World Journal of Gastroenterology

Transcription

1 ISSN 7-97 (print) ISSN 9- (online) World Journl of Gstroenterology World J Gstroenterol Jnury ; (): - Published by Bishideng Publishing Group In

2 S Contents Weekly Volume Number Jnury, MINIREVIEWS Drug-eluting beds trnsrteril hemoemboliztion for heptoellulr rinom: Current stte of the rt Fiorusso A ORIGINAL ARTICLE Bsi Study 7 Antifibrogeni effets of vitmin D derivtives on mouse pnreti stellte ells Wllbum P, Rohde S, Ehlers L, Lnge F, Hohn A, Bergner C, Shwrzenbök SM, Kruse BJ, Jster R 79 Metboli nd hepti effets of lirglutide, obetiholi id nd elfibrnor in diet-indued obese mouse models of biopsy-onfirmed nonloholi stetoheptitis Tølbøl KS, Kristinsen MNB, Hnsen HH, Veidl SS, Rigbolt KTG, Gillum MP, Jelsing J, Vrng N, Feigh M 9 INT-77 improves histopthologil fetures in diet-indued ob/ob mouse model of biopsy-onfirmed nonloholi stetoheptitis Roth JD, Feigh M, Veidl SS, Fensholdt LKD, Rigbolt KT, Hnsen HH, Chen LC, Petitjen M, Friley W, Vrng N, Jelsing J, Young M Novel onept of endosopi devie delivery sttion system for rpid nd tight tthment of polyglyoli id sheet Mori H, Kobr H, Nishiym N, Mski T β-rrestin ttenutes lipopolyshride-indued liver injury vi inhibition of TLR/NF-kB signling pthwy-medited inflmmtion in mie Jing MP, Xu C, Guo YW, Luo QJ, Li L, Liu HL, Jing J, Chen HX, Wei XQ Heptitis C virus ore protein-indued mir-9-p up-regultion inhibits interferon signling pthwy by trgeting IFNAR He CL, Liu M, Tn ZX, Hu YJ, Zhng QY, Kung XM, Kong WL, Mo Q 7 Trnsplnttion of bone mrrow-derived endothelil progenitor ells nd heptoyte stem ells from liver fibrosis rts meliortes liver fibrosis Ln L, Liu R, Qin LY, Cheng P, Liu BW, Zhng BY, Ding SZ, Li XL Cse Control Study Geneti vrints of interferon regultory ftor ssoited with hroni heptitis B infetion Sy BT, Hon NX, Tong HV, Meyer CG, Ton NL, Song LH, Bok CT, Velvn TP Jnury, Volume Issue

3 Contents World Journl of Gstroenterology Volume Number Jnury, Retrospetive Study 7 Timing of surgery fter neodjuvnt hemotherpy for gstri ner: Impt on outomes Liu Y, Zhng KC, Hung XH, Xi HQ, Go YH, Ling WQ, Wng XX, Chen L ditive nd prognosti vlue of serum AFP level nd its dynmi hnges in dvned gstri ner ptients with elevted serum AFP Wng YK, Zhng XT, Jio X, Shen L SYSTEMATIC REVIEWS 7 Neodjuvnt hemotherpy for gstri ner. Is it must or fke? Reddvid R, Sofi S, Chiro P, Colli F, Trpni R, Esposito L, Solej M, Degiuli M CASE REPORT 9 Clinilly dignosed lte-onset fulminnt Wilson s disese without irrhosis: A se report Amno T, Mtsubr T, Nishid T, Shimkoshi H, Shimod A, Sugimoto A, Tkhshi K, Muki K, Ymmoto M, Hyshi S, Nkjim S, Fukui K, Ind M 97 Mss forming hroni pnretitis mimiking pnreti ysti neoplsm: A se report Jee KN Suessful tretment of gint ossified benign mesenteri shwnnom Wu YS, Xu SY, Jin J, Sun K, Hu ZH, Wng WL LETTER TO THE EDITOR Cndid ommodtes non-ulturble Heliobter pylori in its vuole - Koh s postultes ren t pplible Sivoshi F, Sniee P II Jnury, Volume Issue

4 Contents World Journl of Gstroenterology Volume Number Jnury, ABOUT COVER Editoril bord member of World Journl of Gstroenterology, Ginlu Pellino, MD, Reserh Fellow, Surgeon, Unit of Generl nd Geritri Surgery, Università degli Studi dell Cmpni "Luigi Vnvitelli", Nples, Itly AIMS AND SCOPE INDEXING/ABSTRACTING World Journl of Gstroenterology (World J Gstroenterol, WJG, print ISSN 7-97, online ISSN 9-, DOI:.7) is peer-reviewed open ess journl. WJG ws estblished on Otober, 99. It is published weekly on the 7 th, th, st, nd th eh month. The WJG Editoril Bord onsists of experts in gstroenterology nd heptology from 9 ountries. The primry tsk of WJG is to rpidly publish high-qulity originl rtiles, reviews, nd ommentries in the fields of gstroenterology, heptology, gstrointestinl endosopy, gstrointestinl surgery, heptobiliry surgery, gstrointestinl onology, gstrointestinl rdition onology, gstrointestinl imging, gstrointestinl interventionl therpy, gstrointestinl infetious diseses, gstrointestinl phrmology, gstrointestinl pthophysiology, gstrointestinl pthology, evidene-bsed mediine in gstroenterology, pnretology, gstrointestinl lbortory mediine, gstrointestinl moleulr biology, gstrointestinl immunology, gstrointestinl mirobiology, gstrointestinl genetis, gstrointestinl trnsltionl mediine, gstrointestinl dignostis, nd gstrointestinl therpeutis. WJG is dedited to beome n influentil nd prestigious journl in gstroenterology nd heptology, to promote the development of bove disiplines, nd to improve the dignosti nd therpeuti skill nd expertise of liniins. World Journl of Gstroenterology (WJG) is now indexed in Current Contents /Clinil Mediine, Siene Cittion Index Expnded (lso known s SiSerh ), Journl Cittion Reports, Index Medius, MEDLINE, PubMed, PubMed Centrl nd Diretory of Open Aess Journls. The 7 edition of Journl Cittion Reports ites the impt ftor for WJG s. (-yer impt ftor:.7), rnking WJG s 9 th mong 79 journls in gstroenterology nd heptology (qurtile in tegory Q). EDITORS FOR THIS ISSUE Responsible Assistnt Editor: Xing Li Responsible Eletroni Editor: Yu-Jie M Proofing Editor-in-Chief: Lin-Sheng M Responsible Siene Editor: Y-Jun M Proofing Editoril Offie Diretor: Ze-Mo Gong NAME OF JOURNAL World Journl of Gstroenterology ISSN ISSN 7-97 (print) ISSN 9- (online) LAUNCH DATE Otober, 99 FREQUENCY Weekly EDITORS-IN-CHIEF Dmin Gri-Olmo, MD, PhD, Dotor, Professor, Surgeon, Deprtment of Surgery, Universidd Autonom de Mdrid; Deprtment of Generl Surgery, Fundion Jimenez Diz University Hospitl, Mdrid, Spin Stephen C Strom, PhD, Professor, Deprtment of Lbortory Mediine, Division of Pthology, Krolinsk Institutet, Stokholm -, Sweden Andrzej S Trnwski, MD, PhD, DS (Med), Professor of Mediine, Chief Gstroenterology, VA Long Beh Helth Cre System, University of Cliforni, Irvine, CA, 9 E. Seventh Str., Long Beh, CA 9, United Sttes EDITORIAL BOARD MEMBERS All editoril bord members resoures online t EDITORIAL OFFICE Ze-Mo Gong, Diretor World Journl of Gstroenterology Bishideng Publishing Group In 79 Stoneridge Drive, Suite, Plesnton, CA 9, USA Telephone: +-9- Fx: +-9- E-mil: editoriloffie@wjgnet.om Help Desk: PUBLISHER Bishideng Publishing Group In 79 Stoneridge Drive, Suite, Plesnton, CA 9, USA Telephone: +-9- Fx: +-9- E-mil: bpgoffie@wjgnet.om Help Desk: PUBLICATION DATE Jnury, COPYRIGHT Bishideng Publishing Group In. Artiles published by this Open-Aess journl re distributed under the terms of the Cretive Commons Attribution Nonommeril Liense, whih permits use, distribution, nd reprodution in ny medium, provided the originl work is properly ited, the use is non ommeril nd is otherwise in ompline with the liense. SPECIAL STATEMENT All rtiles published in journls owned by the Bishideng Publishing Group (BPG) represent the views nd opinions of their uthors, nd not the views, opinions or poliies of the BPG, exept where otherwise expliitly indited. INSTRUCTIONS TO AUTHORS Full instrutions re vilble online t wjgnet.om/bpg/gerinfo/ ONLINE SUBMISSION III Jnury, Volume Issue

5 Submit Mnusript: DOI:.7/wjg.v.i.79 World J Gstroenterol Jnury ; (): 79-9 ISSN 7-97 (print) ISSN 9- (online) ORIGINAL ARTICLE Bsi Study Metboli nd hepti effets of lirglutide, obetiholi id nd elfibrnor in diet-indued obese mouse models of biopsy-onfirmed nonloholi stetoheptitis Kirstine S Tølbøl, Mri NB Kristinsen, Henrik H Hnsen, Snne S Veidl, Kristoffer TG Rigbolt, Mtthew P Gillum, Job Jelsing, Niels Vrng, Mihel Feigh Kirstine S Tølbøl, Mri NB Kristinsen, Henrik H Hnsen, Snne S Veidl, Kristoffer TG Rigbolt, Job Jelsing, Niels Vrng, Mihel Feigh, Gubr Aps, Hørsholm DK-97, Denmrk Kirstine S Tølbøl, Mri NB Kristinsen, Deprtment of Biomedil Sienes, Fulty of Helth Sienes, University of Copenhgen, Copenhgen DK-, Denmrk Kirstine S Tølbøl, Mtthew P Gillum, Setion for Metboli Imging nd Liver Metbolism, The Novo Nordisk Foundtion Center for Bsi Metboli Reserh, Fulty of Helth Sienes, University of Copenhgen, Copenhgen DK-, Denmrk Niels Vrng, Deprtment of Chemistry, Fulty of Siene, University of Copenhgen, Copenhgen DK-, Denmrk ORCID number: Kirstine S Tølbøl (--7-7); Mri NB Kristinsen ( ); Henrik H Hnsen (--7-); Snne S Veidl (---); Kristoffer TG Rigbolt ( ); Mtthew P Gillum (--9-X); Job Jelsing ( ---); Niels Vrng (--7-9); Mihel Feigh ( ). Author ontributions: Tølbøl KS nd Kristinsen MNB ontributed eqully to the work; Jelsing J, Vrng N nd Feigh M designed the study; Tølbøl KS, Kristinsen MNB, Veidl SS nd Rigbolt KTG quired nd nlysed dt; Tølbøl KS, Kristinsen MNB, Hnsen HH, Veidl SS, Rigbolt KTG, Gillum MP, Jelsing J, Vrng N nd Feigh M interpreted the dt nd ontributed to writing the rtile, editing nd reviewing, ll uthors pproved the finl version of the rtile. Supported by Innovtion Fund Denmrk, KST; no. -B; nd MNBK, no. 9-B. Institutionl niml re nd use ommittee sttement: All niml experiments onformed to the interntionlly epted priniples for the re nd use of lbortory nimls (liene no , The Animl Experiments Inspetorte, Denmrk). Conflit-of-interest sttement: The uthors delre no onflit of interest. Dt shring sttement: No dditionl dt re vilble. Open-Aess: This rtile is n open-ess rtile whih ws seleted by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ordne with the Cretive Commons Attribution Non Commeril (CC BY-NC.) liense, whih permits others to distribute, remix, dpt, build upon this work non-ommerilly, nd liense their derivtive works on different terms, provided the originl work is properly ited nd the use is non-ommeril. See: lienses/by-n/./ Mnusript soure: Unsoliited mnusript Correspondene to: Kirstine S Tølbøl, MS, Reserh Sientist, Gubr Aps, Hørsholm Kongevej B, Hørsholm DK-97, Denmrk. kst@gubr.dk Telephone: +-- Reeived: September, 7 Peer-review strted: September, 7 First deision: November, 7 Revised: November, 7 Aepted: Deember, 7 Artile in press: Deember, 7 Published online: Jnury, Abstrt AIM To evlute the phrmodynmis of ompounds in linil development for nonloholi stetoheptitis (NASH) in obese mouse models of biopsy-onfirmed 79 Jnury, Volume Issue

6 NASH. METHODS Mle wild-type C7BL/J mie (DIO-NASH) nd Lep ob/ob (ob/ob-nash) mie were fed diet high in trns-ft (%), frutose (%) nd holesterol (%) for nd wk, respetively. Prior to tretment, ll mie underwent liver biopsy for onfirmtion nd strtifition of liver stetosis nd fibrosis, using the nonloholi ftty liver disese tivity sore (NAS) nd fibrosis stging system. The mie were kept on the diet nd reeived vehile, lirglutide (. mg/kg, SC, BID), obetiholi id (, mg/kg PO, QD), or elfibrnor ( mg/kg PO, QD) for eight weeks. Within-subjet omprisons were performed on hnges in stetosis, inflmmtion, bllooning degenertion, nd fibrosis sores. In ddition, ompound effets were evluted by quntittive liver histology, inluding perent frtionl re of liver ft, gletin-, nd ollgen. RESULTS Lirglutide nd elfibrnor, but not, redued body weight in both models. Lirglutide improved stetosis sores in DIO-NASH mie only. Elfibrnor nd redued histopthologil sores of hepti stetosis nd inflmmtion in both models, but only elfibrnor redued fibrosis severity. Lirglutide nd redued totl liver ft, ollgen, nd gletin- ontent, driven by signifint redutions in liver weight. The individul drug effets on NASH histologil endpoints were supported by globl gene expression (RNA sequening) nd liver lipid biohemistry. CONCLUSION DIO-NASH nd ob/ob-nash mouse models show distint tretment effets of lirglutide,, nd elfibrnor, being in generl greement with orresponding findings in linil trils for NASH. The present dt therefore further supports the linil trnsltbility nd utility of DIO-NASH nd ob/ob -NASH mouse models of NASH for probing the therpeuti effiy of ompounds in prelinil drug development for NASH. Key words: Nonloholi stetoheptitis; Disese models; Pthology; Fibrosis; Liver biopsy; Trnsriptomis; Phrmodynmis; Glugon-like peptide- reeptor; Peroxisome prolifertor-tivted reeptor; Frnesoid X reeptor The Author(s). Published by Bishideng Publishing Group In. All rights reserved. Core tip: The phrmodynmis of three ompounds in dvned linil development for the tretment of nonloholi stetoheptitis (NASH), inluding lirglutide, elfibrnor nd obetiholi id, were evluted in wild-type nd genetilly (ob/ob ) obese mouse models of NASH. Prior to tretment, ll mie underwent liver biopsy for onfirmtion nd strtifition of liver stetosis nd fibrosis. Withinsubjet omprisons were performed on hnges in liver histopthology. Wild-type nd ob/ob -NASH obese mie showed distint tretment effets of lirglutide,, nd elfibrnor, being in generl greement with orresponding findings in linil trils for NASH. In onlusion, the two obese mouse models of NASH show linil trnsltbility with respet to disese etiology, histopthology nd drug tretment effets, whih supports their utility in prelinil drug development. Tølbøl KS, Kristinsen MN, Hnsen HH, Veidl SS, Rigbolt KT, Gillum MP, Jelsing J, Vrng N, Feigh M. Metboli nd hepti effets of lirglutide, obetiholi id nd elfibrnor in dietindued obese mouse models of biopsy-onfirmed nonloholi stetoheptitis. World J Gstroenterol ; (): 79-9 Avilble from: URL: v/i/79.htm DOI: INTRODUCTION Nonloholi stetoheptitis (NASH) is hrterized by vrying degrees of hepti stetosis, ytoskeletl dmge, nd lobulr inflmmtion with or without fibrosis []. The pthogenesis of NASH is omplex with urrent hypotheses involving ftty id-medited lipotoxiity exhusting heptoyte dptive nd regenertive responses, whereby umulting oxidtive stress n trigger heptoyte neroinflmmtion, fibrosis, nd disruption of hepti ytorhiteture [,]. Immunopthologil ltertions likely lso ply key role in NASH, presumbly driven by mldptive responses in the innte nd dptive immune system [,]. The primry drivers of NASH re obesity nd dibetes, nd the high prevlene of these mjor metboli diseses is expeted to mke NASH one of the most ommon uses of dvned liver disorders in the oming dede []. NASH is therefore rpidly emerging s mjor publi helth problem, however, with urrently no evidene-bsed pproved drug therpies. This hs prompted substntil efforts to identify novel phrmologil onepts for orreting the underlying metboli defiits nd llevite, or prevent, hepti fibrosis in NASH [7,]. To improve the understnding of NASH pthogenesis nd filitte the development of novel therpeutis for NASH, severl mouse models hve been employed []. A number of diet-indued obesity (DIO) models mimi the nturl history of NASH nd hve demonstrted reltively good linil trnsltbility with respet to key metboli nd liver pthologil hnges with mild- to moderte-grde liver fibrosis, nd these models hve therefore been inresingly used in prelinil drug development [9-]. In omprison, non-physiologil diets tht re low or devoid of ertin essentil mino ids promote more severe liver fibrosis, but lso result in signifint weight loss, mking these NASH models more pplible for Jnury, Volume Issue

7 probing drug tretment effiy on hepti injury nd regenertion [-]. Notbly, NASH show spontneous nd unpreditble onset with vrying disese severity nd different rtes of progression []. Tody, liver biopsy-onfirmed pthology is the minsty for linil trils whih llows for strtifition of disese severity/stge nd withinsubjet evlution of endpoint liver histology [7,]. In greement with the linil findings, DIO mouse models of NASH show vrible disese severity. Reent reports hve demonstrted tht ll stges of NASH re represented in mie fed Western-type obesogeni diets for ny period wk, nd signifint proportion of up to % of the nimls fil to develop stetoheptitis nd liver fibrosis [9,,9], inditing tht individul bseline disese stge is lso ritil ftor in the ssessment of tretment effets in DIO mouse models of NASH. However, disese stte heterogeneity is often overlooked in prelinil NASH studies whih my result in unintentionl lrge vribility in endpoint histopthology nd nrrow the window for detetion of therpeuti effets. Another ftor to be onsidered in prelinil drug development for NASH is tht drug tretment periods in mouse models of NASH hve vried from subute to hroni dosing settings with histologil endpoints evluted by different qulittive nd quntittive methods. Comprison of prelinil tretment effets of potentil nti-nash ompounds is therefore misleding in the bsene of hed-to-hed phrmologil studies in experimentl models of NASH. We hve reently reported liver biopsy-onfirmed histopthology in wild-type (DIO-NASH) nd genetilly obese (ob/ob-nash) mouse models of NASH [9]. Both models disply hllmrks of NASH, i.e. stetosis, lobulr inflmmtion, heptoellulr bllooning, but re distinguished histologilly by the representtion of mild (DIO-NASH) or moderte (ob/ob-nash) grde hepti fibrosis [9,9]. The present omprtive study in the DIO-NASH nd ob/ob-nash mouse models of biopsyonfirmed histopthology imed to hrterize withinsubjet tretment responses of ompounds in urrent dvned linil development for NASH, inluding lirglutide [], obetiholi id (, INT-77) [], nd elfibrnor (GFT-) []. Lirglutide is longting glugon-like peptide (GLP-) nlogue with piomolr GLP- reeptor binding poteny [], nd is pproved for the mngement of type dibetes nd obesity. Wheres lirglutide improves glyemi by stimulting pnreti ell funtion, its nti-obesity effets re predominntly entrlly medited through suppression of hypothlmi ppetite signling [,]. is semi-syntheti bile id urrently pproved for the tretment of primry biliry holngitis []. ts s trnsriptionl tivtor through high-ffinity binding to the nuler frnesoid X reeptor (FXR) [7] to regulte bile id synthesis nd trnsport, hepti lipid nd rbohydrte metbolism, s well s immune funtion []. Elfibrnor is seletive dul gonist for peroxisome prolifertor-tivted α/δ reeptors (PPAR-α/δ) [9], nother group of lignd-tivted nuler reeptors with prtiulrly highly bundny in the liver. As for, elfibrnor exerts its mjor effets through trnsriptionl regultion of key genes involved in hepti lipid nd gluose metbolism, but lso modultes hepti inflmmtion nd ollgen turnover []. Lirglutide, nd elfibrnor therefore influenes different disese mehnisms in NASH, mking these ompounds well-suited for ompring the tretment effiy of different therpeuti onepts in the two mouse models of NASH. MATERIALS AND METHODS Animls All niml experiments were onduted ording to interntionlly epted priniples for the re nd use of lbortory nimls (liene no , The Animl Experiments Inspetorte, Denmrk). The niml protool ws designed to minimize pin or disomfort to the nimls. Mle mie were obtined from Jnvier Lbs (Le Genest Sint Isle, Frne) nd housed in ontrolled environment ( h light/drk yle, light on t AM, ±, humidity % ± %). Eh niml ws identified by n implntble mirohip (PetID Mirohip, E-vet, Hderslev, Denmrk). Mie hd d libitum ess to tp wter nd either regulr rodent how (Altromin, Brogrden, Hoersholm, Denmrk), or diet high in ft (%, ontining % trns-ft), % rbohydrtes (% frutose) nd % holesterol (AMLN diet; D9, Reserh Diets, New Brunswik, NJ) [9]. Disese progression ws hrterized in wild-type DIO-NASH mie, wk-old t the rrivl t Gubr, nd fed AMLN diet for up to wk (n = - nimls per time point). Effets of drug tretment were evluted in wild-type C7BL/J nd B.V-Lep ob /JRj (ob/ob) mie - wkold t the rrivl t Gubr. C7BL/J mie were fed how (ontrols) or AMLN diet (DIO-NASH mie) for wk prior to tretment strt. ob/ob mie were fed how (ob/ob ontrols) or AMLN diet (ob/ob-nash mie) for wk prior to tretment strt. Body weight ws mesured dily during the tretment period. Bseline liver biopsy All nimls inluded in the drug tretment experiments underwent liver biopsy, s desribed in detil previously [9,9]. In brief, the mie were nesthetized with isoflurne (Vetflurne, Virb, Kolding, Denmrk) in tmospheri ir. A midline bdominl inision ws mde to expose the left lterl lobe, nd one-shped biopsy of - mg liver tissue ws olleted nd fixed in % prformldehyde overnight nd subsequently used for histologil ssessment. The ut surfes were eletroogulted using n eletrosurgiil unit (ERBE VIO C, ERBE, Mriett, GA, United Sttes), whereupon the liver ws returned to the bdominl Jnury, Volume Issue

8 vity, the bdominl wll ws sutured nd the skin ws stpled. The nimls reeived mg/kg rprofen (Norodyl, SnVet, Fredensborg, Denmrk) prior to surgery nd on post-opertive dy nd. Animls were single-housed fter the proedure nd llowed to reover for wk prior to tretment strt. Only mie with fibrosis stge nd stetosis sore, evluted using the linil riteri outlined by Kleiner et l [], were inluded in the study. Drug tretment Lirglutide (Vitoz pen) ws from Novo Nordisk (Bgsverd, Denmrk), elfibrnor nd were from SunshineChem (Shnghi, Chin). Vehiles were.% rboxymethyl ellulose with. % Tween- (PO dosing) or phosphte-buffered sline with.% bovine serum lbumin (SC dosing), dministered in dosing volume of ml/kg. Animls were strtified (n = 9- per group) bsed on men fibrosis nd stetosis sore, nd treted for wk with vehile (PO, QD), lirglutide (. mg/kg, SC, BID), ( mg/kg, PO, QD), or elfibrnor ( mg/kg, PO, QD). Vehile-dosed howfed mie (PO, QD) served s dditionl ontrols. The ompound doses used in the present study were within the dose rnge reported effiious in other mouse models of diet-indued obesity nd NASH [,,-]. A terminl blood smple ws olleted from the til vein in non-fsted mie nd used for plsm biohemistry. Animls were srified by rdi punture under isoflurne nesthesi. Liver smples were proessed s desribed below. Biohemil nd histologil nlyses Biohemil nd histologil nlyses were performed s reported previously [9]. Plsm nlytes inluded lnine minotrnsferse (ALT), sprtte minotrnsferse (AST), triglyerides (TG) nd totl holesterol (TC). Liver homogentes were nlyzed for TG nd TC. Prformldehyde-fixed liver pre- nd post-biopsies were prffin-embedded, setioned, nd stined with hemtoxylin-eosin (Dko, Glostrup, Denmrk), Piro-Sirius red (Sigm-Aldrih, Broendby, Denmrk), nti-type I ollgen (Col; Southern Bioteh, Birminghm, AL), or nti-gletin- (Biolegend, Sn Diego, CA, United Sttes). The NAFLD tivity sore (NAS) nd fibrosis stging system ws pplied to liver pre-biopies nd terminl smples (drug tretment experiments) or only terminl smples (disese progression experiment) for soring of stetosis, lobulr inflmmtion, heptoyte bllooning, nd fibrosis outlined by Kleiner et l []. All histologil ssessments were performed by pthologist blind to tretment. Beuse ll tretment prdigms ffeted totl liver weight, quntittive dt on liver biohemistry (liver TG, TC) nd histology (liver lipid, gletin-, Col) were expressed s whole-liver mounts by multiplying individul terminl liver weight with the orresponding liver lipid onentrtion (biohemistry dt) or perent frtionl re (histology dt), respetively. RNA sequening Hepti trnsriptome nlysis ws performed by RNA sequening on RNA extrts from terminl liver smples ( mg fresh tissue), s desribed in detil previously [9]. The RNA quntity ws mesured using Qubit (Thermo Sientifi, Eugene, OR, United Sttes). The RNA qulity ws determined using bionlyzer with RNA Nno kit (Agilent, Wldbronn, Germny). RNA sequene librries were prepred with Neop (Illumin, Sn Diego, CA, United Sttes) using Illumin TruSeq strnded mrna Librry kit for Neop (Illumin, Sn Diego, CA, United Sttes) nd sequened on the NextSeq (Illumin, Sn Diego, CA, United Sttes) with NSQ hi-output KT v (7 CYS, Illumin, Sn Diego, CA, United Sttes). Reds were ligned to the GRCm v Ensembl Mus musulus genome using STAR v... with defult prmeters []. Differentil gene expression nlysis ws performed with DEseq [7]. Genes with Benjmini nd Hohberg djusted p. (% Flse Disovery Rte) were regrded s sttistilly signifintly regulted. Enrihment nlysis of KEGG pthwys were performed using the lusterprofiler pkge for R []. Sttistil nlyses Exept from RNA sequening, dt were nlyzed using GrphPd Prism v. softwre (GrphPd, L Joll, CA, United Sttes). All results re shown s men ± SE. A two-wy ANOVA with Bonferroni s post-ho test ws performed for body weight nlysis. Fisher s ext test ws used to test for withinsubjet hnges in histology sores before nd fter tretment, ompred to vehile ontrols. A one-wy ANOVA with Dunnett s post-ho test ws used for ll other prmeters. A P-vlue <. ws onsidered sttistilly signifint. RESULTS Disese progression in wild-type DIO-NASH mie Terminl smples were ssessed for nlysis of plsm nd liver biohemistry, s well s liver morphology nd quntittive histology. Metboli dt re shown in Supplementl Figure. Body weight in DIO-NASH mie grdully inresed over the feeding period nd rehed plteu (pproximtely g) fter pproximtely wk of dieting (Supplementl Figure, pnel A). DIO-NASH mie developed heptomegly nd ftty liver from dietry week nd onwrds (pnel B, F). Plsm ALT nd AST inresed during the dieting period, with the effet on AST being trnsient (pnel C, D). Onset of hyperholesterolemi ws evident from dieting week (pnel E), nd liver TC levels were elevted even erlier (pnel G). Liver, but not plsm, TG levels were inresed from dieting wk - (pnel F, H). Jnury, Volume Issue

9 B Inflmmtion Number of nimls Number of nimls NAFLD tivity sore E 7 Fibrosis stge. ColA ColA Timp.... Sd Cd Fsn Ldlr Srebp Srebp Srb Cl (Mp) Cr Cd Lgls (Lgls) Cd Tnf- α Tlt Cd Col Col Timp Mmp Mmp α-sma Ripk Csp Csp Csp Trf Ripk.. Cell deth RPKM CD. I Fibrosis RPKM K. CD CD. Inflmmtion Liver ollgen (% frtionl re). Lipid hndling Liver gletin (% frtionl re) H J F Number of nimls Number of nimls G D Bllooning degenertion C Number of nimls Stetosis Liver lipid (% frtionl re) A Low High Reltive expression Figure Disese progression in DIO-nonloholi stetoheptitis mie. A-E: Individul nd omposite NAFLD tivity sores (NAS) nd fibrosis stge; F-H: Quntittive nlysis of liver lipid, gletin- nd ollgen frtionl re; I-K: Hetmp on liver trnsriptome hnges nd expression of seleted genes. P <., P <. vs week. NASH: Nonloholi stetoheptitis. Histology dt re presented in Figure. Stetosis developed grdully in DIO-NASH mie nd beme severe fter - wk of dieting (pnel A). Lobulr inflmmtion ws observed fter wk nd progressed in severity with inresing dieting periods (pnel B). Heptoyte bllooning ws deteted in few mie fter wk, inresing in prevlene over time, but did not progress beyond stge (pnel C). Mnifest NASH (NAS -) ws onsistently observed fter - wk of dieting nd inrese in severity during the reminder of the monitoring period (pnel D). Fibrosis (stge ) ws onsistently deteted from wk of dieting. All DIO-NASH mie developed fibrosis fter wk (predominntly stge ), but %-% of the Jnury, Volume Issue

10 A Body weight (g) Vehile Lirglutide Elfibrnor t /d B % Body weight of dy 9 Vehile Lirglutide Elfibrnor t /d C D E F Liver weight (g) b Vehile Lirglutide Elfibrnor Liver TG (totl, mg) Vehile Lirglutide Elfibrnor Liver TC (totl, mg) Vehile Lirglutide Elfibrnor Plsm ALT (U/L) b Vehile Lirglutide Elfibrnor G H I. Plsm AST (U/L) Plsm TG (mmol/l).. Plsm TC (mmol/l) Vehile Lirglutide Elfibrnor. Vehile Lirglutide Elfibrnor Vehile Lirglutide Elfibrnor Figure Metboli effets of lirglutide, obetiholi id, nd elfibrnor tretment in DIO-nonloholi stetoheptitis mie. P <., b P <., P <. vs vehile ontrols; : Obetiholi id; NASH: Nonloholi stetoheptitis. mie remined t stge fter wk of dieting (pnel E). Hepti lipid mounts signifintly inresed from dieting week nd rehed mximl level from week (pnel F). Liver gletin- immunoretivity inresed fter wk of dieting nd progressed further with inresing dieting periods (pnel G). Col immunoretivity ws elevted fter wk of dieting nd did not hnge further (pnel H). Ltestge omplitions were rre in DIO-NASH mie, nd ourred only fter wk on AMLN diet (irrhosis nd hepti nodulr formtion, n = /9; extensive bile dut prolifertion, n = /9, nimls not inluded in dt nlysis). A hetmp ws generted for pnel of prototypil gene ndidtes ssoited with NASH pthology (Supplementry Figure, pnel I). Signtures were mrkedly different during the study period, with n upregultion of genes involved in lipid hndling, inflmmtion, fibrosis nd ell deth ourring over time. Expression of genes involved in lipid hndling were grdully upregulted from week, with highest expression from week wk nd onwrds. Gene groupings representtive of monoyte/mrophge infiltrtion, inflmmtion, extrellulr mtrix (ECM) turnover, nd poptosis exeution peked t - wk of dieting (pnel I-K). Drug tretment in DIO-NASH mie As expeted, DIO-NASH mie gined more weight thn how-fed ontrols (Figure A). Tretment with lirglutide nd elfibrnor, but not, resulted in weight loss (nd loss of terminl whole-body ft, dt not shown) whih rehed mximl effet fter pproximtely wk of tretment (Figure A nd B). Lirglutide nd elfibrnor, but not, indued trnsient redution in dily food intke (from tretment d -), whereupon food intke remined similr to vehile-dosed DIO-NASH mie (dt not shown). Wheres lirglutide nd redued liver weight, elfibrnor hd the opposite effet (Figure C). Biohemil (plsm, liver) nd quntittive histologil (liver) nlyses were pplied. Lirglutide nd redued totl liver TG nd TC ontent (Figure D nd E). Only lirglutide signifintly redued plsm ALT nd AST (Figure F nd G). Lirglutide nd, but not Jnury, Volume Issue

11 A B NAFLD tivity sore Number of nimls Sme Lower Higher b b O CA e El fi br n or lu tid Li r g Ve hi le 7 Vehile NAS 7 Lirglutide NAS Elfibrnor NAS 7 NAS 7 C D Sme Lower Number of nimls E Liver fibrosis stge Higher CA Lirglutide fibrosis Elfibrnor fibrosis fibrosis O or fi br n Vehile fibrosis El tid lu r g Li Ve h il e e Elfibrnor Lirglutide Vehile F Vehile Elfibrnor Lirglutide Figure Histomorphologil effets of lirglutide, obetiholi id, nd elfibrnor tretment in DIO-nonloholi stetoheptitis mie. P <., bp <., P <. vs vehile ontrols. : Obetiholi id; NASH: Nonloholi stetoheptitis. elfibrnor, redued plsm TC levels (Figure I). None of the ompounds ffeted plsm TG levels (Figure H). Liver morphometry ws ompred in pre- vs. posttretment liver biopsies. All DIO-NASH mie inluded in the experiment showed pre-biopsy onfirmed NASH (omposite NAS -) nd liver fibrosis (stge -), see Figure B nd Supplementry Figure. All three ompounds signifintly improved NAS in DIO-NASH mie (Figure A nd B), minly due to redued stetosis sore (Supplementry Figure ). In orrespondene, ll drug tretments lso redued totl liver lipid ontent (Figure A nd B). Only elfibrnor improved fibrosis stge in pre- vs. post-tretment liver biopsies (p <., Figure D-F). In ontrst, totl gletin- nd totl Col levels were redued following lirglutide nd, but not elfibrnor, tretment (Figure C-F). Prinipl omponent nlysis (PCA) indited distint uniform nd stble gene trnsriptionl responses in the experimentl groups. The mjor proportion of vrine orrelted with seprtion of the how-fed nd DIO-NASH ontrols (Figure A). PCA on the minor proportion of vrine yielded more mixed profile with prtil seprtion of lirglutide nd effets (Figure B). Overll, lirglutide nd tretment resulted in trnsriptome signtures prtilly resembling the len ontrol signture. Trnsriptome hnges lso mrkedly overlpped between drug tretment groups. Notbly, elfibrnor indued the highest number of Jnury, Volume Issue

12 A Liver liqid (totl lipid ontent, g).... Vehile Lirglutide Elfibrnor C Liver ol (totl ol ontent, g) Vehile Lirglutide Elfibrnor b E Liver gletin- (totl ontent, g)..... Vehile Lirglutide Elfibrnor B D F Stetosis (HE stin) Col (IHC) Gletin- (IHC) Stetosis (morphometry) Col (morphometry) Gletin- (morphometry) Figure Quntittive histologil ssessment of liver lipids, ollgen deposition nd inflmmtion in DIO-nonloholi stetoheptitis mie. P <., b P <., P <. vs vehile ontrols. : Obetiholi id; NASH: Nonloholi stetoheptitis. differentilly expressed genes nd evoked trnsriptome modifitions distint from ll other experimentl groups (Figure A nd C). Lirglutide indued onspiuous hnges in trnsriptionl pthwys ssoited with gluose metbolism, but hd virtully no impt on gene groupings representing other liver metboli pthwys (Figure D). modulted gene groupings linked to gluose metbolism, but lso ffeted signling pthwys ssoited with NAFLD, ECM-reeptor intertion, PI kinse signling, nd fol dhesion. Pthwys perturbtions promoted by elfibrnor tretment mpped to NAFLD, lipid metbolism, oxidtive phosphoryltion, PPAR nd peroxisome signling. All drug tretments redued expression of gene mrkers involved in inflmmtion, fibrogenesis, nd poptosis (Figure E nd F). Drug tretment in ob/ob-nash mie ob/ob-nash ontrols were obese prior to tretment (.7 ±. g, n = ), but showed lower body weight ompred to ge-mthed how-fed ob/ob ontrols (. ±. g, n = 9) (Figure A). Terminl liver weight ws mrkedly higher in ob/ob-nash ontrol mie ompred to how-fed ontrols (Figure A-C). Lirglutide nd elfibrnor tretment progressively redued body weight with mximl weight loss of pproximtely % ompred to bseline (pproximtely % vs vehile-dosing), see Figure B. did not ffet body weight in ob/ob-nash mie. Lirglutide, but not nd elfibrnor, indued trnsient redution in dily food intke (from tretment d -7), whereupon food intke remined similr to vehile-dosed ob/ob-nash mie (dt not shown). Lirglutide nd, but not elfibrnor, redued terminl liver weight (Figure C). Lirglutide redued totl liver TG ontent (Figure D) s well s plsm TG levels (Figure H), while elfibrnor nd redued totl liver TC ontent (Figure E). All ompounds lowered plsm TC levels (Figure I). Lirglutide nd elfibrnor, but not, redued plsm ALT nd AST (Figure F nd G). All ob/ob- NASH mie inluded in the experiment showed prebiopsy onfirmed NASH (omposite NAS -7) nd liver fibrosis (stge -), see Figure 7B nd Supplementry Figure. Elfibrnor nd signifintly redued omposite NAS, totl liver ft nd gletin- ontent in ob/ob-nash mie (Figure 7A, B, E nd F). Liver morphometry ws ompred in pre- vs. post-tretment liver biopsies. While elfibrnor redued NAS lrgely by improving stetosis nd inflmmtion sores, lso onsistently redued bllooning (Supplementry Figure ). Only elfibrnor signifintly redued hepti fibrosis stge (Figure 7C nd D), but did not hnge totl levels of Col (Figure 7F). Lirglutide hd no effet on ny individul NAS omponent, s well s omposite NAS, Jnury, Volume Issue

13 A PC (%) C F Lirglutide Vehile Lirglutide Elfibrnor 9 7 PC (%) Elfibrnor Col D Arginine nd proline metbolism Arginine biosynthesis Biosynthesis of mino ids Glyolysis/Gluoneogenesis Non-loholi ftty liver disese (NAFLD) Peroxisome Biosynth. of unsturted ftty ids Oxidtive phosphoryltion Ftty id elongtion PPAR signling pthwy AGE-RAGE sign. Pth. in dibeti omp. PlK-kt signling pthwy Fol dhesion Glyoxylte nd dirboxylte metbolism ECM-reeptor intertion B PC (%) - Col Lirglutide Vehile Lirglutide Elfibrnor PC (7%) Elfibrnor High Low α-sma Enrihment E Ldlr Srb Srebf Srebf Sd Fsn Cd Cd Cd Lgls (M) Tnf Cl (Mp) Cr Tlr Cd Timp Col Col Mmp Mmp α-sma Csp Ripk Trf Csp Ripk Csp Low Vehile Lirglutide Elfibrnor Reltive expression Timp High Lipid hndling Inflmmtion Fibrosis Cell deth Expression level (RPKM) Vehile Lirglutide Elfibrnor Vehile Lirglutide Elfibrnor Vehile Lirglutide Elfibrnor Vehile Lirglutide Elfibrnor Expression level (RPKM) CL (Mp) Vehile Lirglutide Elfibrnor b Lgls (Mp) Vehile Lirglutide Elfibrnor Cd Vehile Lirglutide Elfibrnor Cr Vehile Lirglutide Elfibrnor Figure Hepti globl gene expression nlysis in DIO-nonloholi stetoheptitis mie. A nd B: Prinipl omponent nlysis; C: Venn digrm of differentilly expressed genes, ompred to vehile tretment; D: Overview of KEGG pthwy-enrihed gene groups; E: Reltive expression of prototypi NASH genes; F: Reltive expression of seleted genes. P <., b P <., P <. vs vehile ontrols. : Obetiholi id; NASH: Nonloholi stetoheptitis. nd did not lter the fibrosis stge (Figure 7A nd B, Supplementry Figure ). In ontrst, lirglutide signifintly redued totl liver levels of lipids, gletin- nd Col (Figure 7E-G). 7 Jnury, Volume Issue

14 A Body weight (g) 7 Vehile Lirglutide Elfibrnor B % Body weight of dy 9 Vehile Lirglutide Elfibrnor t /d t /d C D E Liver weight (g) 7 Vehile Lirglutide Elfibrnor Liver TG (totl, mg) Vehile Lirglutide Elfibrnor Liver TC (totl, mg) Vehile Lirglutide Elfibrnor F G Plsm ALT (U/L) Plsm AST (U/L) H Plsm TG (mmol/l)... Vehile Lirglutide Elfibrnor Vehile Lirglutide Elfibrnor. Vehile Lirglutide Elfibrnor I Plsm TC (mmol/l) b Vehile Lirglutide Elfibrnor Figure Metboli effets of lirglutide, obetiholi id, nd elfibrnor tretment in ob/ob-nonloholi stetoheptitis mie. P <., b P <., P <. vs vehile ontrols. : Obetiholi id; NASH: Nonloholi stetoheptitis. DISCUSSION We nd others hve previously reported tht DIO-NASH mie develop low-grde liver fibrosis when mintined on trns-ft ontining obesogeni diet [9,9,9]. To obtin further informtion on the pthology in this trnsltionl mouse model of NASH we initilly performed detiled investigtion on the disese onset nd progression in DIO-NASH mie, followed by omprison of the tretment effets of lirglutide, nd elfibrnor in both DIO-NASH nd ob/ob-nash mie. DIO-NASH mie exhibited reltively rpid onset of body weight gin with onurrent liver TG nd TC umultion nd progressive development of heptomegly. DIO-NASH mie developed hyperholesterolemi, but not hypertriglyeridemi. This is onsistent finding DIO-NASH mie [9,9], s well s in ob/ob-nash mie [,9] nd other Western dietbsed mouse models of NASH [,]. In ontrst, highft diets with lower holesterol ontent (.%) re reported to indue ombined hypertriglyeridemi nd hyperholesterolemi in obese mie [,,], suggesting Jnury, Volume Issue

15 A Number of nimls Vehile NAFLD tivity sore Lower Sme Higher Lirglutide Elfibrnor B 7 Vehile NAS 7 Lirglutide NAS 7 Elfibrnor NAS 7 NAS C Number of nimls Vehile Liver fibrosis stge Lower Sme Higher Lirglutide Elfibrnor D Vehile fibrosis Lirglutide fibrosis Elfibrnor fibrosis fibrosis E Liver lipid (totl lipid ontent, g) Vehile Lirglutide Elfibrnor F Liver ol (totl ol ontent, g)..... Vehile Lirglutide Elfibrnor G Liver gletin- (totl ontent, g)..... Vehile Lirglutide Elfibrnor b Figure 7 Histomorphologil effets of lirglutide, obetiholi id, nd elfibrnor tretment in ob/ob-nonloholi stetoheptitis mie. P <., b P <., P <. vs vehile ontrols. : Obetiholi id; NASH: Nonloholi stetoheptitis. tht elevted dietry holesterol influenes heptoyte TG seretion, perhps by modifying holesterol ester nd lipoprotein synthesis [,]. Heptopthology ws further indited by inresed levels of plsm trnsminses, most onsistently observed for ALT. As persistently elevted rtios of AST:ALT hs been reported to ssoite to dvned fibroti NASH but not t milder disese stges [], the moderte hnges in ALT nd AST levels in DIO-NASH (nd ob/ob-nash) mie presumbly reflets the mildto-moderte disese severity in these two models. Histologil morphometry suggested sequentil onset of NASH pthology in DIO-NASH mie, with initil stetosis progressing to inflmmtion nd bllooning degenertion (from week ). Quntittive histology on stetosis (HE stining), inflmmtion (gletin-) nd fibrosis (Col) supported distint temporl sequene of heptopthologil events in DIO-NASH mie. In orrespondene, trnsriptome pthwy nlyses reveled initil gene signtures of upregulted hepti lipid synthesis nd hndling, over enhned inflmmtion signling to reruitment of monoytes/myofibroblsts, stimultion of fibrogenesis nd extrellulr mtrix turnover. These findings re in generl onordne with reent hypotheses tht mounting lipotoxiity onstitutes triggering event for progression of NAFLD into NASH [,]. In greement with previous findings [9,9], ll morphologil hllmrks of NASH were onsistently present in DIO-NASH mie t wk of dieting (NAS -, orresponding to the linil riteri for NASH [] ). NAS ws lrgely driven by progressive hnges in heptostetosis nd inflmmtion s ffeted mie only exhibited mrginl heptoyte bllooning. The bsene of onspiuous heptoyte bllooning is in ordne with reent omprehensive histologil 9 Jnury, Volume Issue

16 soring study onluding tht obese rodent models of NASH do not entirely meet the histomorphologil riteri for humn heptoyte bllooning [7]. As is seen in the lini, DIO-NASH mie represented ll stges of NAFLD nd fibrosis for ny dieting period wk, whih onfirms the highly individul fibrosis progression in this model [9,9]. Notbly, up to % of the nimls filed to develop stetoheptitis nd fibrosis, nd the severity in histopthology did not beome more homogenous by extending AMLN dieting periods. Similr disese vribility hs been reported in other DIO mouse models of NASH [,,], whih therefore likely generlizes to ll obese mouse models of fibroti stetoheptitis. Therefore, the use of prebiopsy for seletion nd strtifition of in DIO-NASH models not only substntites the effiy redouts, but lso llows for the employment of linilly relevnt inlusion riteri nd strtifition proedures. In the present phrmologil study, inherent differenes in NASH pthology were therefore onsidered by introduing liver biopsy-bsed histology for evluting NASH severity in ll individul mie prior to introdution of tretment. Only mie with biopsy-onfirmed fibroti NASH were inluded in the study, nd rndomiztion nd strtifition proedures were pplied to equlize men levels of bseline NAS nd fibrosis stge ross tretment groups. The present study evluted the phrmodynmis of three individul ompounds in urrent dvned linil development for NASH. The seleted ompounds re representtive of different drug lsses nd therpeuti onepts nd inluded gonists for the GLP- reeptor (lirglutide), FXR () nd PPAR-α/δ (elfibrnor) [-]. We hve reently reported omprtive liver biopsy study on DIO-NASH nd ob/ ob-nash mie, whih onfirmed tht ob/ob-nash mie generlly disply more mrked fibroti stetoheptitis [9]. Thus, potentil differenes in therpeuti effiies s funtion of overll severity in heptopthology were ddressed by pplying identil tretment protools to both DIO-NASH nd ob/ob-nash mie. The GLP- nlog, lirglutide, whih is urrently pproved for the tretment of type dibetes nd obesity [,], redued weight gin in both DIO-NASH nd ob/ob-nash mie with onurrent improvements in whole-body ft mss, heptomegly, liver lipid deposition, nd hyperholesterolemi, whih is onsistent with findings in other obese mouse models of NASH [,]. The intrhepti lipid-lowering effets of lirglutide re likely seondry to the progressive weight loss promoted by the GLP- gonist [9]. This is relevnt, s weight loss per se is known to medite benefits on NAFLD endpoints []. The present findings re lso in greement with previous study ssessing the effet of n exendin- nlogue in ob/ob-nash mie []. Lirglutide showed most mrked effets on hepti gene trnsriptionl signtures ssoited to the nonil effets of GLP- reeptor gonists on hepti gluose metbolism nd prodution. The liver trnsriptome nlyses in DIO-NASH mie lso suggested tht lirglutide-indued improvements in lipid metbolism ws linked to tivtion of signling pthwys involved in ftty id degrdtion, oxidtive phosphoryltion nd holesterol hndling. Perturbtions in these signling pthwys ould therefore potentilly trigger gene expression progrms tht fvored redued hepti free ftty id uptke, s well s improving the lerne of holesterol nd ftty ids, ll of whih hve been implited in the pthogenesis of NASH. In ontrst to nd elfibrnor, lirglutide did not improve omposite NAS in ob/ob-nash mie. In ddition, lirglutide tretment did not influene fibrosis nd low-grde heptoyte bllooning in either model, inditing tht GLP- reeptor medited meliortion of hepti lipid overlod ws perhps not suffiient to redue morphologil indies of more dvned NASH. In ontrst, totl hepti gletin- nd Col levels were signifintly lowered in both DIO-NASH nd ob/ob-nash mie fter lirglutide tretment, onsistent with the redution in heptomegly. Overll, the phrmodynmis of lirglutide in the DIO-NASH mie re in generl greement with reent phse- II (LEAN) tril for NASH, where lirglutide tretment promoted signifint weight loss (pproximtely %) nd improved NAS (lowered stetosis sore) without worsening of fibrosis stge []. GLP- reeptor gonists hve not shown signifint effets on liver fibrosis morphology in vrious mouse models of NASH []. Future studies must therefore im to lrify if extended dosing periods re required for deteting ntifibroti effiy of lirglutide, or whether the lk of diret hepti tion ould be limiting ftor for influening liver fibrosis in obese mouse models of NASH. is first-in-lss seletive syntheti FXR gonist tht is pproximtely -fold more potent thn its nturl bile id homologue [7]. Nuler FXRs re bundntly expressed in the liver, intestine, nd dipose tissue, serving s primry sensors for regulting enterohepti bile id flow. In the present study, redued heptomegly nd NAS sores in both DIO-NASH nd ob/ob-nash mie. FXR gonists, inluding, hve shown similr effets in geneti nd methinonine-holine defiient (MCD) dietry models of NASH [,,]. The improvement of stetoheptitis in DIO-NASH mie orresponded to redued expression of genes tht re importnt trnsriptionl trgets of FXRs nd regulte heptoyte lipid metbolism nd mrophge tivity []. Wheres severl genes involved in holesterol nd lipoprotein metbolism re trnsriptionl trgets of heptoyte FXRs [], it is not resolved whether FXR stimultion diretly tivtes mster trnsription ftors tht ontrols the expression of genes involved in ftty id biosynthesis []. As FXRs lso suppress trnsriptionl tivity of severl rbohydrte responsive genes, it is ssumed tht FXR redues TG levels minly through inresing plsm lipoprotein lerne nd improving hepti insulin resistne [-]. The nti-inflmmtory effets of FXR re well-estblished. Aordingly, severl 9 Jnury, Volume Issue

17 studies hve reported tht FXR gonists, inluding, ttenute hepti inflmmtion in high-ft fed obese rodents [,,,], nd Fxr-null mie re highly sensitive to develop severe stetoheptitis when fed n obesogeni diet [7]. tretment lso reversed mild-stge heptoyte bllooning in ffeted ob/ob- NASH mie. hs previously been reported to reverse low-grde heptoyte bllooning in mie fed n therogeni diet [], nd different semisyntheti FXR gonist (INT-77) hs shown similr effet in non-fibroti db/db mie []. Interestingly, enhned heptoyte bllooning is lso reported in FXR-defiient hyperholesterolemi mie [7], thus lending further support to FXR-dependent heptoyte protetive effets. As for lirglutide, tretment did not influene fibrosis morphometry, but redued totl hepti Col expression due to redued liver weight. A reent study in ob/ob-nash mie (fed AMLN diet for wk) nd treted with similr dily dose of for wk lso indited no effet on liver fibrosis []. In ontrst, ntifibroti effets of syntheti FXR gonists, inluding, hve been demonstrted in more ggressive models of fibroti NASH [,9]. It should, however, be noted tht redued ll NAS omponents in ob/ob- NASH mie, suggesting tht prolonged tretment might led to improved fibrosis stge in this model. The present dt re in ordne with reent phse-ii (FLINT) tril on tretment for NASH [], reporting signifint improvements of ll NAS omponents without worsening of fibrosis. The FLINT tril lso indited no nti-obesity potentil of, nlogous to the observtions in the present study. PPARs onstitute nother fmily of lignd-tivted nuler reeptors. PPARs re bundntly expressed in the liver (PPAR-α, PPAR-β/δ) nd dipose tissue (PPAR-δ, PPAR-γ), being prominent regultors of lipid nd gluose metbolism, but re lso funtionl in immune ells, inluding monoytes nd mrophges (PPAR-γ) []. Severl subtype-seletive PPAR gonists hve been demonstrted to redue experimentl fibroti stetoheptitis, prtiulrly in MCD mie []. In ontrst, there is sprsity in studies ddressing effets of PPAR gonists in more trnsltionl mouse models of NASH. In DIO-NASH mie, elfibrnor showed similr effiy in reduing body weight nd stetosis ompred to lirglutide, however, the metboli effets of elfibrnor were not ompnied by improvements in liver weight. Aordingly, heptomegly ws either entuted (DIO-NASH mie) or unltered (ob/ob-nash mie) with elfibrnor tretment. RNA sequening nlysis indited stimultion of PPAR nd peroxisoml signling pthwys in DIO-NASH mie, nd heptoyte peroxisome prolifertion nd tumorigenesis hve been onsistent findings in rodent studies with PPAR-α gonists (inluding elfibrnor) [], but not in humns []. Notbly, elfibrnor improved NAS sores with onomitnt redutions in fibrosis stge, lso supported by orresponding liver trnsriptome hnges. In ontrst, totl Col nd gletin- immunoretivity ws unffeted in elfibrnortreted DIO-NASH mie due to the liver hypertrophi effet. Elfibrnor tretment lso promoted mrked effets on hepti signling pthwys ssoited with lipid metbolism, mitohondril energy hrvesting, inflmmtion, fibrogenesis nd exeution of ell deth progrms. Other PPAR gonists, inluding fibrtes (bezfibrte, Wy-,; PPAR-α lignds), thizolidinediones (rosiglitzone, PPAR-γ lignd) nd GW (PPAR-β/δ lignd), hve been reported to redue fibroti stetoheptitis in MCD mie [,,]. PPAR stimultion is suggested to meliorte NASH primrily by enhning β-oxidtion ssoited hepti ftty id disposl, but nti-inflmmtory nd ntifibroti mehnisms might lso ontribute to improvements in liver histology independent on redued heptoyte lipid umultion [,]. In the present study, dul PPAR-α/δ stimultion by elfibrnor my thus improve fibrosis stge in DIO-NASH nd ob/ob-nash mie by resolving intrhepti lipid deposition but lso through enggement of lipid metbolism-independent signling pthwys. The phrmokinetis of elfibrnor hs not been reported in detil, but preliminry study in rts indited elfibrnor exretion in the bile suggestive of extensive enterohepti yling [9]. Elfibrnor my therefore be onsidered liver-trgeted, whih ould enhne tretment effiy in DIO-NASH nd ob/ob- NASH mie. The present dt re in overll greement with reent linil phse-ii study (GOLDEN-) on elfibrnor tretment for NASH []. Interestingly, the improvements in men NAS (pproximtely. points) nd fibrosis sore (pproximtely. points) in elfibrnor-treted NASH ptients hieving the modified primry outome (no fibrosis worsening) ws on pr with the redutions in NAS nd fibrosis sores determined in DIO-NASH nd ob/ob-nash mie. In ontrst to the weight neutrl effet of elfibrnor in NASH ptients, elfibrnor mrkedly redued body weight in both DIO-NASH nd ob/ob-nash mie. Wheres body weight loss hs not been reported with PPAR gonist tretment in humns [], stimulted PPAR-α nd PPAR-δ funtion hs been ssoited with weight loss, ppetite suppression nd redued tissue lipid deposition in obesity-prone mie [-], whih ould imply speies-dependent metboli effets of PPAR gonists. In onlusion, the present omprtive phrmologil study in diet-indued nd genetilly obese mouse models of biopsy-onfirmed NASH indited both shred nd distint nti-nash effets of lirglutide,, nd elfibrnor. The nti-nash effets of the ompounds orresponded well to their individul modes of tion, nd were lso in onordne to histologil findings in linil trils for NASH. The present dt therefore further supports the linil trnsltbility nd utility of DIO-NASH nd ob/ob-nash mouse models of NASH in prelinil drug development. 9 Jnury, Volume Issue

18 ARTICLE HIGHLIGHTS Reserh bkground Although vrious diet-indued obese mouse models of nonloholi stetoheptitis (NASH) re highly pplible in prelinil drug development, none of these models hve so fr been systemtilly evluted with respet to ompring individul phrmodynmis of severl importnt ompound lsses in urrent linil development for NASH. Reserh motivtion Comprison of prelinil tretment effets of potentil nti-nash ompounds is urrently hmpered by the bsene of hed-to-hed phrmologil studies in experimentl models of NASH. Moreover, bseline NASH disese heterogeneity is often overlooked in phrmologil studies whih my introdue unintentionl vribility in tretment responses, thereby nrrowing the window for detetion of therpeuti effets of potentil nti-nash ompounds. Reserh objetives The present study imed to hrterize within-subjet tretment responses to lirglutide, obetiholi id nd elfibrnor in two obese mouse models of biopsy-onfirmed NASH. Reserh methods Comprtive tretment studies were onduted in mle wild-type mie (DIO- NASH) nd Lep ob/ob (ob/ob-nash) mie fed diet high in trns-ft, frutose nd holesterol. Liver biopsy ws pplied for strtifition of liver stetosis nd fibrosis, using the nonloholi ftty liver disese tivity sore (NAS) nd fibrosis stging system. Individul biopsy-onfirmed histopthology lso llowed for evlution of within-subjet tretment responses bsed on differenes in bseline vs. endpoint histomorphometry. DIO-NASH nd ob/ob-nash mie were treted with vehile, lirglutide, obetiholi id, or elfibrnor for weeks. Metboli, histomorphologil nd quntittive histologil effets of eh ompound were ompred in the two models of biopsy-onfirmed NASH. Reserh results Only lirglutide nd elfibrnor redued body weight in DIO-NASH nd ob/ob- NASH mie. Lirglutide improved stetosis sores in DIO-NASH mie only. Wheres elfibrnor nd both redued hepti stetosis nd inflmmtion sores in both models, only elfibrnor lso improved fibrosis stge. Owing to mrked redution in liver weight, lirglutide nd lowered totl liver ft, ollgen nd gletin- ontent. Individul drug effets on NASH histologil endpoints were supported by globl gene expression (RNA sequening) nd liver lipid biohemistry. Reserh onlusions DIO-NASH nd ob/ob-nash mie show good linil trnsltbility with respet to disese etiology, histopthology nd therpeuti effets of ompounds in lte-stge linil development for NASH. Reserh perspetives The present dt supports the utility of DIO-NASH nd ob/ob-nash mie for prelinil evlution of the tretment effiy of potentil novel nti-nash ompounds. As lso pplied in linil trils for NASH, biopsy-onfirmed histopthology in the two obese mouse models of NASH llows for strtifition of disese severity prior to tretment strt nd improves detetion of tretment effiy of test ompounds by onsidering within-subjet responses. ACKNOWLEDGEMENTS The uthors would like to thnk Louise D Fensholdt, Dn Hemmingsen, Mrtin Illemnn, Rsmus Lind nd Chen Zhng for skillful tehnil ssistne. REFERENCES Bedoss P. Pthology of non-loholi ftty liver disese. Liver Int 7; 7 Suppl : -9 [PMID: 9 DOI:./ liv.] Rosso N, Chvez-Tpi NC, Tiribelli C, Bellentni S. Trnsltionl pprohes: from ftty liver to non-loholi stetoheptitis. World J Gstroenterol ; : 9-99 [PMID: 77 DOI:.7/wjg.v.i7.9] Berlng A, Guiu-Jurdo E, Porrs JA, Auguet T. Moleulr pthwys in non-loholi ftty liver disese. Clin Exp Gstroenterol ; 7: -9 [PMID: 7 DOI:.7/ CEG.S] Sutti S, Jindl A, Bruzzì S, Lotelli I, Bozzol C, Albno E. Is there role for dptive immunity in nonloholi stetoheptitis? World J Heptol ; 7: 7-79 [PMID: 7 DOI:./wjh.v7.i.7] Gnz M, Szbo G. Immune nd inflmmtory pthwys in NASH. Heptol Int ; 7 Suppl : 77-7 [PMID: 77 DOI:.7/s7--9-] Younossi ZM, Koenig AB, Abdeltif D, Fzel Y, Henry L, Wymer M. Globl epidemiology of nonloholi ftty liver disese-metnlyti ssessment of prevlene, inidene, nd outomes. Heptology ; : 7- [PMID: 77 DOI:./ hep.] 7 Rotmn Y, Snyl AJ. Current nd upoming phrmotherpy for non-loholi ftty liver disese. Gut 7; : -9 [PMID: 79 DOI:./gutjnl--] Hnsen HH, Feigh M, Veidl SS, Rigbolt KT, Vrng N, Fosgeru K. Mouse models of nonloholi stetoheptitis in prelinil drug development. Drug Disov Tody 7; : 77-7 [PMID: 79 DOI:./j.drudis.7..7] 9 Clpper JR, Hendriks MD, Gu G, Wittmer C, Dolmn CS, Herih J, Athnio J, Villesz C, Ghosh SS, Heilig JS, Lowe C, Roth JD. Diet-indued mouse model of ftty liver disese nd nonloholi stetoheptitis refleting linil disese progression nd methods of ssessment. Am J Physiol Gstrointest Liver Physiol ; : G-G9 [PMID: DOI:./ jpgi.79.] Trevskis JL, Griffin PS, Wittmer C, Neushwnder-Tetri BA, Brunt EM, Dolmn CS, Erikson MR, Npor J, Prkes DG, Roth JD. Glugon-like peptide- reeptor gonism improves metboli, biohemil, nd histopthologil indies of nonloholi stetoheptitis in mie. Am J Physiol Gstrointest Liver Physiol ; : G7-G77 [PMID: 99 DOI:./ jpgi.7.] Asghrpour A, Cznve SC, Pn T, Seneshw M, Vinent R, Bnini BA, Kumr DP, Dit K, Min HK, Mirshhi F, Bedoss P, Sun X, Hoshid Y, Koduru SV, Contifer D Jr, Wrnke UO, Wijesinghe DS, Snyl AJ. A diet-indued niml model of non-loholi ftty liver disese nd heptoellulr ner. J Heptol ; : 79- [PMID: 7 DOI:./ j.jhep...] Krishnn A, Abdullh TS, Mounjjed T, Hrtono S, MConio A, White T, LeBrsseur N, Lnz I, Nir S, Gores G, Chrlton M. A longitudinl study of whole body, tissue, nd ellulr physiology in mouse model of fibrosing NASH with high fidelity to the humn ondition. Am J Physiol Gstrointest Liver Physiol 7; : G-G [PMID: DOI:./jpgi..] Hzeyni F, Poekes L, Wng H, Mridh AR, Brn V, Geoffrey High W, Ionnou GN, Yeh MM, Lelerq IA, Teoh NC, Frrell GC. Obetiholi id improves dipose morphometry nd inflmmtion nd redues stetosis in dietry but not metboli obesity in mie. Obesity (Silver Spring) 7; : - [PMID: 7 DOI:./oby.7] Ip E, Frrell G, Hll P, Robertson G, Lelerq I. Administrtion 9 Jnury, Volume Issue

19 of the potent PPARlph gonist, Wy-,, reverses nutritionl fibrosis nd stetoheptitis in mie. Heptology ; 9: -9 [PMID: 77 DOI:./hep.7] Zhng S, Wng J, Liu Q, Hrnish DC. Frnesoid X reeptor gonist WAY- ttenutes liver inflmmtion nd fibrosis in murine model of non-loholi stetoheptitis. J Heptol 9; : - [PMID: 997 DOI:./j.jhep.9..] Lee JH, Kng YE, Chng JY, Prk KC, Kim HW, Kim JT, Kim HJ, Yi HS, Shong M, Chung HK, Kim KS. An engineered FGF vrint, LY9, n prevent non-loholi stetoheptitis by enhning hepti mitohondril funtion. Am J Trnsl Res ; : 7-7 [PMID: 7977] 7 Snyl AJ, Friedmn SL, MCullough AJ, Dimik-Sntos L; Amerin Assoition for the Study of Liver Diseses; United Sttes Food nd Drug Administrtion. Chllenges nd opportunities in drug nd biomrker development for nonloholi stetoheptitis: findings nd reommendtions from n Amerin Assoition for the Study of Liver Diseses-U.S. Food nd Drug Administrtion Joint Workshop. Heptology ; : 9- [PMID: 79 DOI:./hep.77] Rtziu V, Bellentni S, Cortez-Pinto H, Dy C, Mrhesini G. A position sttement on NAFLD/NASH bsed on the EASL 9 speil onferene. J Heptol ; : 7- [PMID: 97 DOI:./j.jhep...] 9 Kristinsen MN, Veidl SS, Rigbolt KT, Tølbøl KS, Roth JD, Jelsing J, Vrng N, Feigh M. Obese diet-indued mouse models of nonloholi stetoheptitis-trking disese by liver biopsy. World J Heptol ; : 7- [PMID: 7 DOI:./wjh. v.i.7] Armstrong MJ, Gunt P, Aithl GP, Brton D, Hull D, Prker R, Hzlehurst JM, Guo K; LEAN tril tem, Aboud G, Aldersley MA, Stoken D, Gough SC, Tomlinson JW, Brown RM, Hübsher SG, Newsome PN. Lirglutide sfety nd effiy in ptients with non-loholi stetoheptitis (LEAN): multientre, doubleblind, rndomised, plebo-ontrolled phse study. Lnet ; 7: 79-9 [PMID: DOI:./S- 7()-X] Neushwnder-Tetri BA, Loomb R, Snyl AJ, Lvine JE, Vn Ntt ML, Abdelmlek MF, Chlsni N, Dsrthy S, Diehl AM, Hmeed B, Kowdley KV, MCullough A, Terrult N, Clrk JM, Tonsi J, Brunt EM, Kleiner DE, Doo E; NASH Clinil Reserh Network. Frnesoid X nuler reeptor lignd obetiholi id for non-irrhoti, non-loholi stetoheptitis (FLINT): multientre, rndomised, plebo-ontrolled tril. Lnet ; : 9-9 [PMID: DOI:./ S-7()9-] Rtziu V, Hrrison SA, Frnque S, Bedoss P, Lehert P, Serfty L, Romero-Gomez M, Boursier J, Abdelmlek M, Cldwell S, Drenth J, Anstee QM, Hum D, Hnf R, Roudot A, Megnien S, Stels B, Snyl A; GOLDEN- Investigtor Study Group. Elfibrnor, n Agonist of the Peroxisome Prolifertor-Ativted Reeptor-α nd -δ, Indues Resolution of Nonloholi Stetoheptitis Without Fibrosis Worsening. Gstroenterology ; : 7-9.e [PMID: 77 DOI:./j.gstro...] Mdsen K, Knudsen LB, Agersoe H, Nielsen PF, Thøgersen H, Wilken M, Johnsen NL. Struture-tivity nd protrtion reltionship of long-ting glugon-like peptide- derivtives: importne of ftty id length, polrity, nd bulkiness. J Med Chem 7; : - [PMID: 7979 DOI:./ jm7j] Druker DJ, Dritselis A, Kirkptrik P. Lirglutide. Nt Rev Drug Disov ; 9: 7- [PMID: 7 DOI:./ nrd] Nuffer WA, Trujillo JM. Lirglutide: A New Option for the Tretment of Obesity. Phrmotherpy ; : 9-9 [PMID: 9779 DOI:./phr.9] Mrkhm A, Kem SJ. Obetiholi Aid: First Globl Approvl. Drugs ; 7: - [PMID: 7 DOI:.7/ s---x] 7 Pelliiri R, Fiorui S, Cmioni E, Clerii C, Costntino G, Mloney PR, Morelli A, Prks DJ, Willson TM. lph-ethylhenodeoxyholi id (-ECDCA), potent nd seletive FXR gonist endowed with ntiholestti tivity. J Med Chem ; : 9-7 [PMID: 97 DOI:./jm9g] Asghrpour A, Kumr D, Snyl A. Bile ids: emerging role in mngement of liver diseses. Heptol Int ; 9: 7- [PMID: DOI:.7/s7--9-7] 9 Criou B, Hnf R, Lmbert-Porheron S, Zïr Y, Suvinet V, Noël B, Flet L, Vidl H, Stels B, Lville M. Dul peroxisome prolifertor-tivted reeptor α/δ gonist GFT improves hepti nd peripherl insulin sensitivity in bdominlly obese subjets. Dibetes Cre ; : 9-9 [PMID: 77 DOI:.7/d-] Stels B, Rubenstrunk A, Noel B, Rigou G, Deltille P, Milltt LJ, Bron M, Lus A, Tilleux A, Hum DW, Rtziu V, Criou B, Hnf R. Heptoprotetive effets of the dul peroxisome prolifertortivted reeptor lph/delt gonist, GFT, in rodent models of nonloholi ftty liver disese/nonloholi stetoheptitis. Heptology ; : 9-9 [PMID: 7 DOI:./ hep.] Kleiner DE, Brunt EM, Vn Ntt M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unlp- Arid A, Yeh M, MCullough AJ, Snyl AJ; Nonloholi Stetoheptitis Clinil Reserh Network. Design nd vlidtion of histologil soring system for nonloholi ftty liver disese. Heptology ; : - [PMID: 9 DOI:./ hep.7] Wng XX, Wng D, Luo Y, Mykl K, Dobrinskikh E, Rosenberg AZ, Levi J, Kopp JB, Field A, Hill A, Lui S, Qiu L, Jing T, Peng Y, Orliky D, Gri G, Hermn-Edelstein M, D Agti V, Henriksen K, Adorini L, Pruznski M, Xie C, Krusz KW, Gonzlez FJ, Rnjit S, Dvornikov A, Grtton E, Levi M. FXR/ TGR Dul Agonist vents Progression of Nephropthy in Dibetes nd Obesity. J Am So Nephrol ; 9: -7 [PMID: 997 DOI:./ASN.7] Rhmn K, Liu Y, Kumr P, Smith T, Thorn NE, Frris AB, Anni FA. C/EBP homologous protein modultes lirglutidemedited ttenution of non-loholi stetoheptitis. Lb Invest ; 9: 9-9 [PMID: 797 DOI:./ lbinvest..] Henderson SJ, Konkr A, Hornigold DC, Trevskis JL, Jkson R, Fritsh Fredin M, Jnsson-Löfmrk R, Nylor J, Rossi A, Bednrek MA, Bhgroo N, Slri H, Will S, Oldhm S, Hnsen G, Feigh M, Klein T, Grimsby J, Mguire S, Jermutus L, Rondinone CM, Coghln MP. Robust nti-obesity nd metboli effets of dul GLP-/glugon reeptor peptide gonist in rodents nd non-humn primtes. Dibetes Obes Metb ; : 7-9 [PMID: 777 DOI:./dom.7] Hnf R, Milltt LJ, Criou B, Noel B, Rigou G, Deltille P, Dix V, Hum DW, Stels B. The dul peroxisome prolifertor-tivted reeptor lph/delt gonist GFT exerts nti-dibeti effets in db/db mie without peroxisome prolifertor-tivted reeptor gmm-ssoited dverse rdi effets. Dib Vs Dis Res ; : -7 [PMID: 9 DOI:.77/79 7] Dobin A, Dvis CA, Shlesinger F, Drenkow J, Zleski C, Jh S, Btut P, Chisson M, Gingers TR. STAR: ultrfst universl RNA-seq ligner. Bioinformtis ; 9: - [PMID: DOI:.9/bioinformtis/bts] 7 Love MI, Huber W, Anders S. Moderted estimtion of fold hnge nd dispersion for RNA-seq dt with DESeq. Genome Biol ; : [PMID: DOI:./s9---] Yu G, Wng LG, Hn Y, He QY. lusterprofiler: n R pkge for ompring biologil themes mong gene lusters. OMICS ; : -7 [PMID: DOI:.9/omi..] 9 Hond Y, Imjo K, Kto T, Kessoku T, Ogw Y, Tomeno W, Kto S, Mwtri H, Fujit K, Yoned M, Sito S, Nkjim A. The Seletive SGLT Inhibitor Iprgliflozin Hs Therpeuti Effet on Nonloholi Stetoheptitis in Mie. PLoS One ; : e7 [PMID: 77 DOI:.7/journl.pone.7] Henkel J, Colemn CD, Shrplu A, Johrens, Weber D, Cstro JP, Hugo M, Shulz TJ, Krämer S, Shürmnn A, Püshel GP. Indution of stetoheptitis (NASH) with insulin resistne in wildtype B mie by western-type diet ontining soyben oil 9 Jnury, Volume Issue

20 nd holesterol. Mol Med 7; : Epub hed of print [PMID: 9 DOI:.9/molmed..] Mells JE, Fu PP, Shrm S, Olson D, Cheng L, Hndy JA, Sxen NK, Soresu D, Anni FA. Glp- nlog, lirglutide, meliortes hepti stetosis nd rdi hypertrophy in C7BL/J mie fed Western diet. Am J Physiol Gstrointest Liver Physiol ; : G-G [PMID: 9 DOI:./jpgi.7.] Tetri LH, Bsrnoglu M, Brunt EM, Yerin LM, Neushwnder- Tetri BA. Severe NAFLD with hepti neroinflmmtory hnges in mie fed trns fts nd high-frutose orn syrup equivlent. Am J Physiol Gstrointest Liver Physiol ; 9: G97-G99 [PMID: 77 DOI:./jpgi.97.] M K, Mlhotr P, Soni V, Hedroug O, Annb F, Dudej A, Shen L, Turner JR, Khrmtsov EA, Sksen S, Dudej PK, Gill RK, Alrefi WA. Overtivtion of intestinl SREBP in mie inreses serum holesterol. PLoS One ; 9: e [PMID: 97 DOI:.7/journl.pone.] Rinell ME. Nonloholi ftty liver disese: systemti review. JAMA ; : -7 [PMID: 77 DOI:./ jm..7] Rei I, Kumr J, Akldios C, Virdis F, Pi M, Hbib N, Splding D. Non-loholi ftty liver disese: A sign of systemi disese. Metbolism 7; 7: 9- [PMID: 7 DOI:./ j.metbol.7..] Neushwnder-Tetri BA. Non-loholi ftty liver disese. BMC Med 7; : [PMID: DOI:./ s9-7--] 7 Ling W, Menke AL, Driessen A, Koek GH, Lindemn JH, Stoop R, Hvekes LM, Kleemnn R, vn den Hoek AM. Estblishment of generl NAFLD soring system for rodent models nd omprison to humn liver pthology. PLoS One ; 9: e9 [PMID: 9 DOI:.7/journl.pone.9] Frrell GC, Mridh AR, Yeh MM, Arsov T, Vn Rooyen DM, Brooling J, Nguyen T, Heydet D, Delghingro-Augusto V, Noln CJ, Shkel NA, MLennn SV, Teoh NC, Lrter CZ. Strin dependene of diet-indued NASH nd liver fibrosis in obese mie is linked to dibetes nd inflmmtory phenotype. Liver Int ; : -9 [PMID: 7 DOI:./liv.] 9 Knoski SE, Hyes MR, Skibik KP. GLP- nd weight loss: unrveling the diverse neurl iruitry. Am J Physiol Regul Integr Comp Physiol ; : R-R9 [PMID: 79 DOI:./jpregu..] Ptel NS, Doyhev I, Peterson MR, Hooker J, Kisselv T, Shnbl B, Seki E, Sirlin CB, Loomb R. Effet of weight loss on mgneti resonne imging estimtion of liver ft nd volume in ptients with nonloholi stetoheptitis. Clin Gstroenterol Heptol ; : -.e [PMID: 7 DOI:./j.gh...9] MMhn RH, Wng XX, Cheng LL, Krisko T, Smith M, El Ksmi K, Pruznski M, Adorini L, Golden-Mson L, Levi M, Rosen HR. Bile id reeptor tivtion modultes hepti monoyte tivity nd improves nonloholi ftty liver disese. J Biol Chem ; : 7-77 [PMID: DOI:.7/jb.M.7] Kuipers F, Bloks VW, Groen AK. Beyond intestinl sop--bile ids in metboli ontrol. Nt Rev Endorinol ; : -9 [PMID: DOI:./nrendo..] Fiorui S, Menrelli A, Plldino G, Ciprini S. Bile-idtivted reeptors: trgeting TGR nd frnesoid-x-reeptor in lipid nd gluose disorders. Trends Phrmol Si 9; : 7- [PMID: 977 DOI:./j.tips.9..] Li Y, Jdhv K, Zhng Y. Bile id reeptors in non-loholi ftty liver disese. Biohem Phrmol ; : 7- [PMID: 97 DOI:./j.bp...] M Y, Hung Y, Yn L, Go M, Liu D. Syntheti FXR gonist GW prevents diet-indued hepti stetosis nd insulin resistne. Phrm Res ; : 7-7 [PMID: 77 DOI:.7/s9--9-7] Ciprini S, Menrelli A, Plldino G, Fiorui S. FXR tivtion reverses insulin resistne nd lipid bnormlities nd protets ginst liver stetosis in Zuker (f/f) obese rts. J Lipid Res ; : 77-7 [PMID: 97 DOI:.9/jlr.M] 7 Kong B, Luyendyk JP, Twfik O, Guo GL. Frnesoid X reeptor defiieny indues nonloholi stetoheptitis in lowdensity lipoprotein reeptor-knokout mie fed high-ft diet. J Phrmol Exp Ther 9; : - [PMID: 997 DOI:./jpet..] Jouihn H, Will S, Guionud S, Bolnd ML, Oldhm S, Rvn P, Celeste A, Trevskis JL. Superior redutions in hepti stetosis nd fibrosis with o-dministrtion of glugon-like peptide- reeptor gonist nd obetiholi id in mie. Mol Metb 7; : -7 [PMID: 97 DOI:./ j.molmet.7.9.] 9 Zhng DG, Zhng C, Wng JX, Wng BW, Wng H, Zhng ZH, Chen YH, Lu Y, To L, Wng JQ, Chen X, Xu DX. Obetiholi id protets ginst rbon tetrhloride-indued ute liver injury nd inflmmtion. Toxiol Appl Phrmol 7; : 9-7 [PMID: 7 DOI:./j.tp...] Chinetti G, Fruhrt JC, Stels B. Peroxisome prolifertortivted reeptors: new trgets for the phrmologil modultion of mrophge gene expression nd funtion. Curr Opin Lipidol ; : 9- [PMID: DOI:.97/ --] Gonzlez FJ, Shh YM. PPARlph: mehnism of speies differenes nd heptorinogenesis of peroxisome prolifertors. Toxiology ; : - [PMID: DOI:./ j.tox.7.9.] Ngsw T, Ind Y, Nkno S, Tmur T, Tkhshi T, Mruym K, Ymzki Y, Kurod J, Shibt N. Effets of bezfibrte, PPAR pn-gonist, nd GW, PPARdelt gonist, on development of stetoheptitis in mie fed methionine- nd holine-defiient diet. Eur J Phrmol ; : -9 [PMID: 799 DOI:./j.ejphr...] Pwlk M, Bugé E, Bourguet W, De Bossher K, Llloyer F, Tilleux A, Lebherz C, Lefebvre P, Stels B. The trnsrepressive tivity of peroxisome prolifertor-tivted reeptor lph is neessry nd suffiient to prevent liver fibrosis in mie. Heptology ; : 9- [PMID: 999 DOI:./ hep.797] Tnk N, Aoym T, Kimur S, Gonzlez FJ. Trgeting nuler reeptors for the tretment of ftty liver disese. Phrmol Ther 7; 79: -7 [PMID: DOI:./j.phrmther. 7..] Svkur RS, Miller AR. Investigtionl PPAR-gmm gonists for the tretment of Type dibetes. Expert Opin Investig Drugs ; : 7-77 [PMID: 77 DOI:.7/7..7.7] Wng YX, Zhng CL, Yu RT, Cho HK, Nelson MC, Byug- Ompo CR, Hm J, Kng H, Evns RM. Regultion of musle fiber type nd running endurne by PPARdelt. PLoS Biol ; : e9 [PMID: DOI:.7/journl.pbio.9] 7 Hrrington WW, S Britt C, G Wilson J, O Milliken N, G Binz J, C Lobe D, R Oliver W, C Lewis M, M Ignr D. The Effet of PPARlph, PPARdelt, PPARgmm, nd PPARpn Agonists on Body Weight, Body Mss, nd Serum Lipid Profiles in Diet- Indued Obese AKR/J Mie. PPAR Res 7; 7: 97 [PMID: 777 DOI:./7/97] Rhid TL, Penn-de-Crvlho A, Bringhenti I, Aguil MB, Mndrim-de-Lerd CA, Souz-Mello V. Fenofibrte (PPARlph gonist) indues beige ell formtion in subutneous white dipose tissue from diet-indued mle obese mie. Mol Cell Endorinol ; : -9 [PMID: 7 DOI:./j.me...7] P- Reviewer: Blbn YH, Murotomi K, Pn Q, Sutti S, Tnk N S- Editor: Gong ZM L- Editor: A E- Editor: M YJ 9 Jnury, Volume Issue

21 Published by Bishideng Publishing Group In 79 Stoneridge Drive, Suite, Plesnton, CA 9, USA Telephone: Fx: E-mil: Help Desk: I S S N Bishideng Publishing Group In. All rights reserved.