SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling

LIVER INJURY/REGENERATION

SIRT1 Controls Liver Regeneration by Regulating BileAcid Metabolism Through Farnesoid X Receptor and

Mammalian Target of Rapamycin SignalingJuan L. Garc�ıa-Rodr�ıguez,1 Luc�ıa Barbier-Torres,1 Sara Fern�andez-�Alvarez,1 Virginia Guti�errez-de Juan,1

Mar�ıa J. Monte,2 Emina Halilbasic,3 Daniel Herranz,4 Luis �Alvarez,5 Patricia Aspichueta,6 Jose J.G. Mar�ın,2

Michael Trauner,3 Jose M. Mato,1 Manuel Serrano,4 Naiara Beraza,1,7* and Mar�ıa Luz Mart�ınez-Chantar1*

Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis,gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe thatSIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy(PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRTmice showed increased mortality,impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. Thedamaging phenotype in SIRTmice correlated with impaired farnesoid X receptor (FXR) activitydue to persistent deacetylation and lower protein expression that led to decreased FXR-targetgene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increasedCyp7A1. Next, we show that 24-norUrsodeoxycholic acid (NorUDCA) attenuates SIRT proteinexpression, increases the acetylation of FXR and neighboring histones, restores trimethylationof H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis.Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increasedsurvival and hepatocyte proliferation. Furthermore, a leucine-enriched diet restored mamma-lian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to anoverall lower BA production through SHP-inhibition of Cyp7A1 and higher transport (BSEP)and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found thathuman hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which cor-related with the absence of FXR, suggesting its oncogenic potential. Conclusion: We defineSIRT1 as a key regulator of the regenerative response in the liver through posttranscriptionalmodifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data sug-gest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis bypersistent FXR deacetylation. (HEPATOLOGY 2014;59:1972-1983)

Sirtuin 1 (SIRT1) is a class III NAD1-dependenthistone deacetylase that tightly regulates lipid, glu-cose, and bile acid (BA) metabolism.1,2 SIRT1 is

activated in situations of low energy availability and links

nutritional status with metabolic homeostasis.1 SIRT1regulates adenosine monophosphate activated proteinkinase (AMPK) by deacetylation of LKB1,1 which areinvolved in tissue repair processes.3 Contrary to SIRT1,

Abbreviations: BA, bile acid; BSEP, bile salt export pump; DCA, deoxycholic acid; FXR, farnesoid X receptor; HCC, hepatocellular carcinoma; mTOR, mamma-lian target of rapamycin; NorUDCA, 24-norUrsodeoxycholic acid; NR, nuclear receptor; PH, partial hepatectomy; SHP, small heterodimer partner; SIRT1, Sirtuin1.

From the 1Department of Metabolomics, CIC bioGUNE, Centro de Investigaci�on Biom�edica en Red de Enfermedades Hep�aticas y Digestivas (Ciberehd), Tech-nology Park of Bizkaia, Bizkaia,, Spain; 2Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, CIBERehd,, University of Sala-manca, Salamanca, Spain; 3Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III,Medical University Vienna, Vienna, Austria; 4Spanish National Cancer Research Center (CNIO), Madrid, Spain; 5Pediatric Liver Service, La Paz UniversityHospital, Madrid, Spain; 6Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country UPV/EHU, Leioa, Spain; 7Biochemis-try and Molecular Biology Department, University of the Basque Country (UPV/EHU), Bilbao, Spain.

Received June 13, 2013; accepted December 9, 2013.Supported by grants from the Instituto de Salud Carlos III; FIS, PS12/00402 (to N.B.), NIH AT-1576 (to M.L.M.-C., and J.M.M.), ETORTEK-2010 (to

M.L.M.-C), Educaci�on Gobierno Vasco 2011 (to M.L.M.-C), PI11/01588 (to M.L.M.-C). Plan Nacional SAF2011-29851 (to JMM), Plan Nacional SAF2010-15517 (to JJ.G.-M). Basque Government IT-336-10 (to PA) and UFI 11/20 (to PA). N.B. is funded by the Program Ramon y Cajal (Ministry of Economy andCompetitiveness, Spain). Ciberehd is funded by the Instituto de Salud Carlos III.

*These authors contributed equally to this work.

1972

mammalian target of rapamycin (mTOR) is activated inhigh-energy conditions and controls cell growth and pro-liferation.4 mTORC1 promotes protein synthesis by acti-vating the S6 ribosomal protein and this axis is essentialto regulate the cell cycle during liver regeneration afterpartial hepatectomy (PH).5 BA is also essential for theregeneration of the liver after PH,6 although, when pres-ent in excess, BA can be toxic and promote hepatocytedeath.7 Therefore, a fine regulation of BA metabolism isessential to preserve liver homeostasis and a properresponse to injury. The orphan nuclear receptor (NR)farnesoid X receptor (FXR; NR1H4) is the master regu-lator of BA, lipid, and glucose metabolism.8 SIRT1directly modulates FXR activity by deacetylation of thisNR and neighboring histones that strictly control targetgene transcription.9,10 In the present work we describethat modulation of SIRT1 is essential for the regenerativeresponse in the liver through mechanisms involving theregulation of (1) FXR and (2) mTOR signaling path-ways by dynamic acetylation/deacetylation, which overallmaintain BA homeostasis.

BA-mediated toxicity is also involved in the patho-genesis of cirrhosis from diverse etiologies.11,12 Therole of SIRT during tumorigenesis remains controver-sial, as both pro- and antioncogenic properties havebeen described.13,14 Here we show that increasedSIRT1 expression correlated with a low presence ofFXR in human HCC samples, supporting the signifi-cance of SIRT1 deacetylase activity in regulating BAhomeostasis and the response to liver injury.

Overall, our data defines SIRT1 as a key regulatorof the regenerative response of the liver, controllingBA homeostasis, protein synthesis, and cell prolifera-tion through deacetylation of FXR and histones andregulation of mTOR. Importantly, our results under-score the implication of mTOR in regulating BAmetabolism as a negative regulator of SIRT1.

Materials and Methods

Experimental Procedures in Animals. SIRT1-overexpressing animals (SIRT) were generated asdescribed14 in a C57/Bl6J background. Males from

8-12 weeks of age were treated and used according tothe guidelines of the National Academy of Sciences(National Institutes of Health publication 86-23,revised 1985). SIRT mice were fed with a diet enrichedin 3% leucine or a diet containing 0.5% w/w 24-norursodeoxycholic acid (NorUDCA)15,16 for 2 or 3weeks, respectively, before 2/3 PH.

Materials. Recombinant tumor necrosis factor(TNF) was obtained from Peprotech. Deoxycholic acid(DCA) was purchased from Sigma-Aldrich.

BA Determination. Bile acids were extracted fromliver samples and analyzed by high-performance liquidchromatography-tandem mass spectrometry as previ-ously reported.17

Analysis of Gene Expression. Messenger RNA(mRNA) was extracted from snap-frozen livers usingTriZol Reagent (Invitrogen). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)was performed using SYBR Green reagent (QuantasBiosciences) in a MyiQ single-color real-time PCRdetection system (BioRad). Gene expression was nor-malized with GAPDH and shown in times versuswild-type (WT) expression before PH (basal). Primerscan be provided upon request. Additional Materialsand Methods are provided as Supporting Material.

Results

Overexpression of SIRT1 Impairs Hepatocyte Pro-liferation After PH. Liver damage leads to a regener-ative response that restores organ mass and function.18

During liver regeneration, the expression of SIRT1 wasdown-regulated at the initial stages to recover later at48 hours after PH (Fig. 1A), highlighting the regula-tion of SIRT1 throughout the regenerative response.

To define the role of SIRT1 during liver regenera-tion, we used transgenic mice that moderately overex-press SIRT1 (SIRT) (Supporting Fig. 1A,B). SIRTmice showed poor prognosis after PH, as 40% ofSIRT mice died within the first 48 hours after resec-tion (Fig. 1B). Hepatocyte proliferation was signifi-cantly attenuated in SIRT mice as shown bybromodeoxyuridine (BrdU) staining (Fig. 1C;

Address reprint requests to: Naiara Beraza, Ph.D., Department of Metabolomics, CIC bioGUNE, Centro de Investigaci�on Biom�edica en Red de EnfermedadesHep�aticas y Digestivas (Ciberehd), Technology Park of Bizkaia, 48160-Derio, Bizkaia, Spain. E-mail: [email protected]; fax 0034/944061304.

Copyright VC 2014 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.26971Potential conflict of interest: The authors declare that they have no competing financial interests. The Medical University of Graz has filed a patent on the

medical use of norUDCA and Michael Trauner is listed as co-inventor. Prof. Trauner is also on the speakers’ bureau for and received grants from Falk and Roche.He advises Phenex and Amgen. He is on the speakers’ bureau for MSD and Gilead. He received grants from Albireo and Intercept.

Additional Supporting Information may be found in the online version of this article.

HEPATOLOGY, Vol. 59, No. 5, 2014 GARC�IA-RODR�IGUEZ ET AL. 1973

Supporting Fig. 1C) and analysis of cyclin D1, E, andA2 mRNA expression after PH (Fig. 1D). Additionally,the mitotic response was significantly impaired in SIRTmice 72h after PH as shown by p-histone H3S10 IHC(Fig. 1E; Supporting Fig. 1D). Finally, we foundimpaired activation of the mTOR pathway in SIRTmice before and after PH (Fig. 1F), in agreement withthe recently described inhibitory effect of SIRT1 overmTORC1.19 WT mice showed basal p-mTOR and p-S6 expression that was up-regulated 6 hours after PH(Fig. 1F), whereas SIRT mice showed low basal p-S6levels that were unaffected after PH. SIRT1 activates

AMPK.1 Accordingly, AMPK was basally phosphoryl-ated in SIRT mice compared to WT animals (Fig. 1F;Supporting Fig. 1E). PH triggered phosphorylation ofAMPK in WT mice, whereas in SIRT animals pAMPKexpression remained unchanged after resection (Fig. 1F;Supporting Fig. 1E). Furthermore, we found that cyto-solic shuttling of HuR, a pAMPK target protein, wasattenuated in SIRT mice after PH (Supporting Fig. 1F).HuR stabilizes the mRNA of diverse molecules involvedin cell proliferation after PH.20 Accordingly, MAT2A(Supporting Fig. 1F,G) and NOS2 (Supporting Fig.1G) expression were attenuated in SIRT mice. Overall,

Fig. 1. Overexpression of SIRT1 impairs liver regeneration in mice. (A) qPCR analysis of SIRT expression in WT animals at different timepointsafter PH. (B) Kaplan-Meier curve depicting survival of SIRT and WT mice after PH. (C) IHC using an anti-BrdU antibody (Ab) (red) and stainedwith DAPI (blue) on liver sections after PH. (D) Analysis of Cyclin D1, Cyclin E, and Cyclin A mRNA expression by qPCR. (E) IHC performed inliver sections using a phistone H3 Ab (green) versus DAPI1 nuclei (blue). (F) pmTORC1, pS6, and pAMPK levels in liver extracts analyzed bywestern blot (WB) analysis. Values are mean 6 SD. Survival curve: n 5 20 animals; *P< 0.05, **P< 0.01, ***P< 0.001 (WT versus SIRT).

1974 GARC�IA-RODR�IGUEZ ET AL. HEPATOLOGY, May 2014

these data suggest that persistent phosphorylation ofAMPK renders it nonfunctional in SIRT mice, furthercontributing to impairment of liver regeneration.

Overexpression of SIRT1 Impairs FXR-MediatedSignaling Leading to BA Accumulation and LiverInjury After PH. BA signaling is essential for liverregeneration,6 whereas the excessive presence of BA istoxic for hepatocytes, promoting apoptosis and necro-sis.7 Histological analysis of livers from SIRT miceshowed no parenchymal damage and appeared similarto WT animals prior to PH (Fig. 2A, left panels). BApool size in untreated SIRT livers was similar to WTs(Fig. 2B) but its composition was shifted towards sec-ondary BA (Supporting Table 1). The poor prognosisof SIRT mice within the first 48 hours after PH wasassociated with the presence of bile infarcts, evidencedby profuse presence of necrotic areas and inflammatorycell infiltration in the liver (Fig. 2A, right panels). Inline with bile infarcts, we found a significant increaseof total BA levels, especially conjugated, in SIRT mice48 hours after PH (Fig. 2B). BA metabolism is tightlycontrolled by FXR as it activates small heterodimerpartner (SHP), leading to inhibition of Cyp7a1. InSIRT mice, we found that FXR was strongly deacety-

lated at basal conditions (Fig. 2C; SupportingFig. 2A), which correlated with lower protein expres-sion (Fig. 2C; Supporting Fig. 2A,B). Lower acetyla-tion of FXR was associated with down-regulation ofSHP and increased Cyp7A1 expression, indicatingimpaired FXR-signaling (Fig. 2D). Finally, expressionof the canalicular export pump, bile salt export pump(BSEP) and detoxification-related enzyme Sult2a1 weresignificantly down-regulated in SIRT mice (Fig. 2D).Interestingly, BA metabolism-related gene expression inSIRT mice resembles what was found in WT animalsafter 8 hours of fasting, characterized by BA synthesisand attenuated transport (Supporting Fig. 3A). Thesedata indicate that overexpression of SIRT1 mimics a“fasting-like” status despite the fed-status of SIRTmice. In accordance, we found low glucose levels inserum from SIRT mice at basal conditions (SupportingFig. 3B). SIRT1 regulates glucose metabolism throughPGC1agluconeogenesis.21 In SIRT mice, deacetylationof PGC1a correlated with lower levels of total protein(Supporting Fig. 3C), suggesting its degradation.22

Accordingly, PEPCK and PDK4 were significantlydown-regulated in SIRT mice (Supporting Fig. 3D).Overall, these data suggest that “fasting-like” status

Fig. 2. Overexpression of SIRT1 leads to altered BA and glucose homeostasis due to dysregulation of FXR and PGC1a. (A) H&E staining onliver sections at 0 and 48 hours after PH. (B) Quantification of BA in livers from WT and SIRT mice before and 48 hours after PH. (C) Immuno-precipitation (IP) using an FXR Ab and WB analysis using acetylated lysine. WB of nuclear extracts FXR and b-actin (input). (D) Analysis of SHP,Cyp7a1, BSEP, and Sult2a1 mRNA expression by qPCR in livers at basal conditions. Values are mean 6 SD. n 5 5 animals/timepoint;*P< 0.05, **P< 0.01, ***P< 0.001 (WT versus SIRT).


characterized by dysregulation of gluconeogenesis andBA metabolism may contribute to the poor regenera-tive capacity of the liver in SIRT mice after PH.

Overexpression of SIRT1 Sensitizes the Liver andHepatocytes to BA-Induced Injury. At high concen-trations, certain species of BA induce toxicity and celldeath.7 Accordingly, BA accumulation in the regenerat-ing liver of SIRT mice correlated with profuse hepato-cyte death 48 hours after PH (Fig. 3A). Next, weperformed in vitro experiments to better define theimpact of BA in SIRT1-overexpressing hepatocytes.We observed increased apoptosis, oxidative stress, andpJNK in isolated SIRT hepatocytes when cultured inserum starvation conditions (Fig. 3B-D), supportingthe essential role of SIRT1 in protecting the cellagainst energetic stress. Furthermore, DCA promotedprofuse death of SIRT hepatocytes (Fig. 3B). Addition-ally, reactive oxygen species (ROS) was furtherincreased 4 hours and 6 hours after DCA treatment inSIRT hepatocytes (Fig. 3C). Basal phosphorylation ofJNK in SIRT hepatocytes was further sustained afterDCA, whereas transient and milder activation of JNKwas found in WT cells after treatment (Fig. 3D). Inhi-bition of JNK signaling with SP600125 significantlyreduced DCA-induced hepatocyte death and ROS for-mation in SIRT hepatocytes (Fig. 3E), supporting the

role of JNK in mediating BA-cell damage.7 Overall,these data point out that SIRT1 overexpression sensi-tizes hepatocytes to BA death.

NorUDCA Restores Hepatocyte Proliferation AfterPH in SIRT Mice. NorUDCA has strong beneficialeffects against sclerosing cholangitis and diet-inducednonalcoholic steatohepatitis (NASH) by improvingbile flow and hydrophilicity and detoxification ofBA.15,16 We fed SIRT mice with a diet enriched inNorUDCA for 3 weeks, after which PH was per-formed. Acetylation and protein expression of FXR(Fig. 4A; Supporting Fig. 4A,B) were restored inNorUDCA/SIRT mice and SHP mRNA levelsincreased (Fig. 4B). Interestingly, we found elevatedCyp7A1 mRNA levels in NorUDCA/SIRT mice(Fig. 4B), while protein expression was lower in theseanimals compared to SIRT mice (Supporting Fig.4C). As expected, NorUDCA increased expression ofbile export pumps and detoxification-related mole-cules in SIRT mice (Fig. 4B). Importantly, Nor-UDCA caused only mild restoration of acetylation ofPGC1a (Supporting Fig. 5A) and no significant dif-ferences were observed in total protein levels andPGC1a target gene expression (data not shown), sug-gesting a certain specificity of NorUDCA over FXR-signaling.

Fig. 3. Overexpression of SIRT1 sensitizes the liver and hepatocytes to BA-induced damage. (A) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on liver sections 48 hours after PH. (B) TUNEL assay on primary hepatocytes stimulated withDCA (100 lM) for 24 hours. (C) Primary hepatocytes incubated with DCFH-DA (red) before and 6 hours after stimulation with DCA. (D) WB ofwhole protein extracts using pJNK Ab. (E) Inhibition of pJNK with SP600125-protected SIRT hepatocytes from DCA-induced ROS and apoptosisas shown by DCFH-DA and TUNEL assay. n 5 5 animals/time point; in vitro experiments were performed three times, each in duplicate.


Fig. 4. NorUDCA restores FXR-signaling and promotes liver regeneration in SIRT mice after PH. (A) IP using an FXR Ab and further WB analysisusing acetylated lysine Ab of liver extracts at basal conditions. WB analysis of nuclear extracts using FXR and b-actin Abs (lower panel). (B)Analysis of BA metabolism-related gene expression by qPCR in livers. (C) Quantification of BA in livers from SIRT and NorUDCA/SIRT mice beforeand 48 hours after PH. (D) H&E staining, TUNEL assay, and BrdU staining on liver sections 48 hours after PH. (E) IHC using an anti-SIRT1 Ab.(F) WB analysis of nuclear protein extracts using H3K4me3 and H3K9me3 Ab. Values are mean 6 SD. n 5 5 animals/timepoint; *P< 0.05,**P< 0.01, ***P< 0.001 (SIRT versus NorUDCA/SIRT).


Feeding SIRT mice with NorUDCA shifted thebasal free BA composition, as 90% of the unconju-gated BA content consisted of NorUDCA. After PH,the overall free BA pool size was reduced in Nor-UDCA/SIRT compared to SIRT mice (Fig. 4C). Theconjugated BA pool was significantly reduced beforeand after PH in NorUDCA/SIRT mice (Fig. 4C).Total BA pool was basally higher in NorUDCA/SIRTmice consisting mainly of this modified BA. After PH,NorUDCA treatment reduced the total BA pool (Fig.4C), supporting the well-described impact of Nor-UDCA on promoting BA efflux and detoxification.15

In line with the shift towards a hydrophilic nontoxicBA pool (mainly consisting of NorUDCA), weobserved an absence of necrotic areas, parenchymaldegeneration, and hepatocyte cell death in NorUDCA/SIRT mice (Fig. 4D; Supporting Fig. 6A). BrdU analy-sis and qPCR of cyclins confirmed that NorUDCArestored hepatocyte proliferation 48 hours after PH inSIRT mice (Fig. 4D; Supporting Fig. 6B). mTORC1/pS6 signaling was not restored upon NorUDCA feed-ing (Supporting Fig. 6C), suggesting a potential speci-ficity over FXR-signaling. Importantly, we found thatmiR34a expression was low in SIRT mice but restoredupon NorUDCA feeding (Supporting Fig. 6D).Accordingly, NorUDCA decreased SIRT1 proteinexpression in SIRT mice (Fig. 4E; Supporting Fig. 6E).Moreover, SIRT mice exhibited increased presence ofthe trimethylated forms of both H3K4 and H3K9basally, whereas NorUDCA decreased their expressionback to WT levels (Fig. 4F). Restoration of acetylationby NorUDCA correlated with recovery of mRNAexpression of FXR in NorUDCA/SIRT mice (Support-ing Fig. 6F). Finally, all NorUDCA/SIRT mice sur-vived after PH, confirming the beneficial impact ofNorUDCA during the regenerative response of theliver in the context of SIRT1 overexpression (Support-ing Fig. 6G).

Overall, our data indicates that the beneficial effectsof NorUDCA during the regenerative process are, atleast in part, mediated by the restoration of the func-tionality of FXR through mechanisms involving tran-scriptional and translational regulation, leading to therecovery of FXR mRNA, protein, and dynamicacetylation.

Recovery of mTOR Signaling Restores BA Homeo-stasis and Liver Regeneration in SIRT Mice. m-TORC1 is directly activated by amino acids (Aa)through RagGTPases4 and leucine is the most effectiveAa in inducing this process.23 In order to restoremTOR signaling, we fed SIRT mice with a 3%leucine-rich diet (Leu) for 2 weeks, after which PH

was performed. Leu successfully restored mTORC1signaling to levels comparable to those found in WTanimals; basal S6-phosphorylation was found in Leu/SIRT mice and further increased at 6 hours and 24hours after PH (Supporting Fig. 7A,B) and AMPKactivation was also restored (Supporting Fig. 7A,B). InLeu/SIRT mice, recovery of mTORC1 signaling led toa significant improvement in survival after PH, as themortality rate was only 20% (Supporting Fig. 7C),and to normalization of hepatocyte proliferation (Fig.5A; Supporting Fig. 7D,E).

Interestingly, hematoxylin and eosin (H&E) stainingrevealed significant improvement of the liver paren-chyma in Leu/SIRT mice 48 hours after PH, and nosigns of bile infarcts were detected (Fig. 5B). Leureduced the accumulation of conjugated and total BAat 48 hours after PH in SIRT mice (Fig. 5C) andrestored FXR acetylation and total protein levels (Fig.5D; Supporting Fig. 7F-H), which correlated withincreased SHP and BSEP expression and Cyp7a1down-regulation (Supporting Fig. 7I). Interestingly,Leu lowered SIRT1 protein expression in SIRT mice(Fig. 5E; Supporting Fig. 7J) and restored the expres-sion of H3K4 and H3K9 to WT levels (Fig. 5F).Finally, Leu attenuated the increased apoptosis foundin isolated SIRT hepatocytes when cultured in serumstarvation conditions (Supporting Fig. 7K). Overall,these data indicate that restoration of the mTORC1signaling pathway circumvents the detrimental impactof SIRT1 overexpression on hepatocyte proliferationand BA accumulation. The potential mechanismsunderlying these effects rely on the impact of mTORon SIRT1-regulated acetylation of FXR and neighbor-ing histones. Also, our data point to a link betweenmTORC1 and regulation of BA metabolism.

SIRT1 Is Overexpressed in HCC Patients. Therole of SIRT during tumorigenesis remains controver-sial.13,14 We examined the expression of SIRT1 in acohort of human HCC from different etiologies.SIRT1 mRNA expression was increased in HCC sam-ples (Supporting Fig. 8A). A more detailed study ofdifferent HCC samples by immunohistochemistry(IHC) and further quantification revealed that SIRT1was significantly overexpressed in samples derived fromhepatitis C virus (HCV) and ASH (Fig. 6A). Interest-ingly, FXR expression was significantly down-regulatedin HCC human samples, both from an HCV andASH etiology (Fig. 6B). Overall, these data supportthe implication of SIRT during liver tumorigenesis ina context of BA metabolism misregulation and suggeststhat this oncogenic role might be linked to the abilityof SIRT1 to regulate FXR through deacetylation.


Fig. 5. Leucine-enriched diet recovers mTOR signaling and restores liver regeneration in SIRT mice. (A) IHC using an anti-BrdU Ab (red) andstained with DAPI (blue) showing restored proliferation in Leu/SIRT mice. (B) H&E staining of liver sections 48 hours after PH (203) (C) Bileacids were measured in livers 48 hours after PH. (D) IP using an FXR Ab and further WB analysis using acetylated lysine Ab of liver extracts atbasal conditions. WB analysis of nuclear extracts using FXR and b-actin Abs (lower panel). (E) IHC using an anti-SIRT Ab showing normalizationof SIRT levels in Leu/SIRT livers. (F) IHC showing aberrant tri-methylation of H3K4 and H3K9 in SIRT mice, which was reverted by Leu. Valuesare mean 6 SD. n 5 5 animals/timepoint; *P< 0.05, **P< 0.01, ***P< 0.001 (SIRT versus Leu/SIRT).


Discussion

SIRT1 is essential for cell metabolism control as, insituations of low energy availability (low adenosine tri-phosphate [ATP]), it promotes catalytic processes torestore cell-energy homeostasis.1,2 Here we describethat SIRT1 overexpression has a detrimental impacton liver regeneration, as poor survival and impairedproliferation were found after PH. In apparent contra-diction, recent work showed that reduced SIRT1expression in older mice correlated with impaired liverregeneration, which was restored after normalization ofSIRT levels in these aged mice.24 Interestingly, bothJin et al. and our work emphasize the critical relevanceof the fine-tuned regulation of SIRT deacetylase activ-ity for the regeneration of the liver, as either deficiencyor excess have a detrimental impact on this process.Here we show that SIRT-overexpressing mice have per-sistent deacetylase activity that profoundly alters keymetabolic responses such as (1) BA metabolism and(2) protein synthesis, highlighting the role of SIRT1 incontrolling the regenerative response of the liver.

FXR is the master regulator of BA metabolism.FXR activates SHP that represses the expression ofCyp7A1, the BA synthesis rate-limiting enzyme.8

Additionally, essential BA transporters like BSEP arepositively regulated by FXR. Little is known regardingthe mechanisms by which FXR regulates gene expres-sion, although posttranscriptional modifications(PTM) seem to play a key role. PTM occur at two lev-els: modifying the NR and its cofactors, and/or modi-

fying histones at the promoters of the NR-target genes(reviewed25). FXR is tightly regulated by p300- andSIRT1-mediated acetylation/deacetylation. Whereasacetylation is generally related to gene activation, Kem-per et al.9 showed that acetylation of FXR by p300leads to lower transactivation potential. Accordingly,deacetylation of FXR by SIRT1 allows interaction withRXR, leading to DNA binding or to its degradationby ubiquitination. Interestingly, the FXR-target geneSHP is differently regulated, as histone deacetylation,by SIRT1, of its promoter contributes to silence tran-scription.25 This underlines that the complex mecha-nism by which SIRT1 controls BA metabolismthrough deacetylation must be a dynamic process. Inaddition to the ostensible deacetylation of FXR andlow transcriptional capacity, we found aberrant methyl-ation of histones in SIRT mice, as both H3K4 andH3K9 were tri-methylated. The histone methyltrans-ferase Set9 competes with histone deacetylases tomethylate H3 on the lysine 4. H3K4me3 inhibits themethyltransferase Suv39H1, which (tri-)methylatesH3K9. This regulatory mechanism avoids simultane-ous methylation of H3K4me3 and H3K9me3, as theyhave generally opposing activities: activation andrepression of gene transcription, respectively. Sir2, theSIRT1 analog in yeast, promotes trimethylation ofH3K4 and H3K9.26,27 H3K4m3 relates with FXRactivation of target gene expression such as BSEP,28

whereas acetylation of H3K9 by p300 is essential forSHP-mediated activation of its transcriptionalactivity.25

Fig. 6. SIRT1 is highly expressed in HCC human samples. (A) IHC using SIRT1 and FXR. (B) Antibody in human HCC samples from patientswith HCV (n 5 10) and ASH (n 5 10) etiology. Quantitative assessments of IHC staining were performed using FRIDA image analysis software.Expressed as percent of positive staining per area *P< 0.05, **P< 0.01, ***P< 0.001 (control versus HCC).


Overall, we propose that SIRT1 modulates BAmetabolism through two mechanisms: (1) PTM of NR(FXR) and its cofactors (SHP) that repress target genetranscription (CYp7a1, BSEP), and (2) PTM of histo-nes permitting methylation through deacetylation.Both of these mechanisms are altered when SIRT1 isoverexpressed, leading to dysregulated FXR activityand aberrant methylation of H3K4 and H3K9.

SIRT mice show no signs of liver injury at basalconditions and have comparable levels of liver BAthan WT. However, a closer analysis shows that misre-gulation of FXR-related BA metabolism found basallycorrelates with a shift towards the enhanced presenceof secondary BA in SIRT livers. These data suggestthat the role of the intestine in BA homeostasis mightbe affected in SIRT mice. Although it is out of thescope of this work, the unbalance towards secondaryBA in SIRT mice, despite having a similar BA poolsize to WT animals, may indicate enhanced intestineelimination rate due to reduced reuptake by the ileum.Also, consistent with enhanced intestinal elimination,our data points to a role of intestinal bacteria inchanging the BA pool composition in SIRT mice.

Previous work showed that BA homeostasis is essen-tial for liver regeneration, as FXR2/2 mice showimpaired response after PH.6 Accordingly, our datasuggest that persistent deacetylation of FXR by SIRT1contributes to defective liver regeneration through mis-regulation of BA synthesis, transport, and detoxifica-tion. Consequently, accumulation of BA inregenerating livers of SIRT mice may cause severenecrosis and tissue damage, supporting the toxicity ofBA when present in excess.7 We show that BA-mediated liver injury was attenuated and liver regener-ation was fully restored in NorUDCA/SIRT animals.Our data not only confirms what was previouslydescribed regarding the therapeutic role of NorUDCAduring liver damage,15,16 but also provides newinsights in the mechanisms by which NorUDCA exertsits beneficial effects. Our results suggest that Nor-UDCA controls the acetylation status of the cell byregulating the expression of SIRT1. Thus, in agree-ment with the known regulation of SIRT1 bymiR34a,29 we found that NorUDCA recovered theexpression of this microRNA attenuating SIRT1.Overall, our data suggest that regulation of FXR byNorUDCA undergoes through transcriptional andtranslational mechanisms, involving restoration ofmRNA and protein expression, and by regulating theacetylation status in the cell through SIRT1.

Furthermore, we found that persistent deacetylationby SIRT1 promotes a “fasting-like” status in the body

characterized by increased BA synthesis throughCyp7a1. During fasting, the interaction of SIRT1 andFXR increases, whereas it is reduced upon refeedingand in the presence of BA, the natural FXR ligands.8

During fasting, SIRT1 regulates FXR through variousmechanisms: repressing gene transcription by deacety-lating histones or direct deacetylation of FXR and fur-ther ubiquitination and proteosomal degradation,which may explain the lower protein levels found inSIRT mice.

Fasting promotes activation of PGC1a by SIRT1-mediated deacetylation in order to increase gluconeo-genesis and blood glucose levels. In SIRT mice, wefound that low acetylation of PGC1a correlated withimpaired gluconeogenesis, which may be explained bypersistent deacetylation by SIRT1 and further degrada-tion by ubiquitination.30 Overall, our data regardingthe regulation of FXR and PGC1a further underlinesthe importance of SIRT1 as a regulator of the fine-tuning of the acetylation/deacetylation process, whichseems essential to orchestrate a proper response to tis-sue injury.

SIRT1 regulates the energy status of the cell throughactivation of AMPK/LKB1.1,4,19 Accordingly, SIRTmice showed phosphorylation of AMPK at basal con-ditions that was not further activated after PH, con-trary to what we observed in WT animals. Theseresults suggest that persistent phosphorylation ofAMPK renders it nonfunctional in SIRT mice, as wefound down-regulation of AMPK-target molecules likeHuR, MAT2A, and NOS2. These data support thekey role of LKB1/AMPK to mediate cell growth dur-ing liver regeneration.3

We describe that mTOR signaling was attenuated inSIRT mice throughout the regenerative response, sup-porting the role of SIRT1 as a negative regulator ofmTOR.19 Interestingly, we provide evidence of a feed-back regulation of SIRT1 by mTOR, as Leu attenuatedSIRT1 expression in SIRT mice. Our data links the pre-viously described regulation of SIRT1-deacetylase activ-ity by mTOR-mediated phosphorylation31 with thedegradation of SIRT by phosphorylation.32 Leu restoredmTORC1 signaling that correlated with reduced BA-accumulation and liver injury, pointing to a linkbetween mTOR and BA metabolism. Accordingly,mTOR activation by Leu restored FXR acetylation andits target gene expression in SIRT mice and attenuatedmethylation of H3K4 and H3K9 reaching comparableWT liver levels. We propose that the direct regulationof SIRT1 by mTORC1 influences histone and proteinacetylation, which may explain the potential of mTORto regulate histone acetylation.33


It is worth noting that the beneficial effects of Leuand NorUDCA seem directly related to counteractingthe detrimental effects of SIRT overexpression. Thus,WT animals fed with Leu showed attenuated hepato-cyte proliferation (Supporting Fig. 9A), whereas Nor-UDCA/WT mice showed a comparable number ofproliferating hepatocytes than WT mice after PH (Sup-porting Fig. 9B).

PTM, such as chromatin alterations by acetylation/deacetylation, are involved in carcinogenesis. The roleof SIRT1 during tumorigenesis remains controversial,as it has both pro- and anticarcinogenic effects.34

Here we confirm that SIRT1 is highly expressed inhuman HCC samples, regardless of etiology: HCV orASH. Based on this fact, we propose that the tumori-genic characteristic of SIRT1 relies on its role as aregulator of BA metabolism, as both HCV- andASH-cirrhosis are characterized by dysregulated BAmetabolism.11,12 This might explain the apparentcontradiction with a previous study14 where the pro-tection against inflammation and ROS exerted bySIRT1 overexpression seemed sufficient to circumventtumor development. Moreover, we convincingly showthat SIRT1 overexpression leads to dysregulation ofFXR, a well-known feature of metabolic disease andtumor development, as deletion of FXR leads toHCC development.35 Previous work related hyperace-tylation of FXR with cancer, metabolic-, and aging-related diseases. Importantly, we propose that overex-pression of SIRT1 correlates with an absence of FXRexpression in tumors potentially due to hypoacetyla-tion and further degradation of FXR. These compel-ling data evidence the inverse correlation betweenSIRT1 and FXR during tumorigenesis and points tothe oncogenic role of SIRT1.

Overall, our data underscore the significance ofmaintaining the deacetylation activity of SIRT1 as adynamic process in the liver during the regenerativeresponse after injury. Also, our work highlights theneed to propose the cautious use of SIRT1-activating drugs, as persistent SIRT1 activity mayhave detrimental effects on the liver in response toinjury. Importantly, our findings underline thepotential use of NorUDCA- and mTORC1-activators as therapeutic tools in the context of anaberrant overexpression of SIRT1, to counteract livermetabolic diseases involving dysregulated BA home-ostasis and/or the induction of a regenerativeresponse after injury.

Acknowledgment: We thank Imanol Zubiete-Francofor editing the article.

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SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling