Ji-Min Lee,1 Woo-Young Seo,2 Hye-Sook Han,2 Kyoung-Jin Oh,2 Yong-Soo Lee,1

Don-Kyu Kim,1 Seri Choi,2 Byeong Hun Choi,2 Robert A. Harris,3 Chul-Ho Lee,4

Seung-Hoi Koo,2 and Hueng-Sik Choi1

Insulin-Inducible SMILE InhibitsHepatic GluconeogenesisDiabetes 2016;65:62–73 | DOI: 10.2337/db15-0249

The role of a glucagon/cAMP-dependent protein kinase–inducible coactivator PGC-1a signaling pathway is wellcharacterized in hepatic gluconeogenesis. However, anopposing protein kinase B (PKB)/Akt-inducible corepressorsignaling pathway is unknown. A previous report has de-monstrated that small heterodimer partner–interactingleucine zipper protein (SMILE) regulates the nuclear re-ceptors and transcriptional factors that control hepaticgluconeogenesis. Here, we show that hepatic SMILE ex-pression was induced by feeding in normal mice but notin db/db and high-fat diet (HFD)-fed mice. Interestingly,SMILE expression was induced by insulin in mouse pri-mary hepatocyte and liver. Hepatic SMILE expressionwas not altered by refeeding in liver-specific insulin re-ceptor knockout (LIRKO) or PKB b-deficient (PKBb2/2)mice. At the molecular level, SMILE inhibited hepatocytenuclear factor 4–mediated transcriptional activity via directcompetition with PGC-1a. Moreover, ablation of SMILEaugmented gluconeogenesis and increased blood glucoselevels in mice. Conversely, overexpression of SMILE re-duced hepatic gluconeogenic gene expression and ame-liorated hyperglycemia and glucose intolerance in db/dband HFD-fedmice. Therefore, SMILE is an insulin-induciblecorepressor that suppresses hepatic gluconeogenesis.Small molecules that enhance SMILE expression wouldhave potential for treating hyperglycemia in diabetes.

Insulin induces the insulin receptor tyrosine kinase–mediated activation of the phosphatidylinositol 3-kinase

pathway that controls hepatic glucose production. Ablationof insulin signaling leads to the increased gluconeogenesisin type 2 diabetes (1–3). In the fed condition, insulin inhib-its hepatic gluconeogenesis by downregulating the expres-sion of PEPCK and glucose-6-phosphatase (G6Pase). Thispathway involves phosphorylation of the forkhead tran-scription factor FOXO1 and CREBP (4–7) and recruitmentof coactivators, including PGC-1a and CREB-regulated tran-scription coactivator 2 (8,9).

Small heterodimer partner–interacting leucine zipper pro-tein (SMILE), including two alternative translation-derivedisoforms, SMILE-L (CREBZF: long form of SMILE) andSMILE-S (Zhangfei: short form of SMILE), has been classi-fied as a member of the CREB/ATF family of basic region-leucine zipper transcription factors. However, SMILE cannotbind to DNA as a homodimer (10–12). SMILE has also beenreported to function as a coactivator of activating transcrip-tion factor 4 or as a corepressor of host cell factor-bindingtranscription factor (13,14). Previously, we have reportedthat SMILE is a corepressor of the estrogen receptor–relatedreceptor g, glucocorticoid receptor (GR), constitutive andro-stane receptor, hepatocyte nuclear factor 4a (HNF4a), andCREBH (15–17). A recent study demonstrated that SMILEactivates tumor suppressor p53 and inhibits the function ofbone morphogenetic protein 6 by interacting with Smads(18,19). However, the roles of SMILE in hepatic glucose me-tabolism still need to be clarified.

PGC-1a is a multifunctional transcriptional coactivatorinvolved in diverse physiological metabolisms. In the liver,

1National Creative Research Initiatives Center for Nuclear Receptor Signals andHormone Research Center, School of Biological Sciences and Technology, ChonnamNational University, Gwangju, Republic of Korea2Division of Life Sciences, College of Life Sciences and Biotechnology, KoreaUniversity, Seoul, Republic of Korea3Richard Roudebush Veterans Affairs Medical Center and Department of Bio-chemistry and Molecular Biology, Indiana University School of Medicine, Indian-apolis, IN4Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic ofKorea

Corresponding authors: Hueng-Sik Choi, hsc@chonnam.ac.kr, and Seung-HoiKoo, koohoi@korea.ac.kr.

Received 23 February 2015 and accepted 26 August 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0249/-/DC1.

J.-M.L. and W.-Y.S. contributed equally to this work.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 14.

62 Diabetes Volume 65, January 2016

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PGC-1a expression is induced in the fasting state andincreases the expression of gluconeogenic genes via directinteraction with transcription factors including HNF4a,FOXO1, and GR (8,20). Overexpression of PGC-1a leadsto the increased expression of G6Pase and PEPCK,key enzymes in the hepatic glucose production. Con-versely, knockdown or knockout of PGC-1a results inthe lower blood glucose levels as a result of reducedgluconeogenesis.

In this report, we show that SMILE is tightly regulatedby the nutritional status, displaying an expression patternopposite to that of PGC-1a. Whereas hepatic SMILE ex-pression was elevated in the fed state relative to fasting,this regulation was lost in the absence of insulin signaling.Moreover, SMILE regulates hepatic gluconeogenesis viainhibition of PGC-1a–mediated gluconeogenic genes ex-pression. Collectively, this study indicates that SMILEcounteracts the stimulatory effect of PGC-1a on hepaticgluconeogenesis and plays an important role in insulinaction on hepatic glucose metabolism.

RESEARCH DESIGN AND METHODS

Animal ExperimentsFour-week-old male C57BL/6 mice (for the DIO model)purchased from Orient (Seongnam, South Korea) were feda high-fat diet (HFD) (60 kcal% fat diet, D12492;Research Diets, Inc.) for 12 weeks (12:12 h light:darkcycle). Male 8- to 12-week-old C57BL/6 and db/db micewere provided with a standard rodent diet. Livers ofLIRKO and PKBb2/2 mice were provided by S.B. Biddinger(Harvard Medical School, Boston, MA) and B.A. Hemmings(Friedrich Miescher Institute for Biomedical Research, Basel,Switzerland) as previously described (21). AdenovirusesGFP and SMILE were delivered by tail vein injection onthe 7th day. Seven days later, protein and total RNA wereextracted from liver for Western blot and quantitative PCR(qPCR) analyses, respectively. For measurement of fastingblood glucose levels, animals were fasted for 4 h with freeaccess to water. For activation of the insulin signaling path-way, either PBS (for control) or insulin (0.5 unit/kg body wti.p.) was injected for 1–12 h before the collection of liver forfurther analyses. All procedures were performed in a specificpathogen-free facility at the Korea University College ofMedicine, based on the protocols that were approved bythe Korea University Institutional Animal Care and UseCommittee.

Glucose, Insulin, and Pyruvate Tolerance TestsFor the glucose or pyruvate tolerance tests, mice werefasted for 16 h and then injected with 2 g/kg i.p. glucoseor 1–1.5 g/kg i.p. pyruvate (on day 6 post–adenoviral in-jection). Plasma glucose levels were measured using blooddrawn from the tail vein at designated time points usingan automatic glucose monitor (One Touch, LifeScan Ltd.,Milpitas, CA). For the insulin tolerance test, mice werefasted for 4 h and then injected with 0.75–1 unit/kg i.p.insulin on day 6 after adenoviral injection.

Insulin MeasurementPlasma insulin was measured using plasma collected attime of sacrifice by using mouse insulin ELISA kit (ShibayagiCo., Ltd., Ishihara, Japan).

Glycogen AssayLiver tissue (30 mg) was homogenized in 0.1 mol/L ice-coldCitrate buffer (pH 4.2) plus NaF, and glycogen was measuredusing Enzychrom Glycogen assay kit (Bioassay Systems,Hayward, CA) according to the manufacturer’s instructions.

Glucose Output AssayGlucose production from primary hepatocytes was mea-sured according to the manufacturer’s protocol, usinga colorimetric glucose oxidase assay (Sigma). Primary he-patocytes were seeded and cultured for 24 h, the cellswere washed three times with phosphate-buffered saline,and the medium was then replaced with glucose produc-tion buffer (glucose-free DMEM, pH 7.4, containing20 mmol/L sodium lactate, 1 mmol/L sodium pyruvate,and 15 mmol/L HEPES without phenol red). The glucoseassays were performed in triplicate.

ChemicalsInsulin was purchased from Sigma-Aldrich (St. Louis,MO). Forskolin was purchased from Calbiochem and dis-solved in the recommended solvents.

Cell Culture and Transient Transfection Assaya-Mouse liver 12 (AML12) cells were maintained inDMEM/F-12 (Invitrogen), supplemented with 10% FBS(Cambrex Bio Science Walkersville, Inc., Walkersville,MD) and antibiotics (Invitrogen). Cells were split in 24-well plates (2–8 3 104 cells/well) at the day before trans-fection. Transient transfections were performed using theLipofectamine 2000 transfection reagent (Invitrogen)according to the manufacturer’s instructions. Total DNAused in each transfection was adjusted to 0.8 mg/well byadding appropriate amount of empty vector, and cyto-megalovirus-b-galactosidase plasmids were cotransfectedas an internal control. The cells were harvested 48 h afterthe transfection, and luciferase activity was measured andnormalized to b-galactosidase activity.

Culture of Primary HepatocytesPrimary hepatocytes were prepared from C57BL/6 miceby the collagenase perfusion method as previously de-scribed (22). After attachment, cells were infected withadenoviruses for 18 h. Subsequently, cells were main-tained in the serum-free medium 199 media (Mediatech)overnight and treated with 10 mmol/L forskolin and/or100 nmol/L insulin.

Recombinant AdenovirusAdenoviruses expressing unspecific short hairpin (sh)RNA,shSMILE, control GFP, SMILE, HNF4a, PGC-1a, SREBP-1c,and dominant-negative SREBP-1c (dn-SREBP-1c) were de-scribed previously (17,22–24). All viruses were purified byusing CsCl2 or the Adeno-X Maxi Purification kit (Clontech,Mountain View, CA).

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Chromatin Immunoprecipitation AssayThe chromatin immunoprecipitation (ChIP) assay wasperformed as previously described (17). In brief, mouselivers (40 mg/sample) were fixed with 1% formaldehydeand sonicated. The soluble chromatin was then subjected toimmunoprecipitation (IP) using anti-HA (cat. no. 2367; CellSignaling Technology), anti-FLAG (2368; Cell SignalingTechnology), anti–PGC-1a (sc-13067; Santa Cruz Biotech-nology, Santa Cruz, CA) and anti-SMILE (sc-49329; SantaCruz Biotechnology) followed by treatment with protein A-agarose/salmon sperm DNA (Upstate Biotechnology, Up-state, NY). DNA samples were quantified by quantitativereal-time PCR using two pairs primers of the mousePepck promoter, forward (59-GATGTAGACTCTGTCCTG-39)and reverse (59-GATTGCTCTGCTATGAGT-39); mouseG6Pase promoter, forward (59-CCTTGCCCCTGTTTTATATGCC-39) and reverse (59-CGTAAATCACCCTGAACATGTTTG-39); or mouse PGC-1a promoter, forward (59-GCCTATGAGCACGAAAGGCT-39) and reverse (59-GCGCTCTTCAATTGCTTTCT-39).

Western Blot AnalysisWhole-cell extracts were prepared using RIPA buffer (Elpis-Biotech, Korea). Proteins from whole-cell lysates wereseparated by 10% SDS-PAGE and then transferred tonitrocellulose membranes. The membranes were probed

with indicated antibodies. Immunoreactive proteins werevisualized using an Amersham ECL kit (GE Healthcare,Piscataway, NJ), according to the manufacturer’s instructions.

qPCRTotal RNA from primary hepatocytes or liver was extractedusing an RNeasy mini-kit (Qiagen). All of the samples werecombined for the experiment. cDNA was generated bySuperscript II enzyme (Invitrogen) and analyzed by qPCRusing an SYBR green PCR kit and a TP800 Thermal CyclerDICE Real Time system (Takara). All data were normal-ized to ribosomal L32 expression.

Statistical AnalysesAll values are expressed as means6 SEM. The significancebetween mean values was evaluated by a two-tailed un-paired Student t test.

RESULTS

Hepatic SMILE Gene Expression Is Elevated byRefeeding in Normal Mice but Not in Insulin-ResistantMiceTo investigate whether SMILE plays a role in glucosehomeostasis, we monitored the effect of different nutri-tional conditions on hepatic SMILE gene expression inmouse liver. Ad libitum feeding increased SMILE expression

Figure 1—Hepatic SMILE expression is induced in the feeding condition but not in the insulin-resistant condition. A: qPCR analysis wasperformed using total RNA from mice liver samples after ad libitum feeding or 6-h fasting. SMILE, Pgc-1a, Pepck, G6pase, and b-actin geneswere amplified by qPCR using corresponding primers. B: C57BL/6 mice (n = 4) were fasted for 6 h and refed for 1–6 h. Western blot analysis(left) showing SMILE expression during refed time course. Graphical representation (right). All of the samples were combined for the Westernblot analysis. C and D: Expression of SMILE and Pgc-1a. Normal and db/db mice were fasted for 6 h and refed for 2 h. qPCR analysis wasperformed to measure the expression of SMILE and Pgc-1a using total RNA from mice liver samples (C), and Western blot analysis wasperformed using SMILE, PGC-1a, and a-tubulin antibodies (D). All of the samples were combined for the Western blot analysis. E and F:C57BL/6 mice were maintained on normal chow or HFD for 12 weeks. qPCR analysis was performed using total RNA frommice liver of normaland HFD mice to measure SMILE, Pgc-1a, and L32 expression (E), and Western blot analysis was performed using SMILE, PGC-1a, anda-tubulin antibodies (F). Error bars show 6 SEM. *P < 0.05, **P < 0.01 by two-tailed Student t test. NCD, normal chow diet.

64 SMILE Inhibits Gluconeogenesis Diabetes Volume 65, January 2016

in liver compared with 6-h-fasted mice (Fig. 1A). For furtherconfirmation of the induction of SMILE expression inthe refed condition, protein levels of SMILE were mea-sured in the liver from fasted and refed mice. Asexpected, hepatic SMILE protein levels were increasedby refeeding in a time-dependent manner, while PGC-1aprotein levels were decreased (Fig. 1B). These resultsindicated that SMILE expression may be elevated bya signal induced by feeding. On the basis of SMILEgene expression in different nutritional conditions,we investigated whether insulin signaling is responsi-ble for the modulation of hepatic SMILE gene expres-sion in vivo. To confirm this, we examined whetherSMILE expression is also increased by refeeding ininsulin-resistant mouse models. qPCR analysis showedthat the expression of hepatic SMILE was significantlyhigher in the refed conditions in normal mice but notin db/db mice (Fig. 1C). A similar pattern of SMILE pro-tein levels was observed in response to fasting and refeed-ing conditions in normal versus db/db mice (Fig. 1D).Furthermore, SMILE mRNA (Fig. 1E) and protein levels(Fig. 1F) were decreased in the livers of HFD mice com-pared with those of normal chow diet mice. Collectively,

these results demonstrate that hepatic expression ofSMILE was tightly regulated by the nutritional condi-tion in normal mice but not in insulin-resistant mousemodels.

Insulin Receptor/PKB Signaling Pathway RegulatesSMILE Gene Expression During RefeedingOn the basis of different SMILE gene expression innormal mice and insulin-resistant mouse models, insulinsignaling would be expected to modulate hepatic SMILEgene expression in vivo. To examine this hypothesis, weinjected wild-type mice with insulin and measured hepaticSMILE gene expression levels. As expected, insulin in-jection led to a significant induction of hepatic SMILEmRNA and protein in wild-type mice (Fig. 2A and B).Therefore, we hypothesized that if insulin activatesSMILE gene expression during refeeding condition, thenliver-specific insulin receptor knockout (LIRKO) mice andprotein kinase B (PKB) b-deficient (PKBb2/2) miceshould be resistant to these effects. To examine this hy-pothesis, we measured the expression levels of SMILEafter fasting and refeeding in wild-type and LIRKOmice. LIRKO and PKBb2/2 mice exhibit elevated levels

Figure 2—Insulin receptor signaling is required for SMILE gene induction during refeeding. A and B: Expression of hepatic SMILE in mice(n = 4) injected with insulin. qPCR analysis was performed using total RNA from mice liver injected with insulin. SMILE and b-actin geneswere amplified using specific primers for PCR (A). Western blot analysis (left) and graphical representation (right) showing SMILE expressionin liver and phosphorylated PKB (p-PKB) levels were used for positive control of insulin signaling (B). C and D: LIRKO mice exhibit markedreduction of hepatic SMILE gene expression. Normal and LIRKO mice were fasted for 6 h and refed for 2 h. SMILE and Pgc-1a geneexpression was analyzed using qPCR analysis (C) and Western blot (D). E and F: Hepatic expression of SMILE in PKBb2/2 mice. Wild-typeand PKBb2/2 mice were fasted for 6 h and refed for 2 h. SMILE and Pgc-1a gene expression was analyzed using qPCR analysis (E) andWestern blot analysis (F ). All of the samples were combined for the Western blot analysis. Error bars show6 SEM. n.s, not significant. *P<0.05, **P < 0.01 by two-tailed Student t test.

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of plasma insulin, glucose, and hepatic gluconeogenicgene expression during refeeding compared with WT mice(21). Although hepatic SMILE gene expression was signif-icantly increased by refeeding in wild-type mice, the he-patic SMILE mRNA and protein levels were not changedby fasting or refeeding in LIRKO mice (Fig. 2C and D). Incontrast, the basal expression of hepatic PGC-1a washigher in LIRKO mice than in wild-type mice but notaltered during fasting and refeeding. To further verifywhether PKB signal pathway is involved in the inductionof hepatic SMILE gene expression in vivo, we measuredthe expression levels of SMILE after fasting and refeedingin both PKBb+/+ and PKBb2/2 mice. Consistent with SMILEgene expression pattern in LIRKO mice, the hepaticSMILE mRNA and protein levels were not altered byrefeeding in PKBb2/2 mice compared with PKBb+/+

mice (Fig. 2E and F). Taken together, these results dem-onstrate that insulin signaling via PKB is responsible forthe induction of hepatic SMILE gene expression duringrefeeding.

Insulin Signaling Activates the SMILE Expression at theTranscriptional LevelTo verify whether insulin activates SMILE expression atthe transcriptional level, we performed transient trans-fection assay. Insulin treatment significantly activatedSMILE promoter activity in a dose-dependent manner(Fig. 3A). We also found that insulin triggered induction

of SMILE levels in a time-dependent manner (Fig. 3B).Consistent with the increased SMILE mRNA expres-sion, exposure of mouse primary hepatocytes to insulinalso increased the protein levels of SMILE in a time-dependent manner (Fig. 3C). Based on the ability of in-sulin to enhance the hepatic SMILE gene expression, weexamined whether PKB increases SMILE gene expressionusing a constitutively active form of PKB adenovirus. Ad-enoviral overexpression of constitutively active PKB stronglyincreased SMILE mRNA and protein levels (Fig. 3D and E).Because insulin-induced protein modification such as phos-phorylation often changes protein amounts by triggeringtheir nuclear export (5–8), we examined the effects ofinsulin on subcellular localization of SMILE. Insulin treat-ment triggered increased accumulation of SMILE in thenucleus in a time-dependent manner, whereas the amountsof SMILE in the cytoplasm were not altered (Fig. 3F).Interestingly, forskolin treatment decreased the basaland insulin-stimulated SMILE gene expression (Fig.3G). A previous report demonstrated that SREBP-1cincreased SMILE gene expression in rat insulinoma cells(INS-1) (24). Consistent with that report, adenoviral over-expression of SREBP-1c dose dependently increasedSMILE gene expression in mouse primary hepatocytes(Supplementary Fig. 1A). Moreover, insulin-inducedSMILE expression was blocked by overexpression of dn-SREBP-1c (Supplementary Fig. 1B). Collectively, these re-sults indicated that insulin/PKB signal pathway increases

Figure 3—Insulin increases SMILE gene expression at a transcriptional level. A: AML12 cells were cotransfected with SMILE promoter andtreated with vehicle or insulin. B and C: Insulin effects on SMILE gene expression in primary hepatocytes. Primary hepatocytes werecultured for 12 h under serum starvation. The cells were then treated with insulin (100 nmol/L) for 0.5–9 h. SMILE gene expression wasanalyzed using qPCR analysis (B). Western blot analysis (left) and graphical representation (right) showing hepatic SMILE expression andphosphorylated PKB (p-PKB) levels were used for positive control of insulin signaling (C ). D and E: Overexpression of PKB results in theinduction of SMILE gene expression. Ad-GFP and Ad-PKBca (constitutive active form) were infected in primary hepatocytes. SMILE geneexpression was analyzed using qPCR analysis (D). Western blot analysis (left) and graphical representation (right) showing SMILE expres-sion levels (E ). F and G: Nuclear expression of SMILE by insulin. Immunoblot of SMILE in cytosolic (CE) and nuclear (NE) fraction fromAML12 cells exposed to insulin (100 nmol/L) and forskolin (Fsk) (10 mmol/L). Cytoplasmic (CE) and nuclear (NE) fraction was indicated.a-tubulin, cytoplasmic protein loading control; LaminA, nuclear protein loading control. Error bars show 6 SEM. *P < 0.05, **P < 0.01 bytwo-tailed Student t test.

66 SMILE Inhibits Gluconeogenesis Diabetes Volume 65, January 2016

hepatic SMILE gene expression at the transcriptional levelrather than at the level of protein modification.

SMILE Decreases HNF4a-Induced GluconeogenicGene Expression via Competing With PGC-1aPGC-1a is induced by cAMP/cAMP-dependent protein ki-nase (PKA) signaling in hepatocytes and is strongly increasedin the liver by fasting (25). Here, we found that hepatic PGC-1a was expressed in pattern opposite that of SMILE andwas significantly elevated in both LIRKO and PKBb2/2

mice. Having seen the opposite expression pattern be-tween SMILE and PGC-1a, we then examined the effect ofSMILE on PGC-1a transcriptional activity. SMILE inhibitedPGC-1a–mediated G6Pase and Pepck promoter activity(Fig. 4A). Moreover, PGC-1a–mediated G6Pase and Pepckexpression and hepatic glucose production were signifi-cantly decreased by SMILE (Fig. 4B and C). A previousreport demonstrated that PGC-1a activates G6Pase andPepck gene expression by functional interaction withHNF4a (8). We have also reported that SMILE interactswith HNF4a and inhibits transcriptional activity of HNF4a(16). For exploration of whether PGC-1a–induced HNF4atranscriptional activity is repressed by SMILE, AML12 cellswere cotransfected with HNF4a, PGC-1a, and SMILEexpression vector together with gluconeogenic genepromoter-reporter constructs. HNF4a-mediated induc-tion of G6Pase and PEPCK promoter activity was signifi-cantly decreased by SMILE but was recovered by coexpressionof PGC-1a (Fig. 4D).

Next, to examine whether SMILE can affect the PGC-1aoccupancy on the HNF4a binding region of the G6Pase andPepck promoter in different nutritional conditions, we per-formed ChIP assays using fasted or refed mouse liver tis-sues. We observed increased occupancy of PGC-1a in theHNF4a binding region of the G6Pase and Pepck promoterin the fasted condition compared with the refed condi-tion, whereas the occupancy of SMILE was significantlyincreased in the refed condition, which is completely oppo-site the occupancy of PGC-1a (Fig. 4E). Moreover, over-expression of SMILE significantly decreased PGC-1aoccupancy in the HNF4a binding region of the G6Paseand Pepck promoter. In contrast, overexpression of PGC-1a decreased SMILE occupancy in the HNF4a binding re-gion (Fig. 4F). These results suggest that SMILE competeswith PGC-1a in forming a complex with HNF4a on G6Paseand Pepck promoter. Moreover, adenoviral overexpression ofSMILE markedly inhibited HNF4a-mediated G6Pase andPepck gene expression (Fig. 4G) or hepatic glucose produc-tion (Fig. 4H). We have also assessed the effect of SMILE onglycolytic gene expression. Overexpression of HNF4a in-creased the glucokinase (Gck) gene expression, whereasHNF4a-induced Gck gene expression was not affected bySMILE overexpression. Moreover, insulin-induced Gck ex-pression was not altered by knockdown of SMILE (Supple-mentary Fig. 2A and B). These results indicate that SMILEspecifically affects gluconeogenesis in the liver. A previousreport showed that activation of FOXO1 by PGC-1a is

required for gluconeogenic gene expression (20). We foundthat SMILE inhibits FOXO1-mediated hepatic gluconeo-genic gene promoter activity or expression. Moreover,SMILE directly interacts with FOXO1 and competes withPGC-1a to inhibit FOXO1 transcriptional activity (Supple-mentary Fig. 3A–D). Taken together, these results demon-strate that SMILE regulates the HNF4a-mediated PEPCKand G6Pase gene expression via competing with PGC-1aand that stimulatory effect of PGC-1a on FOXO1 is alsoinhibited by SMILE.

Knockdown of SMILE Gene Expression CausesHyperglycemia in MiceThe action of insulin in suppression of gluconeogenesisoccurs rapidly via phosphorylation or dephosphorylationof its target molecules, whereas SMILE is regulated byinsulin through gene transcription level. We assessed theeffect of insulin-mediated SMILE gene expression onhepatic gluconeogenesis in early or delayed response toinsulin. In the early response to insulin, knockdownof SMILE did not show any significant effect on insulin-mediated repression, whereas insulin-mediated repressionof hepatic glucose output (Fig. 5A) and gluconeogenic geneexpression (Fig. 5B) were significantly relieved byknockdown of SMILE in the delayed time. These resultsindicate that prolonged inhibition of hepatic gluconeo-genesis by insulin occurs via induction of SMILE expres-sion at the later time point. Next, we hypothesized thatalteration in SMILE gene expression would affect hepaticglucose metabolism in vivo. To explore the action ofhepatic SMILE in vivo, we injected mice with an adeno-virus expressing (Ad-)shRNA against SMILE. As expected,knockdown of endogenous SMILE in the liver eliciteda marked increase of fasting blood glucose levels in mice(Fig. 5C). However, plasma insulin levels were unaltered inAd-shSMILE–injected mice compared with Ad-unspecificcontrol–injected mice (Fig. 5D). Moreover, we confirmeda marked induction of hepatic gluconeogenic gene expres-sion such as Pepck and G6Pase upon SMILE knockdown(Fig. 5E). Next, we further investigated whether ablationof endogenous SMILE affect glucose excursion, insulin sen-sitivity, and hepatic glucose production. During an intra-peritoneal glucose tolerance test, glucose tolerance wassignificantly attenuated in Ad-shSMILE–injected micecompared with control mice (Fig. 5F). However, ablationof endogenous SMILE did not lead to the significantchanges in insulin sensitivity as observed by insulin toler-ance test in vivo (Fig. 5G). To examine the impact of abla-tion of endogenous SMILE on hepatic glucose production,we also performed a pyruvate tolerance test. Ablation ofendogenous SMILE elevated the pyruvate-dependent in-crease of blood glucose levels (Fig. 5H).

Overexpression of SMILE in Normal Mice ImprovesGlucose Tolerance Without Markedly Affecting FastingBlood Glucose LevelBased on the fact that ablation of SMILE increasesblood glucose levels or hepatic gluconeogenesis, we

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suspected that induction of SMILE would have effectson hepatic glucose metabolism in normal mice. Adeno-virus-mediated expression of SMILE did not elicita significant change in overnight fasting blood glucoselevels or liver glycogen contents (Fig. 6A and B). On the

contrary, overexpression of SMILE significantly im-proved glucose tolerance and reduced gluconeogenesisas shown by improved pyruvate tolerance and reducedgluconeogenic gene expression (Fig. 6C–E). In addition,overexpression of SMILE did not cause significant

Figure 4—SMILE inhibits HNF4a-mediated hepatic gluconeogenesis via competing with PGC-1a. A: SMILE decreases PGC-1a–inducedPEPCK and G6Pase promoter activity. AML12 cells were cotransfected with PEPCK-Luc, G6Pase-Luc, PGC-1a, and SMILE expressionvector. B: Primary hepatocytes were infected with Ad-GFP, Ad-PGC-1a, and Ad-SMILE for 24 h before cell harvest. Expression of Pepckand G6pase was analyzed by qPCR. C: SMILE decreases PGC-1a–induced hepatic glucose production. Glucose output assay wasperformed using Ad-PGC-1a– and Ad-SMILE–infected primary hepatocytes. D: SMILE competes with PGC-1a to inhibit HNF4a transcrip-tional activity. AML12 cells were cotransfected with PEPCK-Luc, G6Pase-Luc, HNF4a, PGC-1a, and SMILE expression vector. E: C57BL/6mice (n = 3) were fasted or fed for 12 h, and ChIP assay was performed using mice liver sample. F: AML12 cells were cotransfected withHA-PGC-1a, and FLAG-SMILE and ChIP assay were performed. G: Overexpression of SMILE results in the decrease of HNF4a-inducedgluconeogenic gene expression. Primary hepatocytes were infected with Ad-GFP, Ad-HNF4a, Ad-PGC-1a, and Ad-SMILE for 24 h, andgluconeogenic gene expression was analyzed by qPCR. H: SMILE decreases HNF4a-induced hepatic glucose production. Primary he-patocytes were infected with Ad-HNF4a and Ad-SMILE for 24 h, and glucose output assay was performed using glucose-free mediasupplemented with gluconeogenic substrate sodium lactate (20 mmol/L) and sodium pyruvate (1 mmol/L). Error bars show 6 SEM. *P <0.05 by two-tailed Student t test.

68 SMILE Inhibits Gluconeogenesis Diabetes Volume 65, January 2016

induction of insulin signaling in the liver or in the mus-cle of normal chow-fed mice (Supplementary Fig. 4Aand B). These results indicate that SMILE significantlyimproved glucose tolerance or pyruvate tolerance with-out affecting glycogen levels in normal chow diet–fedmice.

SMILE Decreases Gluconeogenic Gene Expressionand Improves Hyperglycemia in Diabetic Mice ModelsWe next hypothesized that alteration in SMILE expres-sion would affect hepatic glucose metabolism in di-abetic mice. To further assess the functional consequencesof SMILE-mediated repression in gluconeogenic geneexpression and improvement of blood glucose level,we performed adenoviral overexpression of SMILEin db/db and HFD mice. The Ad-GFP– and Ad-SMILE–injected mice showed similar body weights (Fig. 7A),

but overexpression of SMILE significantly reduced fastingblood glucose levels in db/db and HFD mice (Fig. 7B).Moreover, we observed a significant improvement of glu-cose tolerance and pyruvate tolerance without affectinginsulin sensitivity in Ad-SMILE–injected db/db or HFDmice compared with Ad-GFP–injected db/db or HFDmice (Fig. 7C-E). Concomitantly, there was a significantreduction in mRNA levels of hepatic gluconeogenic gene,such as G6Pase, Pepck, and Pgc-1a, in Ad-SMILE–injecteddb/db or HFD mice compared with Ad-GFP–injected db/dbor HFD mice (Fig. 7F and G). However, overexpression ofSMILE did not lead to significant enhanced insulin signal-ing either in the liver or in the muscle of HFD mice(Supplementary Fig. 4C and D). Taken together, theseresults suggest that SMILE directly regulate hepatic glu-cose metabolism in diabetic mice. Therefore, activation ofSMILE could be a new therapeutic approach to treat

Figure 5—Ablation of SMILE leads to hyperglycemia in mice. A and B: Knockdown of SMILE rescues insulin-mediated repression hepaticglucose production or gluconeogenic gene expression. Primary hepatocytes were infected with Ad-US or Ad-shSMILE for 24 h and treatedwith insulin for 2–12 h with glucagon. Glucose output assay was performed using glucose-free media supplemented with gluconeogenicsubstrate sodium lactate (20 mmol/L) and sodium pyruvate (1 mmol/L) (A). Gluconeogenic gene expression was analyzed by qPCR (B).C–H: Knockdown of SMILE induces hepatic gluconeogenic genes and fasting blood glucose levels. Ad-US or Ad-shSMILE was injected viathe tail vein into mice (n = 5–9). Blood glucose levels measured at day 4 after Ad-US or Ad-shSMILE injection (C). Insulin levels measured atday 4 after Ad-US or Ad-shSMILE injection (D). qPCR analysis of gluconeogenic gene expression using total RNA isolated from mouse liverafter Ad-US or Ad-shSMILE injection (E ). Mice were injected with 2 g/kg i.p. glucose, 1 g/kg i.p. pyruvate, or 1 unit/kg i.p. insulin on day 6after adenoviral injection for glucose tolerance test (GTT) (F ), insulin tolerance test (ITT) (G), and pyruvate tolerance test (PTT) (H). Plasmaglucose or insulin was measured on blood drawn from the tail vein at designated time points. Error bars show 6 SEM. n.s, not significant.*P < 0.05, **P < 0.01, ***P < 0.005 by two-tailed Student t test.

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diabetes by suppressing gluconeogenesis and amelioratinghyperglycemia.

DISCUSSION

Insulin is the major hormone for the regulation of hepaticglucose metabolism by suppressing the expression ofgluconeogenic enzyme genes (7,9,20). SMILE expressionwas increased in the refeeding condition but not in thefasting condition or insulin-resistant status, i.e., in HFDand db/db mice. Thus, it seemed that the insulin/PKBpathway plays a major role in the regulation of SMILEexpression. In support of this hypothesis, we found thatinsulin increases hepatic expression of SMILE both invitro and in vivo. However, the identity of the transcrip-tion factor that mediates activation of SMILE gene ex-pression by insulin signaling is still elusive and requiresfurther exploration.

One potential candidate transcription factor for medi-ating insulin signaling is SREBP-1c, a well-known factor inthe transcriptional activation of lipogenesis. Indeed, SREBP-1c increased SMILE gene expression in rat insulinoma cells(INS-1) (24). In primary hepatocytes, adenoviral overex-pression of SREBP-1c increased SMILE gene expressionand insulin-induced SMILE expression was blocked bydn-SREBP-1c. These results suggest that insulin-mediatedinduction of SREBP-1c expression could increase SMILEgene expression in hepatocytes. Meanwhile, our previousstudy suggested that AMPK-induced SMILE decreasesSREBP-1c gene expression (26). We speculated that thephysiological induction of SMILE expression mainly re-presses gluconeogenesis under feeding conditions, whilesupraphysiological activation of SMILE expression couldalso reduce SREBP-1c expression and the subsequent

lipogenic program. Alternatively, SMILE might be criticalin fine-tuning the levels of fatty acid biosynthesis in theliver under feeding conditions. Reduced expression ofSMILE in the livers of insulin resistance models couldthus be associated with increased triacylglycerol synthe-sis in this setting. Taken together, insulin-mediated in-duction of SREBP-1c increases SMILE gene expression,which could decrease SREBP-1c expression via negativefeedback mechanism. Under normal physiological condi-tions, SREBP-1c–mediated SMILE induction may inhibitthe SREBP-1c gene expression to maintain lipid and glu-cose homeostasis.

While SMILE gene expression was significantly in-creased by refeeding in normal mice, LIRKO and PKBb2/2

mice did not display induction of SMILE gene expressionduring refeeding. Thus, SMILE could be the nutritionalstatus sensing nuclear corepressor by insulin signalingthat represses gluconeogenesis in the fed status. Collec-tively, we suggest that enhanced expression of SMILE inthe fed state is another critical mechanism by which in-sulin regulates hepatic gluconeogenesis. We have alsoshown that activation of AMPK by curcumin, a polyphenolcompound, increases SMILE gene expression, which leadsto the SMILE-mediated repression of CREBH transcrip-tional activity via competing with PGC-1a (17). Thesedata suggest that pharmacological activation of SMILEexpression can be used as a new therapeutic strategy torelieve hyperglycemia. Genetically altered animal modelsof SMILE are also needed to fully understand the physi-ological and pathological role of SMILE in hepaticgluconeogenesis.

Gluconeogenic enzyme gene expression is regulated byboth transcription factors and coactivator, such as PGC-1a

Figure 6—Minimal effect of SMILE overexpression in normal mice. A–E: C57BL/6 8-week-old mice (n = 6–8) injected with Ad-GFP or Ad-SMILE. Overnight fasting blood glucose levels (A) and glucagon levels (B) were measured in adenovirus GFP– or SMILE-injected normalmice. Mice were injected with 2 g/kg i.p. glucose or 2 g/kg i.p. pyruvate on day 6 after adenoviral injection for glucose tolerance test (GTT)(C) and pyruvate tolerance test (PTT) (D). Expression of Pepck and G6pase was analyzed by real-time quantitative RT-PCR in Ad-GFP– orAd-SMILE–injected mice (E). Error bars show 6 SEM. n.s, not significant. *P < 0.05, **P < 0.01 by two-tailed Student t test.

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(8). In this study, SMILE inhibits HNF4a- or FOXO1-mediated hepatic gluconeogenic genes promoter activityor expression via competing with PGC-1a. In addition,previous report revealed that SMILE inhibits GR, whichplays an important role in regulation of gluconeogenicgenes expression such as PEPCK and G6Pase (16). Thus,SMILE could function as a universal transcriptional co-repressor for PGC-1a–interacting transcription factorssuch as HNF4a, FOXO1, and GR. A previous reportshowed that insulin and PKB inhibit FOXO1 and PGC-1a

activity by a phosphorylation-dependent mechanism (5,20,27).In the immediate early response of insulin, gluconeogenesisis regulated by classical phosphorylation mechanism.Here, we found the role of the delayed effect of insulin onthe regulation of gluconeogenesis via induction of core-pressor SMILE. These results suggest that SMILE has animportant role in delayed action of insulin, and theinduction of SMILE is another way by which insulincounteracts the action of PGC-1a in the regulation ofhepatic glucose metabolism. In this study, we used the

Figure 7—SMILE ameliorates hyperglycemia in db/db and HFD mice. A–G: Recombinant Ad-GFP or Ad-SMILE was delivered by tail veininjection to db/db and HFD mice (n = 4–7). Mice were killed at day 7 after Ad-GFP or Ad-SMILE injection. Body weight (A) or blood glucoselevels (B) were measured in Ad-GFP– or Ad-SMILE–injected mice. Ad-GFP– or Ad-SMILE–injected mice were fasted for 4 h and injectedwith 2 g/kg i.p. glucose, 1 unit/kg i.p. insulin, or 1.5 g/kg i.p. pyruvate for glucose tolerance test (GTT) (C ), pyruvate tolerance test (PTT) (D),or insulin tolerance test (ITT) (E). Expression of Pepck, G6pase, Pgc-1a, and SMILE was analyzed by real-time quantitative RT-PCR in Ad-GFP– or Ad-SMILE–injected db/db (F ) or HFD (G) mice. All data were normalized to ribosomal L32 expression. Error bars show 6 SEM.*P < 0.05, **P < 0.01, ***P < 0.005 by two-tailed Student t test.

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adenoviral approach to explore the regulatory role ofSMILE in glucose homeostasis. Thus, genetically alteredanimal models of SMILE are also needed to understandthe physiological and pathological role of SMILE in he-patic gluconeogenesis.

In the fasting condition, PKA/PGC-1a pathway in-creases hepatic gluconeogenesis. In the fed condition,the PKB/SMILE pathway turns off the PGC-1a–inducedhepatic gluconeogenesis via a competitive binding mech-anism (Fig. 8). Overall, our observations provide insightinto a novel mechanism for insulin-mediated regulationin hepatic glucose metabolism that can be used as a noveltherapeutic approach for the treatment of hyperglycemiain diabetes.

Acknowledgments. This paper is dedicated to the memory of the late Dr.Richard W. Hanson (Case Western Reserve University) in recognition of his manygreat contributions to science.Funding. This work was supported by a National Creative Research Initiativesgrant through the National Research Foundation of Korea (NRF) (20110018305)funded by the Korean government (Ministry of Science, ICT and Future Planning).S.-H.K. is supported by the NRF (grant nos. NRF-2012M3A9B6055345, NRF-2015R1A5A1009024, and NRF-2015R1A2A1A01006687) and Korea University.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.-M.L., W.-Y.S., H.-S.C., and S.-H.K. designedmost of the experiments. J.-M.L., W.-Y.S., H.-S.H., K.-J.O., Y.-S.L., D.-K.K., S.C.,and B.H.C. performed the experiments. J.-M.L. wrote the manuscript. R.A.H.,C.-H.L., S.-H.K., and H.-S.C. contributed to discussion and review and editing ofthe manuscript. S.-H.K. and H.-S.C. are the guarantors of this work and, as such,

had full access to all the data in the study and take responsibility for the integrityof the data and the accuracy of the data analysis.

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