OrphanNuclearReceptorEstrogen-RelatedReceptor (ERR ... · ERR and ERR have the potential to regulate mitochondrial programs involved in oxidative phosphorylation, and gene expression

Orphan Nuclear Receptor Estrogen-Related Receptor �(ERR�) Is Key Regulator of Hepatic Gluconeogenesis*□S

Received for publication, October 19, 2011, and in revised form, April 10, 2012 Published, JBC Papers in Press, May 1, 2012, DOI 10.1074/jbc.M111.315168

Don-Kyu Kim‡1, Dongryeol Ryu§1, Minseob Koh¶2, Min-Woo Lee§, Donghyun Lim�, Min-Jung Kim§,Yong-Hoon Kim**, Won-Jea Cho‡‡, Chul-Ho Lee**, Seung Bum Park¶�3, Seung-Hoi Koo§4,and Hueng-Sik Choi‡§§5

From the ‡National Creative Research Initiatives Center for Nuclear Receptor Signals, Hormone Research Center, School ofBiological Sciences and Technology and ‡‡College of Pharmacy and Research Institute of Drug Development, Chonnam NationalUniversity, Gwangju 500-757, Republic of Korea, §Department of Molecular Cell Biology and Samsung Biomedical ResearchInstitute, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republicof Korea, Departments of ¶Chemistry and �Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University,Seoul 151-747, Republic of Korea, **Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea,and §§Research Institute of Medical Sciences, Department of Biomedical Sciences, Chonnam National University Medical School,Gwangju 501-746, Republic of Korea

Background: Dysregulation of glucose homeostasis is often associated with insulin resistance and diabetes.Results:Hepatic ERR� expression is increased by fasting-dependent activation of theCREB-CRTC2pathway, which leads to theinduction of hepatic gluconeogenesis.Conclusion:Orphan nuclear receptor ERR� is a novel transcriptional regulator of hepatic gluconeogenesis.Significance: An ERR� inverse agonist could be a new potential therapeutic approach for the treatment of type 2 diabetes.

Glucose homeostasis is tightly controlled by hormonal regu-lation of hepatic glucose production. Dysregulation of this sys-tem is often associated with insulin resistance and diabetes,resulting in hyperglycemia in mammals. Here, we show that theorphan nuclear receptor estrogen-related receptor� (ERR�) is anovel downstream mediator of glucagon action in hepatic glu-coneogenesis anddemonstrate a beneficial impact of the inverseagonist GSK5182. Hepatic ERR� expression was increased byfasting-dependent activation of the cAMP-response element-binding protein-CRTC2 pathway. Overexpression of ERR�induced Pck1 and G6PC gene expression and glucose produc-tion in primary hepatocytes, whereas abolition of ERR� gene

expression attenuated forskolin-mediated induction of gluco-neogenic gene expression. Deletion and mutation analyses ofthePck1promoter showed that ERR�directly regulates thePck1gene transcription via ERR response elements of the Pck1 pro-moter as confirmed by ChIP assay and in vivo imaging analysis.We also demonstrate thatGSK5182, an inverse agonist of ERR�,specifically inhibits the transcriptional activity of ERR� in aPGC-1� dependent manner. Finally, the ERR� inverse agonistameliorated hyperglycemia through inhibition of hepatic gluco-neogenesis in db/db mice. Control of hepatic glucose produc-tion by an ERR�-specific inverse agonist is a new potential ther-apeutic approach for the treatment of type 2 diabetes.

Glucose homeostasis is tightly controlled by hormonal regu-lation during fasting and feeding periods. As an initial responseto fasting, the pancreatic hormone glucagon triggers the break-down of glycogen stored in the liver via glycogenolysis. In timesof prolonged fasting, however, glucagon stimulatesde novo syn-thesis of additional glucose through gluconeogenesis in theliver (1). A rise in hepatic cAMP levels occurs in response toglucagon, and hepatic glucose production is mainly controlledby cAMP-response element-binding protein (CREB),6 peroxi-

* This work was supported in part by National Creative Research InitiativesGrant 20110018305 from the Korean Ministry of Education, Science andTechnology and Korea Healthcare Technology Research and DevelopmentProject and Future-based Technology Development Program (BIO Fields)Grant 20100019512 through the National Research Foundation of Korea(NRF) from the Ministry of Education, Science and Technology.

□S This article contains supplemental Methods and Figs. 1–3.1 Both authors contributed equally to this work.2 Supported by a Brain Korea 21 Program fellowship award and a Seoul Sci-

ence fellowship award.3 Supported by the NRF and the MarineBio Technology Program funded by

the Ministry of Land, Transport and Maritime Affairs (MLTM), Korea. Towhom correspondence may be addressed: Dept. of Chemistry, College ofNatural Sciences, Seoul National University, Seoul 151-747, Republic ofKorea. Tel.: 82-2-880-9090; Fax: 82-2-884-4025; E-mail: [email protected].

4 Supported by NRF Grants 2011-0016454 and 2011-0019448 funded by theKorea government (Ministry of Education, Science and Technology) and bythe Marine Biotechnology Program funded by the MLTM, Korea. To whomcorrespondence may be addressed: Dept. of Molecular Cell Biology, Sung-kyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu,Suwon, Gyeonggi-do 440-746, Republic of Korea. Tel.: 82-31-299-6122;Fax: 82-31-299-6239; E-mail: [email protected].

5 To whom correspondence may be addressed: Hormone Research Center,School of Biological Sciences and Technology, Chonnam National Univer-sity, Gwangju 500-757, Republic of Korea. Tel.: 82-62-530-0503; Fax: 82-62-530-0506; E-mail: [email protected].

6 The abbreviations used are: CREB, cAMP-response element-binding protein;ERR, estrogen-related receptor, PGC-1�, peroxisome proliferator-activatedreceptor � coactivator-1�; Pck1, phosphoenolpyruvate carboxykinase;G6PC, glucose-6-phosphatase; CRTC2, CREB-regulated transcriptioncoactivator 2; Q-PCR, real time quantitative PCR; PDK, pyruvate dehydro-genase kinase; SIK, salt-inducible kinase; CPT-1�, carnitine palmitoyltrans-ferase 1A; PEPCK, phosphoenolpyruvate carboxykinase; ERE, estrogenresponse element; ERRE, ERR response element; SHP, small heterodimerpartner; 8-Br-cAMP, 8-bromoadenosine 3�,5�-cyclic monophosphate; CRE,cAMP-responsive element; ER�, estrogen receptor �; Luc, luciferase; mut,mutant; FSK, forskolin; CHX, cycloheximide; A-CREB, acidic CREB; Ad, ade-novirus; 4-OHT, 4-hydroxytamoxifen; HNF4, hepatocyte nuclear factor 4.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 26, pp. 21628 –21639, June 22, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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some proliferator-activated receptor � coactivator-1� (PGC-1�), and CREB-regulated transcription coactivator 2 (CRTC2),which contribute to the expression of key gluconeogenic genes,such as phosphoenolpyruvate carboxykinase (PEPCK) and glu-cose-6-phosphatase (G6PC) (2–6). In contrast, insulin sup-presses hepatic glucose production by inhibiting the gluconeo-genic genes through inactivation of FOXO1, PGC-1�, andCRTC2 by the Akt-mediated phosphorylation under feeding(7–9). Dysregulation of insulin action on these factors is oftenassociated with the pathogenesis of insulin resistance and dia-betes (10).The estrogen receptor-related receptor subfamily consists of

threemembers, ERR�, -�, and� (NR3B1–3), which bind to clas-sic estrogen response elements (EREs) and to extended half-sitecore sequences (TNAAGGTCA; ERR response element(ERRE)) as either monomers or dimers (11). Structural studiesindicate that ERR� is constitutively active in the absence ofendogenous ligands, but small molecule ligands that could fur-ther transcriptionally activate or repress ERR� have beenreported to date (12–15). The ligand-independent transcrip-tional activity of ERR� depends on nuclear receptor coregula-tors, such as steroid receptor coactivator 2, PGC-1�, receptor-interacting protein 140, and small heterodimer partner (SHP),all of which are involved in the regulation of hepatic glucosemetabolism (16–20). It has been reported that ERRs areexpressed in tissues with highmetabolic demand and regulatedby peripheral circadian clock in key metabolic tissues, such aswhite and brown adipose tissues, muscle, and liver (21). Inter-estingly, ERR� has been reported to play an important role inthe regulation of a nucleus-encodedmitochondrial genetic net-work that coordinates postnatal metabolic transition in cardiacmuscle as evidenced by phenotype analyses of perinatally lethalERR�-null mice (22). Recently, chromatin immunoprecipita-tion (ChIP)-on-chip analysis in cardiomyocytes has shown thatERR� and ERR� have the potential to regulate mitochondrialprograms involved in oxidative phosphorylation, and geneexpression analysis in ERR�-null mice has demonstrated thatthis receptor plays an important role in regulation of potassiumhomeostasis in the heart, kidney, and stomach (23, 24). How-ever, the role of ERR� in hepatic glucose metabolism in adultsremains largely unknown. In this study, we have demonstratedthat orphan nuclear receptor ERR� is a novel transcriptionalregulator of hepatic gluconeogenesis, and its inverse agonistcould ameliorate hyperglycemia in mouse models of type 2diabetes.

EXPERIMENTAL PROCEDURES

Chemicals—8-Br-cAMP and insulin were purchased fromSigma-Aldrich and dissolved in the recommended solvents.GSK5182 was synthesized as described previously (15). Syn-thetic method of D4 is described in the supplemental Methods.GSK5182 was used as in HCl salt form and dissolved in sterilefiltered 30% PEG-400 aqueous solution to give a 40 mg/kg con-centration for in vivo experiments.Plasmids and DNA Constructs—The reporter plasmids

encoding rat Pck1-Luc (�2371 to �73) were described previ-ously (25). Mouse ERR� promoter was PCR-amplified frommouse genomic DNA (Novagen, Merck KGaA) and inserted

into the pGL3 Basic vector (Promega, Madison, WI) using theMluI and XhoI restriction enzyme sites. Pck1 ERRE1 � 2 mut-Luc, Pck1 cAMP-responsive element (CRE) mut-Luc(�93TTACGTCA�86 to �93TTAAAACA�86; underlined nu-cleotides were changed), Pck1 CRE/ERREs mut-Luc, and ERR�CRE mut-Luc were generated with the QuikChange II site-di-rected mutagenesis kit (Stratagene, La Jolla, CA). The vectorsexpressing ERE-Luc, ER�, ERR�, ERR�, ERR�, SHP, PGC-1�,CRTC2, salt-inducible kinase (SIK) 1, and SIK2 were describedpreviously (6, 17). ERR� was also subcloned in pEBG (GST)vector using SpeI and NotI enzyme sites for the GST pull downassay. All plasmids usedwere confirmed by automatic sequenceanalysis.Cell Culture and Transient Transfection Assay—HepG2,

H4IIE, and AML12 cells were maintained as described previ-ously (25). Transient transfection was performed using Lipo-fectamine 2000 (Invitrogen) or SuperFect (Qiagen, Hilden,Germany) according to the manufacturers’ instructions. Thecells were treated with 10 �M forskolin for 6 h, 500 �M 8-Br-cAMP for 6 h, 10 �M dexamethasone for 18 h, and/or 10 �M

GSK5182 for 24 h unless noted otherwise. The cells then wereharvested 48 h after transfection, and luciferase activity wasmeasured. Luciferase activity was normalized to �-galactosid-ase activity. The data are representative of at least three to fiveindependent experiments.Recombinant Adenovirus—Adenoviruses expressing unspe-

cific (US) shRNA, shERR�, shCRTC2, control GFP, SIK1, SIK2,CRTC2 S171A, A-CREB, and ERR� were described previously(6, 26, 27). All viruses were purified by using CsCl or anAdeno-X Maxi Purification kit (Clontech).Culture of Primary Hepatocytes—Primary hepatocytes were

isolated from Sprague-Dawley rats (male, 180–300 g) by colla-genase perfusion (28) and seeded with Medium 199 (Cellgro).After 3–6 h of attachment, cells were infected with the indi-cated adenoviruses for overexpression and/or treated with 10�M forskolin for the final 6 h and 10 �M GSK5182 for the final24 h unless noted otherwise.Western Blot Analysis—Whole cell extracts were prepared

using radioimmune precipitation assay buffer (Elpis-Biotech).Proteins from whole cell lysates were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes. Themembranes were probed with monoclonal ERR� antibodies(Perseus Proteomics, Tokyo, Japan), PGC-1� (Santa Cruz Bio-technology, Santa Cruz, CA), and SHP (Santa Cruz Biotechnol-ogy). Immunoreactive proteins were visualized using an Amer-sham Biosciences ECL kit (GE Healthcare) according to themanufacturer’s instructions.ChIP Assay—Nuclear isolation and cross-linking on cell

lines, primary hepatocytes, and liver sampleswere performed asdescribed previously (23, 29). After sonication, soluble chroma-tinwas subjected to immunoprecipitation using anti-FLAGM2(Stratagene), anti-ERR� (Perseus Proteomics), anti-CRTC2(SantaCruz Biotechnology), and anti-PGC-1� (SantaCruz Bio-technology). DNA was recovered by phenol/chloroformextraction and analyzed by PCR or Q-PCR using primersagainst relevant promoters.In Vivo GST Pulldown Assay—HepG2 cells were transfected

with pEBG (GST), pEBG-ERR�, and HA-PGC-1� vectors and

Regulation of Hepatic Gluconeogenesis by ERR�

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then treated with 1 and 10 �M GSK5182 for 24 h. Cell lysateswere co-immunoprecipitated with GST beads and anti-HA(RocheApplied Science) and anti-GST (SantaCruzBiotechnol-ogy) antibodies as described previously (29).Animal Experiments—Male 7–12-week-old C57BL/6J and

db/dbmice (Charles River Laboratories) were maintained on a12-h/12-h light/dark cycle and fed ad libitum. GSK5182 (40mg/kg/day as a final dose) and corn oil emulsionwere sonicatedagain immediately before injection of db/dbmice. After 14 h offasting, intraperitoneal injections were performed for 5 days.After the injections, blood glucose levels were monitored after4 h of fasting. All experiments were conducted following theguidelines of the SungkyunkwanUniversity School ofMedicineInstitutional Animal Care And Use Committee.In Vivo Imaging—C57BL/6J mice were infected with

Ad-Pck1WT-Luc (�2371/�73) orAd-Pck1 ERREmut-Luc viatail vein injections. Three days postinjection, mice were eitherfasted for 16 h or fasted for 16 h and refed for 4 h. Mice wereimaged using an IVIS 100 imaging system (Xenogen) asdescribed previously (30).Glucose Output Assay—Twenty-four hours after seeding of

rat primary hepatocytes, the medium were replaced withKrebs-Ringer buffer (115 mM NaCl, 5.9 mM KCl, 1.2 m MgCl2,1.2 mM NAH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, pH 7.4)supplemented with 10mM lactate and 1mM pyruvate. The cellswere treated with 10 �M forskolin for 12 h, 100 nM insulin for18 h, and/or 10 �M GSK5182 for 18 h. The glucose level in themedium was measured using a QuantiChrom glucose assay kit(Bioassay Systems, Hayward, CA).Electrophoretic Mobility Shift Assay (EMSA)—Double-

stranded oligonucleotides containing a CRE site on ERR� pro-moter or the ERR� binding sites (ERREs) on Pck1 promoterwere generated and labeled with [�-32P]dCTP using the Kle-now fragment of DNA polymerase I. The oligonucleotidesequences were as follows: sense 5�-GGGGCGGCT-CGCGTCGCCTCCCTCC-3� and antisense 5�-GGGGGG-AGGGAGGCGACGCGAGCCG-3� for CRE; sense 5�-GGGG-TGGACCTCCAGGTCATTTCGT-3� and antisense 5�-GGG-GACGAAATGACCTGGAGGTCCA-3� for ERRE1; and sense5�-GGGGGCCTCCCTGACCTAAGGGA-3� and antisense 5�-GGGGTCCCTTAGGTCAGGGAGGC-3� for ERRE2. Under-lined sequenceswere substituted toAA formutantCRE andTTfor mutant ERRE1 and ERRE2. Purified recombinant proteinfor CREB or ERR� was generated by in vitro translation usingthe TNT-coupled reticulocyte lysate system (Promega). Unpro-grammed TNT-coupled reticulocyte lysate was used as a nega-tive control, and 32P-labeled double-stranded oligonucleotidescontaining the ERR� binding site on DAX-1 promoter wereused as a positive control (31). Unlabeled oligonucleotides wereadded as cold competitors at 50–100-fold molar excess whereindicated. DNA-protein complexes were separated on a 4%polyacrylamide gel in 0.5� Tris borate-EDTA. The gels weredried and then analyzed by autoradiography.Surface Plasmon Resonance Analysis—The dissociation con-

stant of GSK5182 with ERR� ligand binding domain was deter-mined by surface plasmon resonance spectroscopy using a Bia-core T100 instrument (GE Healthcare). The surface carboxylgroup of a CM5 sensor chip was activated with a mixture of

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hy-droxysuccinimide in flow cells 3 and 4 to generate the reactivesuccinimide ester on the CM5 chip. ERR� ligand bindingdomain (100 �g/ml in 10 mM NaOAc, pH 5.0) was passedthrough flow cell 4 (5200 resonance units) and immobilized viaamide bond formationwith succinimide ester on theCM5 chip.The remaining succinimide ester on flow cells 3 and 4 wasquenched by an injection of 1 M ethanolamine HCl, pH 8.0.Phosphate-buffered saline (PBS) was used as a running bufferthroughout the immobilization process. After the immobiliza-tion, various concentrations of GSK5182 ranging from 50 nM to5�Mwere injected for 60 swith a flow rate of 30�l/min, and thedissociation of GSK5182 from ERR� ligand binding domainimmobilized on the sensor chip surfacewasmonitored for 600 sat the same flow rate. The running buffer was 10 mM HEPESbuffer, pH 7.5 containing 5%DMSO, 50mMNaCl, 2mMMgCl2,1 mM EDTA, 1 mM DTT, and 0.005% P20. The binding eventswere measured at 25 °C, and a DMSO correction was per-formed during the binding assay. The data were analyzed usingBiacore T100 Evaluation software. Final sensorgrams wereobtained by eliminating responses from flow cell 3 and buffer-only control. The dissociation constant (KD) was calculated byfitting the sensorgrams to a 1:1 binding model.Quantitative PCR—Total RNA from either primary hepato-

cytes or liver was extracted using an RNeasy minikit (Qiagen).cDNA generated by Superscript II enzyme (Invitrogen) wasanalyzed by Q-PCR using a SYBR Green PCR kit and a TP800Thermal CyclerDICEReal Time system (Takara). All data werenormalized to ribosomal L32 expression.Statistical Analyses—All values are expressed as means �

S.E. The significance between mean values was evaluated bytwo-tailed unpaired Student’s t test.

RESULTS

Hepatic ERR� Expression Is Induced by cAMP Signalingunder Fasting—It has been reported that hepatic expression ofERR� rhythmically oscillates in the daily light/dark cycle and isinduced during fasting (21, 32), suggesting that it could be reg-ulated by nutritional status. Because glucagon stimulateshepatic glucose productionmainly through the cAMP signalingpathway under fasting (33), we first investigated whether ERR�expression is induced by the adenylate cyclase activator forsko-lin (FSK) in AML12 cells and rat primary hepatocytes. ThemRNA and protein levels of ERR� were rapidly increased 1 and3 h, respectively, after the addition of FSK (Fig. 1, A and B),whereas the induction of Pck1mRNA occurred after 1-h treat-ment with FSK. However, expression of ERR� was not signifi-cantly changed (Fig. 1A). Notably, the expression of Pck1 andG6PC occurred after 30 and 45 min, respectively, whereas theinduction of ERR� mRNA was only increased after 1-h treat-ment with FSK (Fig. 1C). These results indicate that FSK stim-ulation led to the initial induction of Pck1 and G6PC, which isfaster than that of ERR�. To further test the correlationbetween ERR� and gluconeogenic gene expression in vivo, weanalyzed mRNA levels for these genes in wild type mice underfasting over time. Similar to the results in cultured cell lines,Pck1 and G6PCmRNA levels were rapidly induced after 1-h offasting and were strongly induced after 6-h fasting conditions,




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whereas ERR� gene expression was only enhanced 3 h afterfasting and further elevated until 12 h post-food deprivation(Fig. 1D). These results suggest that, in addition to the initialinduction of gluconeogenic gene expression by fasting, ERR�expression precedes that of additional gluconeogenic geneexpression in vivo.In an attempt to determine whether ERR� expression is

involved in the additive effect of cAMP on Pck1 gene expres-sion, H4IIE cells were treated with cAMP alone or cAMP pluscycloheximide (CHX), a protein synthesis inhibitor, in a time-dependent manner. Pck1 gene expression was significantlyincreased by cAMP stimulation for 1 h and was furtherenhanced by 6-h stimulation with cAMP (Fig. 1E). Interest-ingly,Pck1 gene induction by 1-h treatmentwith cAMPwas notaffected by CHX treatment, whereas the additional Pck1 geneinduction by 6-h treatment with cAMP was blocked by CHXtreatment, clearly suggesting that cAMP-mediated ERR�expression contributes to the additional pathway of cAMP-me-diated Pck1 gene expression.Regulation of ERR� Expression Is Mediated by CREB-

CRTC2—Wenext explored the potential mechanisms underly-ing the induction of ERR� by cAMP during fasting. We firstinvestigated the role of CRTC2, a mediator of the cAMP-de-pendent transcriptional program in hepatocytes (6, 34), in FSK-mediated elevation of ERR� and its target genes. Hepaticexpression of constitutively active CRTC2 (CRTC2 S171A) sig-nificantly increased mRNA levels of ERR�, Pck1, PGC-1�, andCPT-1� but not of SCD-1 and ERR� (Fig. 2A and data not

shown). Conversely, shRNA-mediated knockdown of CRTC2in mouse liver considerably reduced ERR� and gluconeogenicgene expression (Fig. 2B). Adenovirus-mediated expression ofSIK1, a known inhibitor of CRTC2 (6), also reduced hepaticERR� and gluconeogenic gene expression in mouse primaryhepatocytes, but kinase-inactive SIK1 T182A did not (Fig. 2C),further supporting the notion that endogenousCRTC2 inducesERR� expression.

Investigation of human, mouse, and rat ERR� promotersequences revealed the presence of a potential CRE (Fig. 2E,bottom). In transient transfection, we demonstrated that FSKand CREB-CRTC2 significantly increased the ERR� promoteractivity, and this effect was abolished either by the co-transfec-tion of A-CREB or SIK kinases or by mutation of the ERR�promoter CRE (Fig. 2, D and E). Furthermore, ChIP demon-strated thatCRTC2was recruited to theCREofERR�promoteras well as that of Pck1 or G6PC promoters in the presence ofFSK inmouse primary hepatocytes (Fig. 2F). In addition, EMSArevealed that CREB directly binds to CRE on ERR� promoter(Fig. 2G), showing that CREB-CRTC2 directly regulates ERR�at the transcriptional level.ERR� Regulates Gluconeogenic Gene Expression—The

response of ERR� to fasting in liver suggested that this receptormight directly regulate hepatic gluconeogenesis. Indeed, infec-tion with adenovirus expressing ERR� (Ad-ERR�) significantlyinduced the mRNA levels of Pck1 and G6PC as well as theirpromoter activities in cultured cells (Fig. 3,A andB).Moreover,ERR� expression led to the induction of PGC-1� expression

FIGURE 1. Hepatic ERR� expression is regulated by cAMP signaling under fasting. A, time course of ERR�, ERR�, and Pck1 mRNA induction by FSK. Ratprimary hepatocytes were stimulated with FSK (10 �M) for the indicated time, and isolated total RNAs were analyzed by Q-PCR. *, p � 0.05; **, p � 0.01. B, ERR�expression in AML12 cells stimulated by FSK (10 �M) for the indicated time. Five independent experiments were performed, and pooled proteins wereanalyzed. C, time course of ERR�, Pck1, and G6PC mRNA induction by FSK. AML12 cells were stimulated with FSK (10 �M) for the indicated time. *, p � 0.05; **,p � 0.01; ***, p � 0.001. D, hepatic expression of ERR�, Pck1, and G6PC mRNA at the indicated time of fasting in wild type C57BL/6J mice (n � 5). *, p � 0.05; **,p � 0.01; ***, p � 0.001. E, Pck1 mRNA levels in H4IIE cells treated with cAMP (500 �M) or cAMP plus CHX (10 �M) for the indicated time. **, p � 0.01; ***, p � 0.001.Error bars show �S.E. *, p � 0.05; **, p � 0.01; ***, p � 0.001 by two-tailed Student’s t test.




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(Fig. 3A, right). Interestingly, the induction of Pck1 promoteractivity was observed with co-transfection of expression vectorfor ERR� but not with that of other ERR isoforms (Fig. 3B).However, ERR�-mediatedPck1 promoter activitywas inhibitedby ERR� (Fig. 3C). Consistent with previous reports regardingthe roles of PGC-1� and SHP in the transcriptional activity ofERR� (17, 19), PGC-1� potentiated and SHP inhibited ERR�-mediated induction of gluconeogenic gene expression (Fig. 3A).We confirmed the competition between PGC-1� and SHP forthe transcriptional activity of ERR� onPck1 promoter (Fig. 3D),suggesting that the regulation of hepatic gluconeogenesis byPGC-1� and SHP may be achieved in part by competitionbetween these factors for the association with ERR� on gluco-neogenic promoters.ERR� Directly Activates Pck1 Gene Expression at Transcrip-

tion Level—PEPCK is the key enzyme controlling the rate ofhepatic gluconeogenesis and is regulated at the transcriptionallevel (35). We identified two potential conserved ERREs in thehuman, mouse, and rat Pck1 promoters (supplemental Fig. 1).We confirmed the functional significance of these sites usingserial deletion (Fig. 4A) or ERREmutants of the Pck1 promoterin transfection assays (Fig. 4B). In addition, double ERREmuta-tions of the Pck1 promoter largely blunted cAMP- or PGC-1�-

mediated induction of wild type promoter activity (Fig. 4,C andE), whereas CRE-mutated Pck1 promoter activity was signifi-cantly induced by ERR�, suggesting a significant role of thePck1 ERREs in cAMP- or PGC-1�-mediated induction of Pck1promoter activity. However, interestingly, Ad-shERR� led tomarked reduction of basal and FSK-induced gluconeogenicgene expression in rat primary hepatocytes (Fig. 4D), indicatingthat ERR� has indirect effects on Pck1 gene expression becauseit completely abolished the effect of cAMP, whereas mutationof the ERRE did not.FSK-dependent occupancy of ERR�, but not ERR�, on the

Pck1 ERREs was confirmed by EMSA and ChIP assay (Fig. 4, Fand G). An in vivo ChIP assay demonstrated that PGC-1� wasrecruited to the Pck1 ERREs in livers of fasted mice but not offed mice (Fig. 4H). To further examine the importance ofERREs in the Pck1 transcription by fasting signals in vivo, weperformed in vivo imaging analysis with an adenoviral reporterconstruct carrying either wild type or ERRE mutant Pck1 pro-moter fused to luciferase. Fasting increased wild type Pck1 pro-moter activity 45-fold over feeding controls (Fig. 4I). However,the stimulatory effect of fasting was largely ablated inmice withthe double ERRE mutant promoter, strongly indicating thatERR�, rather than theCRE, is a critical downstreammediator of

FIGURE 2. ERR� expression is induced by CREB-CRTC2. A, induction of ERR� by Ad-CRTC2 S171A in liver of wild type mice (n � 3) fasted for 16 h (top). *, p �0.05. Western blot analysis shows CRTC2 S171A overexpression (bottom). B, expression of ERR� by Ad-shCRTC2 in liver of wild type mice (n � 3) fasted for 16 h(top). **, p � 0.01. Western blot analysis shows knockdown of CRTC2 (bottom). C, Ad-SIK1 decreases hepatic ERR� expression. Q-PCR analysis of total RNAs frommouse primary hepatocytes infected with Ad-SIK1 WT or Ad-SIK1 T182A is shown. *, p � 0.05; **, p � 0.01. D, FSK- and CREB-CRTC2-mediated ERR� promoteractivity is inhibited by A-CREB and SIK kinase. *, p � 0.05; **, p � 0.01. HepG2 cells were transfected using ERR�-Luc along with expression vectors for CRTC2,CREB, A-CREB, SIK1, and/or SIK2 followed by treatment with FSK (10 �M) for the final 6 h. E, activation of ERR� promoter by CREB-CRTC2 depends on CRE. *, p �0.05; **, p � 0.01. A transient transfection assay was performed in HepG2 cells using expression vectors for ERR�-Luc, ERR� CRE mut-Luc, CREB, and CRTC2followed by treatment with FSK (10 �M) for the final 6 h (top). The alignment of potential CRE sequences in human, mouse, and rat ERR� promoters is shown(bottom). F, ChIP assay showing the occupancy of CRTC2 on ERR�, Pck1, and G6PC promoters in mouse primary hepatocytes in the presence of FSK. ACTB,�-actin. G, EMSA showing binding of CREB to CRE of ERR� promoter. In vitro translated proteins were incubated with 32P-labeled double-stranded oligonu-cleotides containing CRE of ERR� promoter. Error bars show �S.E. *, p � 0.05; **, p � 0.01 by two-tailed Student’s t test. mt, mutant; US, unspecific shRNA.




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fasting signals, mediating the effect of fasting on Pck1 promoteractivity in vivo.Finally, to elucidate whether ERR�-dependent induction of

gluconeogenic gene expression results in increased glucoseproduction, we assayed glucose output in rat primary hepato-cytes. As expected, Ad-ERR� increased glucose production inprimary hepatocytes, and its effect was greatly decreased by theaddition of insulin (Fig. 4J). Taken together, these data suggestthat ERR� exerts its effects on hepatic glucose productionthrough direct transcriptional regulation of gluconeogenicgenes.GSK5182 Specifically Inhibits Transcriptional Activity of

ERR�—GSK5182 is a 4-hydroxytamoxifen (4-OHT) analog anda selective inverse agonist of ERR� relative to ER� due to itsadditional non-covalent interaction with Tyr-326 and Asn-346of ERR� (15) (supplemental Fig. 2). Indeed, using the mamma-lian one-hybrid assay, we confirmed that GSK5182, but not D4(a non-functional synthetic analog of 4-OHT), directly inhib-ited transcriptional activity of ERR� (Fig. 5A). Interestingly, thisinhibitory effect of GSK5182 depends on the interaction withTyr-326 rather thanAsn-346 of ERR�.We then performed bio-physical binding analysis ofGSK5182with ERR� ligand bindingdomain using surface plasmon resonance spectroscopy andobserved high binding affinity (KD � 65 nM), which confirmsthe direct and specific interaction of GSK5182 with ERR� (Fig.5B). Unlike 4-OHT, GSK5182 had no effect on estradiol-in-

duced transactivation by ER� or other nuclear receptors (Fig. 5,C and D), indicating that the inverse agonist GSK5182 specifi-cally inhibits the transcriptional activity of ERR�.

Previous studies revealed that binding of 4-OHT to ERR�leads to a conformational change in the AF-2 domain thatblocks PGC-1� binding (13, 36). Similarly, transient transfec-tion and in vivo GST pulldown assays showed that treatmentwith GSK5182 inhibited the PGC-1�-potentiated ERR� tran-scriptional activity and disrupted the interaction of ERR� withPGC-1�, effects not observed with D4 (Fig. 5E and supplemen-tal Fig. 3). cAMP- or PGC-1�-induced Pck1 promoter activitywas also significantly inhibited by GSK5182 (Fig. 5F). To testwhether GSK5182 specifically inhibits the recruitment ofPGC-1� to ERREs on the Pck1 promoter, ChIP assays wereperformed in HepG2 cells treated with GSK5182. Indeed,GSK5182 inhibited the occupancy of PGC-1� over ERREs onPck1 promoter without affecting the binding of ERR� on thesame region (Fig. 5G). These results demonstrate thatGSK5182suppresses the expression of gluconeogenic genes by disruptingthe interaction between ERR� and PGC-1� without affectingthe DNA binding ability of ERR�.Inverse Agonist of ERR� Improves Hyperglycemia in db/db

Mice—Based on the ability of ERR� to regulate the gluconeo-genic program, we next examined the effect of this inverseagonist on gluconeogenic gene expression and glucose outputin rat primary hepatocytes. Indeed, GSK5182 treatment

FIGURE 3. ERR� mediates induction of gluconeogenic genes. A, expression of gluconeogenic genes by Ad-ERR�, Ad-PGC-1�, and/or Ad-SHP in rat primaryhepatocytes (left). *, p � 0.05; **, p � 0.01; ***, p � 0.001. Protein levels of ERR�, PGC-1�, and SHP are shown (right). B, ERR�-specific activation of Pck1 promoter.293T cells were transfected using Pck1-Luc along with expression vectors for ERR�, ERR�, and ERR�. *, p � 0.05; **, p � 0.01. C, ERR� effect on ERR�-mediatedPck1 promoter activity in 293T cells (2, 200 ng; 4, 400 ng). *, p � 0.05. D, functional competition between PGC-1� and SHP for the transcriptional activity of ERR�.*, p � 0.05; **, p � 0.01. A transient transfection assay was performed in AML12 cells using expression vectors for ERR�, PGC-1�, and SHP (�, 200 ng; ��, 400ng). Error bars show �S.E. *, p � 0.05; **, p � 0.01; ***, p � 0.001 by two-tailed Student’s t test.




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decreased FSK-induced gluconeogenic gene expression to 40%without a change in ERR� mRNA levels in rat primary hepato-cytes (Fig. 6A). Consistent with the reduction of gluconeogenicgenes, GSK5182 also attenuated FSK-induced glucose produc-tion in primary hepatocytes (Fig. 6B).Finally, to assess whether GSK5182 directly affects hepatic

glucose metabolism in vivo, fasting blood glucose levels weremeasured in db/db mice that were injected intraperitoneallywith GSK5182. Indeed, GSK5182-injected mice showed amarked reduction in fasting blood glucose levels comparedwith control groups (Fig. 6C). Consistent with this result, theexpression of gluconeogenic genes and PGC-1� was markedlydecreased in GSK5182-treated db/db mice (Fig. 6D). In addi-tion, GSK5182 significantly reduced the occupancy of PGC-1�on the Pck1HNF4 regulatory element as well as the Pck1 ERREs

(Fig. 6E). No significant changes were shown in plasma insulin,triglyceride, or total cholesterol levels with GSK5182 treatment(Fig. 6, F–H). Taken together, these results suggest that inverseagonist-mediated inactivation of ERR� ameliorates the hyper-glycemic phenotype in type 2 diabeticmice via direct regulationof hepatic gluconeogenesis.

DISCUSSION

In the current study, we identified the orphan nuclear recep-tor ERR� as a novel alternative downstreammediator of cAMP-CREB in hepatic glucose metabolism (Fig. 7), which is evi-denced by the results showing that FSK-mediated inductionofPck1 andG6PCmRNA expression was faster than the induc-tion of ERR�mRNA expression in vivo and in vitro. In addition,CHX treatment did not block the initial induction of Pck1

FIGURE 4. ERR� is direct regulator of Pck1 gene transcription. A, mapping of ERR� binding sites of Pck1 promoter. *, p � 0.05; **, p � 0.01. Serial deletionconstructs of Pck1 promoter were transfected in 293T cells along with expression vectors for ERR�. B, ERRE-dependent activation of Pck1 promoter by ERR� in293T cells (top). *, p � 0.05. ERRE1 and -2 of Pck1 promoter are shown (bottom). C, involvement of ERR� in cAMP-induced Pck1 promoter activity in AML12 cells.*, p � 0.05; **, p � 0.01. D, effect of Ad-shERR� on basal and FSK-induced gluconeogenic genes in rat primary hepatocytes (left). *, p � 0.05; **, p � 0.01. Westernblot analysis and a graphical representation showing ERR� expression in Ad-US- and Ad-shERR�-infected rat primary hepatocytes are shown (right).E, ERRE-dependent regulation of Pck1 promoter activity by PGC-1� in 293T cells (2, 200 ng; 4, 400 ng). *, p � 0.05. F, EMSA showing binding of ERR� to ERRE1and -2 of Pck1 promoter. In vitro translated proteins were incubated with radiolabeled oligonucleotides containing ERRE1 or ERRE2 on Pck1 promoter.32P-Labeled double-stranded oligonucleotides containing the ERR� binding site on DAX-1 promoter were used as a positive control. G, ChIP assay showing theoccupancy of ERR� and ERR� on both ERRE1 and -2 of Pck1 promoter from FSK-treated rat primary hepatocytes. **, p � 0.01. N.D., not detected. H, in vivo ChIPassay showing the occupancy of PGC-1� on ERREs of Pck1 promoter in normal mouse livers (n � 3) fasted for 6 h. Soluble chromatin was immunoprecipitatedwith �-PGC-1� or IgG. 10% of the soluble chromatin was used as input. I, in vivo imaging of hepatic Pck1 WT-luciferase (Ad-Pck1 WT-Luc) and Pck1 ERREmut-luciferase (Ad-Pck1 ERRE mut-Luc) activity in fasted or fed mice (n � 4 –5) (left). Quantitation of luciferase activity (right) is also shown. **, p � 0.01. J,glucose output assay in rat primary hepatocytes exposed to FSK for 12 h or insulin (INS) for 16 h after infection with Ad-GFP or Ad-ERR�. **, p � 0.01. Error barsshow �S.E. *, p � 0.05; **, p � 0.01 by two-tailed Student’s t test. mt, mutant; US, unspecific shRNA.

FIGURE 5. GSK5182 specifically inhibits transcriptional activity of ERR�. A, inhibitory effect of GSK5182 on ERR� depends on Tyr-326 rather than Asn-346.***, p � 0.001. B, direct binding analysis of GSK5182 with ERR� ligand binding domain using surface plasmon resonance spectroscopy. C, specificity of GSK5182for nuclear receptors. **, p � 0.01. D, GSK5182 has no effect on estradiol (E2)-mediated activation of ER�. **, p � 0.01. Transient transfection was conducted in293T cells using expression vectors for ERE-Luc and ER� followed by treatment with estradiol, 4-OHT, GSK5182, and D4 for the final 24 h. E, in vivo GST pulldownassay showing the disruption of the interaction between ERR� and PGC-1� by GSK5182. F, GSK5182 decreases cAMP- or PGC-1�-induced Pck1 promoteractivity. *, p � 0.05. G, ChIP assay showing the effect of GSK5182 on occupancy of PGC-1� and ERR� on ERREs of Pck1 promoter. Experiments in A, C, andE–G were performed in HepG2 cells using transient transfection. Error bars show �S.E. n.s., not significant. *, p � 0.05; **, p � 0.01; ***, p � 0.001 by two-tailedStudent’s t test. RU, resonance units; CON, control; Ctrl, control; CAR, constitutively active receptor.




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mRNA expression, whereas the additional Pck1 gene expres-sion by 6-h treatment with cAMP was blocked by CHX treat-ment. These results are further supported by the ChIP assayshowing that ERR� occupancy was weakly increased by 1-hstimulation with FSK, and the occupancy on Pck1 ERREs wasstrongly enhanced by 6-h treatment with FSK. We also foundthat mutation of the Pck1 CRE markedly reduces the effect ofcAMP, whereasmutation of the Pck1 ERREs did not abolish theeffect of cAMP. Overall, cAMP-mediated activation of CREB-CRTC2 triggers the initial induction of hepatic gluconeogenicgene expression in response to fasting, and then ERR� expres-sion contributes to the additional gluconeogenic geneexpression.We have also demonstrated that ERR� contributes signifi-

cantly to the fasting-mediated glucose production throughhepatic gluconeogenesis. FSK-induced Pck1 and G6PC geneexpression was reduced up to 80% by knockdown of ERR� inhepatocytes. Moreover, the fasting-mediated induction of Pck1promoter activity was completely abolished by double ERREmutations compared with that of wild type promoter activity invivo, suggesting a more important role for ERR� induction,rather than the CRE, in mediating the effect of fasting on Pck1promoter activity in vivo. In addition, GSK5182-mediated inhi-bition of ERR� transcriptional activity decreased gluconeo-genic gene expression and improved hyperglycemia in db/dbmice. On the other hand, it is known that ERR� induces expres-

sion of pyruvate dehydrogenase kinase 4 (PDK4),which inhibitsthe activity of pyruvate dehydrogenase complex, a key regula-tory enzyme in the oxidation of glucose to acetyl-CoA (37, 38).It has been reported that expression of PDK4 is elevated indiabetes and starvation, leading to decreased oxidation of pyru-vate to acetyl-CoA (39, 40), and inhibition of PDKs by theirinhibitors reduces hyperglycemia in type 2 diabetic rats (41).

FIGURE 6. Inverse agonist of ERR� lowers hyperglycemia in db/db mice. A, GSK5182 inhibits FSK-mediated gluconeogenic gene expression in rat primaryhepatocytes. *, p � 0.05; **, p � 0.01. B, GSK5182 decreases FSK-mediated glucose production in rat primary hepatocytes. *, p � 0.05. C and D, GSK5182 lowersblood glucose levels and gluconeogenic gene expression. After 14 h of fasting, GSK5182 was injected intraperitoneally at 40 mg/kg/day for 5 days in db/db mice(n � 6). After the injections, blood glucose was measured after 4 h (C). Q-PCR analysis of gluconeogenic gene expression in liver of db/db mice is shown (D). *,p � 0.05; **, p � 0.01. E, ChIP assay showing the occupancy of PGC-1� on ERREs or HNF4 regulatory element (HNF4RE) of Pck1 promoter in liver of db/db miceas in C. **, p � 0.01. F–H, effect of GSK5182 on insulin (F), plasma triglyceride (TG) (G), and total cholesterol (H) levels. An ELISA and colorimetric assay wereperformed using the blood of control or GSK5182-injected db/db mice. Error bars show �S.E. n.s., not significant. *, p � 0.05; **, p � 0.01 by two-tailed Student’st test. CON, control.

FIGURE 7. ERR� regulates hepatic gluconeogenesis. Glucagon triggershepatic gluconeogenic gene expression though activation of CREB-CRTC2 inresponse to fasting. In addition, hepatic ERR� expression is also increased byglucagon-mediated activation of CREB-CRTC2, which in turn leads to addi-tional gluconeogenic gene expression through cooperation with PGC-1�.ERR�-dependent induction of gluconeogenic genes is directly inhibited by itsinverse agonist GSK5182 in a PGC-1�-dependent manner.




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Taken together, these observations suggest that ERR�mayhavea dual role by inhibiting glucose oxidation and inducing gluco-neogenic flux.It has been reported that PGC-1�, a master regulator of

hepatic glucose metabolism, is closely associated with the tran-scriptional activity of ERR� and ERR� (42). For example, theregulation of key metabolic processes, such as mitochondrialbiogenesis and oxidative phosphorylation by PGC-1�, wasshown to be dependent on the presence of ERR� (43). Severallines of evidence have also shown that the transcriptional reg-ulation of PDK4 expression by PGC-1� is mediated by ERR� orERR� (37, 44). Similar to these reports, we also found thatPGC-1� plays an important role in ERR�-mediated regulationof gluconeogenic gene expression as evidenced by an in vivoChIP assay showing strong recruitment of PGC-1� onERREs ofPck1 promoter during fasting. Moreover, PGC-1� expressionwas also induced by ERR�, suggesting that they reciprocallycooperate for the regulation of gluconeogenesis. Notably,mutation of the ERREs of the Pck1 promoter largely attenuatedPGC-1�-mediated induction of wild type promoter activity,further supporting the important role of the ERREs for theeffect of PGC-1�. To date, most studies have been focused onthe factors that control the �500 region from the start site ofPck1 gene transcription because this region containsmany crit-ical elements that regulate the response of the gene to diet andhormones (1, 2). However, a number of researches have sug-gested that a part of Pck1 promoter that extends considerablyupstream from the better characterized downstream region isinvolved in both the hormonal and tissue-specific control ofPck1 gene transcription (35, 45). Indeed, a new extended gluco-corticoid regulatory unit that is present at �1365 in the Pck1promoter has been characterized, and this extended glucocor-ticoid regulatory unit was shown to be liver-specific and to playan important role in the regulation of Pck1 gene transcriptionby glucocorticoids (46).ERR� has been shown to repress the PGC-1�-mediated glu-

coneogenic program through inhibition of recruitment ofPGC-1� to Pck1 promoter (47). We found that ERR� inhibitsthe ERR� transcriptional activity for Pck1 promoter, suggestingthat ERR� could also inhibit the association between ERR� andPGC-1�. However, ERR� did not affect the FSK-stimulatedDNA binding ability of ERR� on Pck1 ERREs. Similarly, it hasbeen shown that ERR� can inhibit ERR� transcriptional activitywithout affecting the DNA binding ability of ERR� and sug-gested that a mechanism for ERR� repression is the het-erodimerization between the ERRs as evidenced by the reportsshowing that heterodimerization between ERR� and ERR�inhibits transactivation of each other, whereas homodimeriza-tion is needed for their transcriptional activity (32, 48, 49). Onthe other hand, their functions in terms of transcriptional reg-ulation of each ERR for downstream targets are more compli-cated. This notion is supported by previous reports that thetranscriptional induction of SHP gene expression is regulatedby ERR� but not ERR� or ERR� (17) and that ERR� and ERR�could directly control the same target genes (23). In addition, ithas been reported that ERR� and ERR� in breast cancer exhib-ited correlative opposing functions as negative and positive bio-markers and lead to different responses of specific metabolic

genes (50, 51). Therefore, the molecular mechanism for thedifferent transcriptional regulation or output of each ERR iso-form needs further characterization.ERR� and ERR� are known to have the potential to regulate

mitochondrial programs involved in fatty acid oxidation in car-diac muscle (23). It has been also reported that muscle-specificERR� transgenic mice exhibited increased muscle mitochon-drial activity and oxidative capacity through induction of slowtwitchmuscle fiber properties characterized by highmitochon-drial content, fatigue-resistant fibers, and dense vascularity (52,53). Consequently, these mice showed enhanced oxygen con-sumption and running endurance and reduced weight gainupon high fat diet compared with control. Interestingly, mus-cle-specific PEPCK transgenic mice also displayed greatlyenhanced physical activity, running endurance, and longevitydue in part to an increased the number of mitochondria (54), aphenotype similar to that of ERR� transgenic mice. Based onour results that ERR� regulates Pck1 gene expression as dem-onstrated by in vitro and in vivo studies, the alteration of energymetabolism by overexpression of ERR� in skeletal muscle mayoccur through induction of PEPCK.A number of other nuclear receptors directly regulate

hepatic gluconeogenesis. The orphan nuclear receptor HNF4�has been recognized to stimulate the gluconeogenic genes in aPGC-1�-dependent manner in response to fasting (5), whereasthe NR4A pathway is PGC-1�-independent (55). The orphannuclear receptors TR4 and retinoid-related orphan receptor �have also been reported to induce hepatic gluconeogenesis dur-ing fasting (56, 57). However, none of these orphan receptorsare ligand-responsive. We have confirmed that GSK5182, aninverse agonist of ERR�, strongly suppresses hepatic gluconeo-genesis via direct and specific inhibition of the transcriptionalactivity of ERR� by blocking its association with PGC-1�.Recently, we also reported that GSK5182 inhibited the tran-scriptional activity of ERR� on PDK4 expression by recruit-ment of the transcriptional corepressor SMILE (small het-erodimer partner-interacting leucine zipper protein) (38, 58),suggesting that GSK5182 actively mediates an exchange ofcoactivator for corepressor on transcriptional activity of ERR�.In conclusion, we have demonstrated that the reduction of

ERR� activity by inverse agonist treatment inhibits gluconeo-genic gene expression and lowers blood glucose levels in dia-betic mice. Moreover, we have revealed that GSK5182improves the impaired hepatic insulin signaling induced by dia-cylglycerol-mediated protein kinase C � (PKC�) activation (59).Inhibition of hepatic gluconeogenesis is emerging as a promis-ing intervention strategy in type 2 diabetes, and the generationof ERR�-specific inverse agonists provides a new therapeuticstrategy for the treatment of type 2 diabetic patients.

Acknowledgments—We are grateful to D. Moore and S. Y. Choi forcritical reading of the manuscript and M. Montminy for helpful dis-cussions and providing materials. We thank S. M. Park, D. H. Choi,and J. S. Moon for technical assistance.

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Seung-Hoi Koo and Hueng-Sik ChoiMin-Jung Kim, Yong-Hoon Kim, Won-Jea Cho, Chul-Ho Lee, Seung Bum Park, Don-Kyu Kim, Dongryeol Ryu, Minseob Koh, Min-Woo Lee, Donghyun Lim,

of Hepatic Gluconeogenesis) Is Key Regulatorγ (ERRγOrphan Nuclear Receptor Estrogen-Related Receptor

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