREBP-1c promoter (Fig. 5A). Bycontrast, TRAP80 was not recruited to ABCA1 LXRE regardless ofthe presence or absence of T0901317 and/or PPT in primarymouse hepatocytes (Fig. 5B). Consistent with the ChIP assay,the interaction between LXRa and TRAP80 was shown to beinhibited by PPT by mammalian two hybrid system (Fig. 6A).Furthermore, TRAP80 overexpression derepressed the PPT-mediated inhibition of SREBP-1c promoter activity and tran-script level (Fig. 6: B and C).

and ABCA1 (B). After treatment of primary hepatocytes with 1 mM T0901317 and 10 mg/mmunoprecipitated SREBP-1c LXRE- or ABCA1 LXRE-containing DNA were determinedthyl sulfoxide; T1317, T0901317. *, P < 0.05 vs. DMSO; ##, P < 0.01 vs. T1317.

Fig. 6. Derepression of inhibitory PPT effects on T0901317-dependent activation of SREBP-1c by TRAP80 overexpression. (A) Mammalian two hybrid assay was performed betweenGal4-TRAP80 and VP16-hLXRa LBD in the presence or absence of PPT using a Gal4-TK-luciferase assay (n ¼ 9). Luciferase activity was normalized to cotransfected b-galactosidaseactivity. (B, C) The effects of TRAP80 overexpression on the inhibition of (B) SREBP-1c promoter activity (n ¼ 6) and (C) SREBP-1c transcript levels (n ¼ 3) by PPT. Increasing amountsof TRAP80 cDNA (50, 100, and 200 ng DNA) were transfected into HepG2 cells and primary hepatocytes for each experiment (B) and (C), respectively. Data are presented asmeans ± SD. DMSO, dimethyl sulfoxide; T1317, T0901317. **, P < 0.01 vs. DMSO; ***, P < 0.001 vs. DMSO; #, P < 0.05 vs. T1317; ##, P < 0.01 vs. T1317; ###, P < 0.001 vs. T1317.

G.-S. Oh et al. / Journal of Pharmacological Sciences 128 (2015) 71e7776

4. Discussion

Our current study aimed to identify phytochemicals with anti-lipogenic activity from P. ginseng by screening for Gal4-LXRainhibitors. Excessive hepatic lipogenesis is one of the major path-ogenic pathways in the development of non-alcoholic fatty liverdisease (NAFLD). Fat accumulation in hepatocytes is caused byincreased fatty acid uptake, increased de novo lipogenesis, impairedfatty acid oxidation, and reduced secretion of very low densitylipoprotein (26). Among these pathogenic pathways, de novo lipo-genesis reportedly contributes to ~26% of the hepatic fat accumu-lation in NAFLD patients, whereas fatty acid uptake by diet onlycontributes to ~15% (27). There is currently no satisfactory agent tocontrol excess lipogenesis for the treatment of NAFLD. Herein, weshow that PPT reduces LXRa-mediated fat accumulation in primaryhepatocytes.

PPT is an aglycone of the PPT-type ginsenosides, which includethe ginsenosides Re, Rf, Rg1, Rg2, and Rh1. The ginsenoside Re hasbeen reported to inhibit the transcription of SREBP-1c through theactivation of AMP-activated protein kinase in HepG2 cells (28).Gu et al. showed that the ginsenoside Rh1, another PPT-type gin-senoside, reduces the expression of PPARg, CCAAT/enhancer-binding proteina, FAS, and adipocyte fatty acidebinding protein,and inhibits adipocyte differentiation in 3T3-L1 cells (14). However,to date, no study of ginsenosides related to LXRa-mediated lipo-genesis has been reported. Herein, the ginsenoside Rh1 was foundto exert inhibitory activity against the autonomous transactivationof LXRa, but could not significantly inhibit LXRa-mediated tran-scription of SREBP-1c. Importantly, aglycone PPT significantlyinhibited both the autonomous transactivation of LXRa and LXRa-stimulated transcription of SREBP-1c. Notably, PPD, an aglycone ofthe PPD-type ginsenosides, did not inhibit LXRa-related activities.

Recently, Zhang et al. reported that PPT is a novel PPARgantagonist that can inhibit rosiglitazone-enhanced adipocyte dif-ferentiation of 3T3-L1 cells by repressing the expression oflipogenesis-related genes (29). They also showed that PPT allevi-ated steatosis and improved serum lipid profile in high-fat diet-induced obesity mice and ob/ob mice (29). In that same study, theauthors reported that PPT did not inhibit LXRa transactivation, butthey did not present LXRa-related data. They employed a Gal4-UASsystem in HEK293T cells to test the effects of PPT on the trans-activation of several nuclear hormone receptors, including PPARa,

PPARg, and PPARd. Whereas we used the Gal4-UAS system inHepG2 hepatoma cells to determine the effect of PPT on LXRatransactivation. We then confirmed the effects of PPT using quan-titative RT-PCR analysis of LXRa target gene expression levels, aChIP assay for RNA polymerase II, and measurements of cellularlipid accumulation in primary mouse hepatocytes.

Interestingly, we found that PPT can inhibit the recruitment ofTRAP80 to the LXRE region of the SREBP-1c promoter. TRAP80 waspreviously identified as an LXRa- interacting protein using HepG2nuclear extracts with GST-LXRa LBD as bait (25). TRAP80 canstimulate the transcription of lipogenic genes, including SREBP-1c,but does not affect the transcription of RCT-related genes, such asABCA1 (25). This selective activity of TRAP80 is consistent with thedifferential effects of PPT on the transcription of SREBP-1c andABCA1. PPT inhibited the LXRa-dependent transcription of SREBP-1c, but not ABCA1. RCT can act to help maintain low levels ofLDL-cholesterol. Furthermore, it has been established that PPT re-duces the recruitment of TRAP80 to the LXRE of the SREBP-1c gene.These findings suggest that TRAP80 could potentially be used as atarget protein for the development of anti-lipogenic therapeuticagents, such as PPT.

In conclusion, we find that PPT has inhibitory activity againstLXRa-mediated lipogenesis in primary mouse hepatocytes byrepressing the recruitment of RNA polymerase II and TRAP80 to theLXRE of the SREBP-1c gene. Our present findings suggest that PPTcould represent a potential anti-lipogenic agent for treating NAFLD.

Conflicts of interest

The authors declare no potential conflicts of interest.

Acknowledgments

This research was supported by a grant (A084335) of the KoreaHealth Technology R&D Project through the Korea Health IndustryDevelopment Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea; by a grant (NRF-2006-2005402) fromthe National Research Foundation of Korea Grants funded by theKorean Government; and by intramural grants (2010-352 and2015-352) from the Asan Institute for Life Sciences, Seoul, Koreato SWK.

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