inhibition of ileal bile acid uptake protects against nonalcoholic fatty

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Inhibition of ileal bile acid uptake protectsagainst nonalcoholic fatty liver disease in high-fatdiet–fed miceAnuradha Rao,1 Astrid Kosters,1 Jamie E. Mells,1 Wujuan Zhang,2 Kenneth D. R. Setchell,2

Angelica M. Amanso,1 Grace M. Wynn,1 Tianlei Xu,3 Brad T. Keller,4 Hong Yin,5 Sophia Banton,6

Dean P. Jones,6,7 Hao Wu,8 Paul A. Dawson,1,5 Saul J. Karpen1,5*

Nonalcoholic fatty liver disease (NAFLD) is themost common chronic liver disease in theWestern world, and safe andeffective therapies are needed. Bile acids (BAs) and their receptors [including the nuclear receptor for BAs, farnesoid Xreceptor (FXR)] play integral roles in regulating whole-body metabolism and hepatic lipid homeostasis. We hypothe-sized that interruption of the enterohepatic BA circulation using a luminally restricted apical sodium-dependent BAtransporter (ASBT) inhibitor (ASBTi; SC-435) wouldmodify signaling in the gut-liver axis and reduce steatohepatitis inhigh-fat diet (HFD)–fed mice. Administration of this ASBTi increased fecal BA excretion and messenger RNA (mRNA)expression of BA synthesis genes in liver and reduced mRNA expression of ileal BA-responsive genes, including thenegative feedback regulator of BA synthesis, fibroblast growth factor 15. ASBT inhibition resulted in amarked shift inhepatic BA composition, with a reduction in hydrophilic, FXR antagonistic species and an increase in FXR agonisticBAs. ASBT inhibition restoredglucose tolerance, reducedhepatic triglyceride and total cholesterol concentrations, andimproved NAFLD activity score in HFD-fed mice. These changes were associated with reduced hepatic expression oflipid synthesis genes (including liver X receptor target genes) and normalized expression of the central lipogenictranscription factor, Srebp1c. Accumulation of hepatic lipids and SREBP1 protein were markedly reduced in HFD-fed Asbt−/− mice, providing genetic evidence for a protective role mediated by interruption of the enterohepaticBA circulation. Together, these studies suggest that blocking ASBT function with a luminally restricted inhibitorcan improve both hepatic and whole body aspects of NAFLD.

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INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is one of the most commonliver diseases in the Western world, with an increasing prevalencearound the globe (1, 2). NAFLD encompasses a pathophysiologicalspectrum ranging from steatosis to nonalcoholic steatohepatitis (NASH),cirrhosis, and liver carcinoma, along with substantial liver and whole-body metabolic derangements. Effective medical therapies to slow or re-verse aspects of this progression are limited. Inadequate or no responsewas observed for pioglitazone, vitamin E, and omega-3 fatty acid in clin-ical trials for NASH, although a recent trial with the farnesoid X receptor(FXR) agonistic bile acid (BA) analog obeticholic acid (OCA) showedpromising results (3–6). Dysregulated hepatic lipid, sterol, and insulin-mediatedmetabolic pathways appear to bemajor pathophysiological con-tributors, but an incomplete understanding of themechanismsunderlyingthe development of NAFLD and its progression to NASH continue to af-fect the development of rational therapeutics.

BAs, their receptors (FXR, TGR5, and S1PR2), and gut luminal BA-metabolizing bacteria have emerged as important regulators of hepatic

1Department of Pediatrics, Emory University School of Medicine, 1760 Haygood DriveNortheast, Atlanta, GA 30322, USA. 2Department of Pathology and Laboratory Medicine,Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229,USA. 3Department of Mathematics and Computer Science, Emory University, Atlanta, GA30322, USA. 4Vasculox Inc., St. Louis, MO 63108, USA. 5Children’s Healthcare of Atlanta, 2015Uppergate Drive Northeast, Atlanta, GA 30322, USA. 6Department of Biochemistry, EmoryUniversity, 1510 Clifton Road Northeast, Atlanta, GA 30322, USA. 7Department of Medicine,Emory University School of Medicine, 100Woodruff Circle, Atlanta, GA 30322, USA. 8Depart-ment of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University,Atlanta, GA 30322, USA.*Corresponding author. Email: [email protected]

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lipid and glucose metabolism (7–9). BAs are synthesized from choles-terol in the liver, are secreted into bile as the major solute, and functionto facilitate lipid absorption in the intestine. About 95% of intestinalBAs are reabsorbed in the ileum by the apical sodium-dependent BAtransporter (ASBT) and conveyed through the portal vein to the liver,where they are taken up by hepatocytes to be resecreted into bile (10).This efficient enterohepatic circulation serves to maintain the BA pooland predominantly restrict BAs to intestinal and hepatobiliary com-partments. The size and composition of the BA pool is dependent onmany factors, including FXR-dependent feedback regulatory pathwaysin both liver and ileum, enterohepatic cycling frequency, and metabo-lism by gutmicrobiota (10, 11). Changes in compartmentalization, con-centration, and composition of the BA pool may have metabolicregulatory consequences, particularly in light of the spectrum of FXRagonistic and antagonistic potencies possessed by individual BAs. How-ever, confounding our understanding of the metabolic effects of BAsthrough FXR and other receptors is their complex relationship withthe gut microbiota, whereby the BA pool size and composition appearto be amajor regulator of themicrobiome community and vice versa (9).

BA signaling in the intestine and liver has a role in the regulation oflipid, glucose, and energy homeostasis and is a potential target for treat-ment of obesity and NAFLD (12, 13). However, the underlying mecha-nisms remain unclear because interventions that increase, aswell as thosethat decrease BA (and FXR) signaling may yield metabolic benefits(8, 14–20). Here, we focused on examining the effects of pharmaco-logical and genetic inhibition of ileal BA absorption on the developmentof fatty liver in the American lifestyle–induced obesity syndrome(ALIOS) high-fat diet (HFD)–fed mouse model of NAFLD (21). Oral

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administration of SC-435, a potent luminally restricted ASBT inhibitor(ASBTi), restored glucose tolerance, reduced the steatohepatitic pathol-ogy, and altered liver gene expression in HFD-fed mice. Analysis ofpotential mechanisms in this dietary model and in HFD-fed Asbt−/−

mice revealed that the improved features of NAFLD correlated in partwith reductions in the expression of liver X receptor (LXR) target genesalongwith a shift toward amore hydrophobic and FXR agonistic profileof the hepatic BA pool.

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RESULTS

Administration of an ASBTi impairs ileal BA uptake inHFD-fed miceTo determinewhether interrupting the enterohepatic circulation of BAswith an ASBTi can improve HFD-induced hepatic steatosis, maleC57Bl/6J mice were fed one of four diets for 16 weeks, as outlined inFig. 1A: chow, HFD [modified ALIOS, 45% kcal fat plus 0.2% (w/w)cholesterol], HFD plus ASBTi for 16 weeks (HFD/ASBTi16w; 60 partsper million SC-435, ~11mg/kg per day), or HFD for 12 weeks followedby HFD/ASBTi for the final 4 weeks (HFD/ASBTi4w; to determinewhether the HFD-induced changes could be reversed). All HFD groupswere given 4% sucrose water ad libitum (21). For clarity, data related tothe 16-week arm are discussed first, followed by a section describing thefinal 4-week treatment arm. In agreement with previous studies(22–24), ileal BA absorption was impaired by administration of the


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ASBTi, as indicated by a fourfold increasein fecal BA excretion, induction of hepaticsentinel BA synthesis target genes Cyp7a1and Cyp8b1 [by 2.4 ± 1.0–fold (P<0.0001)and 2.3 ± 1.0–fold (P < 0.0001), respectively;see table S1 for P values], suppression ofFXR-activated Fgf15 mRNA expression inileum [by 93 ± 7% (P < 0.0001)], andincreased FXR-responsive Ibabp mRNAexpression in colon [7.0 ± 2.0–fold (P <0.0001); Fig. 1, B to E]. Asbt mRNA ex-pression in ileum and colon was un-changed by administration of the ASBTi(fig. S1, A and B). In comparison to un-treated HFD-fed mice, administration ofthe ASBTi induced colonic expression ofAkr1b7, an FXR target gene involved inBA metabolism in mice (fig. S1C). Thus,luminally restricted inhibition of ASBTin HFD-fed mice showed the anticipatedeffects in ileum, colon, and liver with re-spect to BAmetabolism.Because BAshavebeen reported to induce injury (25, 26), wealso examined the colonic expression ofgenes involved in cell stress and oncogenicsignaling. There were no differences be-tween HFD and HFD/ASBTi16w mice incolonic expression of genes involved in en-doplasmic reticulum stress (Atf6, Ddit3,Hspa1a, Hspa1b, Xbp1, and Xbp1p; fig.S1D), oncogenic signaling genes (Cdkn2a,Ctnnb1, Ccnd1, Myc, Mmp7, and Ogg1;

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fig. S1E), or genes involved in innate immunity and inflammation (Ccl2,Il1b, Il6, Il12a, Il12b, and Tnf; fig. S1F).

Administration of an ASBTi restores glucose tolerance inHFD-fed miceHFD feeding slightly increased final body weight, as compared to chow-fedmice, in agreementwith the small increase in daily caloric intake (Fig. 2,A toC).However, the final body weights of the HFD/ASBTi16wmicewere not different from those of the HFDmice, despite consumingmorecalories eachday (Fig. 2,A toC, and fig. S1,GandH). Liverweight to bodyweight ratio was increased in the HFDmice versus chow-fedmice, whichwas still apparent in the 16-week HFD/ASBTi–treated group (Fig. 2D).Markers of hepatocellular injury, such as serum alanine aminotransferaseand aspartate aminotransferase were not different between the groups,but a minor increase in serum alkaline phosphatase was observed inthe HFD mice but not in the HFD/ASBTi16w mice (fig. S1I). To deter-mine the effects of the ASBTi on glucose and systemic insulin sensitivity,weperformed glucose and insulin tolerance tests. FeedingmicewithHFDfor 16weeks resulted in glucose intolerance,whereas additionof anASBTito the HFD during this entire time restored glucose tolerance and im-proved systemic insulin sensitivity (Fig. 2, E and F, and fig. S2, A and B).

Administration of an ASBTi prevents hepatic steatosis inHFD-fed miceAfter 16 weeks of feeding mice with the HFD, many characteristic his-tological features ofNASHwere observed in the animals’ livers, including

Fig. 1. Administration of an ASBTi increases fecal BA excretion and hepatic BA synthesis. (A) Studydesign showing diets and durations of ASBTi (SC-435) treatments: chow (n = 12), HFD (n = 12), HFD/ASB-
Ti16w (n = 16), and HFD/ASBTi4w (n = 8). (B) Fecal BA excretion in HFD and HFD/ASBTi16w feeding groups.(C) Hepatic expression of Cyp7a1 and Cyp8b1mRNA. (D and E) Ileal Fgf15mRNA expression (D) and colonicIbabp mRNA expression (E). The labeling scheme for each group is indicated in the embedded legend.Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individualP values are provided in table S1.
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steatosis, lobular inflammation, hepatocyte ballooning, and Mallorybodies (Fig. 3, A and B). Compared to HFD-fed mice, lipid depositionwas markedly decreased in the HFD/ASBTi16w mice and to a lesser ex-tent in HFD/ASBTi4w mice (Fig. 3, B to D). Blinded histologic assess-

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ment of slides by an experienced hepatopathologist (H.Y.) indicatedthat HFD/ASBTi16w treatment reduced the NAFLD activity score(NAS) (27) from 4.8 ± 1.0 to 2.8 ± 0.9, primarily driven by improvementsin the steatosis score (Fig. 3, E and F; see table S2 for individual compo-

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nents of the NAS). Biochemical lipid mea-surements confirmed severe triglyceride(TG) and cholesteryl ester deposition inlivers of HFD-fed mice, as compared tochow-fed animals (~12- and 15-fold in-creases, respectively; Fig. 3, G and H).The hepatic concentrations of TG, choles-teryl ester, and total cholesterol weremark-edly reduced in the HFD/ASBTi16w micecompared to those in theHFD-fedmice, toa degree whereby the liver lipid content inHFD/ASBTi16w mice was similar to theliver lipid content in chow-fed mice (Fig. 3,GandH, and fig. S2C). Onlyminor effectswere noted for hepatic free cholesterolconcentrations in diet and treatmentgroupswhen compared to the chowgroup(fig. S2D). Liver fibrosis was also assessed,but neither histologic assessment by SiriusRed staining nor biochemical quantitationof hydroxyproline content revealed anysignificant hepatic fibrosis in the HFD-fed mice. As such, there was little effecton these fibrosis markers in any of thetreatment groups (fig. S3).

Administration of an ASBTialters hepatic BA pool compositionand FXR regulatory properties inHFD-fed miceThe total BA content was not different be-tween chow, HFD, and ASBTi-treatedlivers (Fig. 4A), and HFD feeding hadno discernible impact on hepatic BA poolcomposition when compared to chow(Fig. 4, B to D). However, blocking intes-tinal BA uptake altered the hepatic BApool composition (Fig. 4B), changing itschemical and predicted FXR signalingproperties. Ileal ASBT inhibition reducedthe hepatic concentration of 6-hydroxylatedTMCA species (w, a, and b by > 50%) andincreased the amount of the more hydro-phobicBAs,TCDCAandTDCA,bygreaterthan 10-fold (Fig. 4B).As a result, the hepat-ic BA content shifted from a typical ~0.8:1balanced ratio of non–6-hydroxylated BAspecies to 6-hydroxylated BA species inchow-fed and HFD-fed mice to a >4:1 ratiowith a predominance of the non–6-hydroxylated species in the livers of ASBTimice (Fig. 4B). This is reflected by an in-crease in the calculated hydrophobicityindex, which rose from −0.406 to +0.088

Fig. 2. Administration of an ASBTi restores glucose tolerance. (A) Body weight gain in the indicated adlibitum–fed groups. (B) Final body weight. (C) Average caloric intake over 16 weeks of ad libitum feeding.
(D) Liver weight to body weight ratio after 16 weeks. (E) Glucose tolerance tests (GTTs). * indicates valuessignificantly different between chowandHFD; # indicates values significantly different between chowandHFD/ASBTi4w. Individual P values are provided in table S1. (F) GTT area under the curve (AUC) (gram perliter per minute). The labeling scheme for each group is indicated in the embedded legend. Means + SDare shown. Distinct lowercase letters indicate significant differences between groups).
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(Fig. 4C). In addition to changing the physicochemical properties of thehepatic BA pool, administration of the ASBTi also yielded a BA poolpredicted to be, in sum, substantially more FXR agonistic (16, 28–30) (Fig.4, B and D). Because FXR signaling BAs are important physiologicalregulators of multiple hepatic functions including lipid metabolism,the combined amounts of FXR agonistic (TCA, TCDCA, TDCA,TLCA, and CA) and antagonistic (MCAs) BA species were comparedto the hepatic TG and cholesterol content in livers of individual HFD,HFD/ASBTi16w, and HFD/ASBTi4w mice. Hepatic TG and choles-terol content directly correlated with the amount of FXR antagonisticmuricholates (r = 0.4980, P = 0.0023; r = 0.5829, P = 0.0002) andinversely correlated with TCDCA+TDCA content (r = −0.5381, P =0.0009; r = −0.5630, P = 0.0004) (fig. S4). Overall, the marked altera-tion of the hepatic BA pool toward FXR agonism correlated with thehistologic and biochemical improvements driven by pharmacologicalinhibition of ileal ASBT function.

Previous in vitro studies using primarily individual BAs foundTCDCA and TDCA to be potent FXR agonists, whereas TUDCAand TMCA species failed to activate FXR andmay function as FXR an-tagonists (16, 30–32). To beginmodeling the FXR activity of the distincthepatic BA pools in HFD andHFD/ASBTimice, we examined the abil-ity of BA mixtures to activate an FXR reporter plasmid in transfected

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human liver–derived HepG2 cells. In vitro, bTMCA antagonizes FXRactivation by TCA or TCDCA (Fig. 4, E and F). However, given thein vivo findings, we thought it relevant to examine the in vitro FXR antag-onistic properties of the muricholates in more complex mixtures of BAs,paralleling the composition of BAs present in liver. Mixtures of the sixmost abundant taurine-conjugated BAs were reconstituted at the ratiospresent in HFD or HFD/ASBTi livers (Fig. 4B and table S3) to modelthe in vivo exposure to multiple BAs and their combined effect on FXRactivity. TheBA composition present inHFD/ASBTi livers activated theFXR reporter plasmid to a greater extent than did the reconstituted BAmixture from HFD livers (Fig. 4G). These findings suggest that theconsequences of thismarked shift in liver BA compositionmay underliesome of the hepatic responses to ileal ASBT inhibition.

Administration of an ASBTi alters hepatic metabolic geneexpression in HFD-fed miceTo explore the gene regulatory mechanisms underlying the hepaticeffects of the luminally active ASBTi on HFD-fed mice, we performedRNA sequencing (RNA-seq) analyses using livers from mice fed withchow, HFD, and HFD/ASBTi16w. Comparison of the chow and HFDgroups identified 5033 RNA species that were differentially expressed inlivers ofHFD-fedmice (3186 genes up-regulated and 1847 genes down-



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regulated, P < 0.05; Fig. 5A, fig. S5A, and
table S4). Subsequent comparison of livergene expression in HFD versus HFD/ASBTi16w mice identified 1483 differ-entially expressed genes (764 genes up-regulated and 719 down-regulated,P<0.05;Fig. 5A and table S4). Cross-comparisonof the lists of genes differentially expressedin chow versus HFDmice and in HFD ver-sus HFD/ASBTi16w mice revealed 675genes that are common in the twodata sets.For 650 of these genes (96%), ASBTi treat-ment of the HFD mice changed their ex-pression toward levels seen in chow-fedmouse livers, including several key genesinvolved in fatty acid andcholesterolmetab-olism (Srebp1c, Srebp2, Abcg5, Abcg8, andCidec; Fig. 5, A and B, and table S4). Hierar-chical clustering of RNAexpression andon-tological and pathway analyses revealedmarkedly distinct patterns between groups(Fig. 5, B and C, and fig. S5, B and C),confirming major changes in hepatic geneexpression induced both by HFD and byconcurrent ileal ASBT inhibition. Adminis-tration of theASBTi changed hepatic geneexpression in a variety of lipid, fatty acid,and cholesterolmetabolic pathways, aswellas intracellular transport and signaling (Fig.5C and table S4). Together, these RNA-seqstudies indicate marked shifts in hepaticgene transcription, intracellular signaling,andmetabolism inHFD-fedmice as a con-sequence of ileal ASBT inhibition.

Real-time polymerase chain reaction(PCR)was used tomeasure the expression

Fig. 3. Administration of an ASBTi reduces hepatic steatosis and accumulation of TGs and cholesterol.(A toD) Hematoxylin and eosin–stained liver sections from (A) chow-, (B) HFD-, (C) HFD/ASBTi16w-, and (D)
HFD/ASBTi4w-fed mice. (E) NAS. (F) Steatosis score. (G) Hepatic TG content. (H) Hepatic cholesteryl estercontent. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown.Different lowercase letters indicate significant differences between groups; individual P values are providedin table S1. Scale bars, 100 mm.



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of critical genes in a variety of NAFLD-related pathways in the liver as well as inthe ileum and colon, with the analysesfocused primarily on HFD versus HFD/ASBTi16w mice (Fig. 6, A to C, and fig.S6, A to E). With regard to BA signaling,minimal effects were observed for severalof the known hepatic FXR target genes in-cluding Abcb11 and Shp, whereas mRNAexpression of the BA-activated G protein–coupled receptor (GPCR) S1pr2 and itsdownstream target Sphk2 were increasedtwo- to threefold in HFD-fed mice and re-duced with ASBT inhibition (Fig. 6A). Ex-pression of FXR target genes was generallydecreased in ileum and increased in colon(Fig. 1, D and E, and Fig. 6, B and C), re-flecting the block in ileal BA absorption.Expression of the BA-activated GPCRTgr5wasunaffected in liver, ileum, or colonby administrationof theASBTi (Fig. 6,A toC). Therewere no changes in ileal or colon-ic expression of the TGR5 target gene Gcg,the GLP-1 precursor (fig. S6, B and C).

The increases in hepatic BA synthesiswith inhibition of the ileal ASBT resultedin the expected compensatory increase inexpression of cholesterol biosyntheticgenes (Srepb2 and Hmgr; Fig. 6A), andthe reduced hepatic cholesterol contentin HFD/ASBTi16w mice was associatedwith lower expression of Ch25oh (an en-dogenous source of the LXRa ligand,25-hydroxycholesterol) and LXRa targetgenes (Abcg5,Abcg8, and Srebp1c). Inhibi-tion of ileal ASBT also markedly alteredthe expression of hepatic genes involvedin fatty acid and TG metabolism. Expres-sion of a variety of key biosynthetic andregulatory genes (Srebp1c, Scd1, Fads1,Fads2, and Pparg) was increased two- tofourfold by HFD, and all were reducedtoward chow-fed levels with ASBT inhibi-tion (Fig. 6A and fig. S6D). Fatty acidoxidation–related gene expression wasminimally affected by the ASBTi (Fgf21,Ppara, and Acaa1b; Fig. 6A and fig.S6D). In line with histological data andchanges in lipogenic gene expression,RNA levels of the lipid droplet formationgenes Cidea and Fsp27/Cidec were in-duced >50-fold by HFD and reduced by70 ± 30% and 86 ± 10% with the ASBTi(Fig. 6A and fig. S6E).

The presence of inflammation and fi-brosis with steatosis are hallmarks of pro-gression to NASH, and HFD-inducedproinflammatory gene expression (Tnf,

Fig. 4. Administration of anASBTi shifts hepatic BA composition toward FXRagonism. (A andB) Totalhepatic BA content (A) and composition (B) (TMCA, tauromuricholic acid; MCA, muricholic acid, THDCA,taurohyodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurocheno-deoxycholic acid; TDCA, taurodeoxycholic acid; TLCA, taurolithocholic acid; CA, cholic acid). (C) Hydropho-bicity index. (D) Pie charts for hepatic FXR antagonist/non-agonist (black) and agonist (white) BA content.(E) bTMCA inhibits TCA-induced FXR activation in HepG2 cells. (F) bTMCA inhibits TCDCA-induced FXR activa-tion in HepG2 cells. The concentration of the individual BAs is indicated on the x axis. (G) Activation of an FXRreporter plasmid (pECRELuc) in transfected HepG2 cells by composite mixtures of the six major BAs (3 mMfinal concentration) modeling the in vivo hepatic BA content from the indicated groups. The vehicle (Veh)usedwasmethanol, andpositive control consisted of 3 mMTCDCA. The composition of the BAmixtures usedin these in vitro studies is shown in table S3. The labeling scheme for each group is indicated in the em-bedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences betweengroups; individual P values are provided in table S1.




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Il1b, Ccl2, Cxcl9, andCxcl10) was reduced in the presence of the ASBTi,whereas mRNA expression of Lcn2, a small secreted adipokine with aprotective role in hepatic inflammation, was induced (Fig. 6A and fig.S6E). RNA expression of fibrosis genes Col1a1, Msln, and Gli2 wereincreased by HFD and reduced to baseline with ASBT inhibition, sug-gesting an antifibrotic effect of the ASBTi (Fig. 6A and fig. S6E), al-though there was no significant fibrosis detected in any of theexperimental conditions in this study (fig. S3). Together, 16 weeks ofileal ASBT inhibition resulted in substantial alterations in gene expres-sion in livers of HFD-fed mice, which is related principally to BA, cho-lesterol, and fatty acidmetabolism, but also to a reduction inmarkers ofinflammation and response to stress.

Administration of an ASBTi does not alter ceramides inHFD-fed mouse ileum or liverRecent studies in a different HFD-fed mouse model (8, 18) support arole for ileal FXR signaling–associated changes in ceramide synthesisand secretion into the portal circulation in the development of fatty liver

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and obesity-related metabolic dysfunction. However, targeted massspectrometry analysis of both liver and ileal ceramide concentrationsin response to HFD orHFD/ASBTi16w revealed no significant changesin the amounts of total ceramide or individual ceramide species in eithertissue (fig. S7). These findings suggest that different mechanisms areoperative in this model, wherein the ASBTi blocks both ileal FXRsignaling and ileal BA internalization.

Administration of an ASBTi partially reverses establishedNAFLD after 12 weeks of HFDTo determine whether inhibition of ileal BA uptake also affectsestablished NAFLD, mice were fed the HFD for 12 weeks, followed by4 weeks of HFD/ASBTi. The responses of sentinel FXR-responsive genesin the liver, intestine, and colonwere similar in the 4- and 16-weekASBTitreatments (Fig. 1, C to E). This short treatment period induced somechanges not seen in the full 16-week treatment, such as a decrease in theliverweight to bodyweight ratio (Fig. 2D), but glucose tolerance,NAS, andsteatosis score were not significantly affected (Fig. 2, E and F, and Fig. 3, E



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and F) in HFD/ASBTi4w when comparedto those in HFD mice. However, hepaticTG and cholesterol contents weremarkedlyreduced compared toHFD-fedmice (Fig. 3,G and H, and fig. S2C), and visually, thereappeared to be zonally restricted reductionsin lipid deposition with the HFD/ASBTi4wtreatment (Fig. 3, B andD). In addition, he-patic BA composition was similarly alteredwith 4 weeks of ASBTi as with 16 weeks oftreatment (Fig. 4, B to D). Overall, theseresults indicate a partial reversal of the he-patic effects of 12 weeks of HFD by ASBTitreatment during the ensuing 4 weeks ofHFD.

Asbt−/− mice are resistant tohepatic steatosis with short-termHFD feedingTo confirm these findings and explorepotential mechanisms involving the roleof ASBT in the hepatic response to HFD,short-term (1 week) HFD was admin-istered to Asbt−/− and wild-type (WT)(Asbt+/+) mice followed by histologic, bio-chemical, and molecular analyses of theliver and ileum.After 1weekofHFD,whencompared toWTmice,Asbt−/−mice gainedless weight, despite ingesting essentiallythe same amount of calories as WT mice(Fig. 7, A and B). Similarly, liver weight inHFD-fed Asbt−/− mice was reduced com-pared to HFD-fedWTmice (Fig. 7C), butliverweight to bodyweight ratioswere un-changed (Fig. 7D). Histological and bio-chemical analyses showed that the liversof HFD-fedAsbt−/−mice, but not the liversof WT mice, were protected against lipidaccumulation after 1 week of HFD (Fig. 7,E to L).

Fig. 5. Inhibition of ileal ASBT function alters global hepatic gene expression. (A) Venn diagram ofRNA-seq analysis of the two comparisons: HFD versus chow and HFD versus HFD/ASBTi16w. (B) Heat map
comparing liver gene expression in HFD versus HFD/ASBTi16w groups. (C) Ontology analysis/pathway(Panther) analysis of HFD versus HFD/ASBTi16w as down-regulated (white bars) and up-regulated (blackbars) pathways.



Asbt−/− mice have reduced total hepatic BAs, with a BAcomposition similar to those treated with ASBTiUnlike inmice treated with the luminally restricted ASBTi, total hepaticBA content was reduced in Asbt−/− mice (by ~50%; Fig. 8A). However,

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the hepatic BA compositional changes were similar in both models(Figs. 4B and 8B), shifting toward a more hydrophobic, FXR agonisticpool (Fig. 8, C andD). Gene expression consequences of 1 week ofHFDinAsbt−/−mouse livers were notable for changes similar to those seen in


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the longer-term ASBTi treatments (Fig. 8Eand fig. S8).Notably forBAand cholesterolmetabolism,Cyp7a1mRNAwas increasedapproximately fivefold, expression of BA-regulated genes (Shp, Abcb11, and Mafg)was reduced in HFD-fed Asbt−/− mice,and the down-regulation of Hmgr andSrebp2 mRNA observed in the HFD-fedWT mice was blocked in HFD-fed Asbt−/−

mice. Increases in lipid metabolism–associated genes (Abcg8, Lxra, Srebp1c,Scd1, Ppara, and Ehhadh) in HFD-fedWT mice were abrogated in HFD-fedAsbt−/−mice (Fig. 8E and fig. S8), compa-rable to changes induced by ASBT inhibi-tion. Immunoblot analysis showed thatthe amount of liver SREBP1 protein in-creased after 1 week of HFD feeding inWT mice (P = 0.0084) but not in Asbt−/−

mice (P = 0.913; Fig. 8F). Thus, absence ofASBT function markedly impairs the ac-cretion of features of NAFLD in mice inas short a period as 1 week of HFD and en-gages pathways associatedwith amore FXRagonistic hepatic BA pool. Together, the al-terations in liver BA composition, ileal andcolonic gene expression, hepatic TG andcholesterol content, andresolutionof insulinresistance support inhibition of ileal ASBTfunction as a rational target for the hepaticandmetabolic consequences of diet-inducedfatty liver.

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DISCUSSION

In the current study of a mouse model ofHFD-induced NASH, the presence of aluminally restricted specific inhibitor ofintestinal BA uptake (ASBTi) preventedhepatic accumulation of TGs and choles-terol and restoredwhole-body insulin sen-sitivity. Ileal ASBT inhibition in HFD-fedmice was associated with substantial he-patic transcriptional and metabolic re-programming, suggesting that hepatocellularBA signaling was markedly altered by thisgut-restricted pharmacological intervention.Ileal ASBT inhibition prevented hepaticcholesterol accumulation by inducing BAbiosynthesis from cholesterol stores andshifting the liver BA pool compositiontowardone enriched inhydrophobic speciesthat are more FXR agonistic in nature. This

Fig. 6. Administration of an ASBTi alters hepatic and intestinal gene expression. (A) Hepatic mRNAexpression of genes involved in BA signaling and transport, cholesterol synthesis, lipid synthesis, lipid drop-
let formation, fatty acid oxidation, inflammation, and fibrosis. (B and C) mRNA expression of genes involvedin BA signaling and transport in the ileum (B) and (C) colon. Quantitative real-time PCR was performed onchow, HFD, HFD/ASBTi16w, and HFD/ASBTi4w groups, and expression shown relative to chow (set as 1). Forclarity, gene expression in the HFD and HFD/ASBTi16w groups are displayed. Cyclophilin was used as ahousekeeping gene to normalize mRNA expression. The labeling scheme for each group is indicated inthe embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differencesbetween groups; individual P values are provided in table S1.



included a dramatic reduction in hydrophilic, FXR antagonistic tauro-muricholates (16, 30) associated with increases in TCDCA, TDCA, andother FXR agonistic BAs. The reduction in muricholates is likely due todecreases in chenodeoxycholic acid synthesis via the alternative pathway,secondary to reductions in hepatic cholesterol content and to suppressionof hepatic 6-hydroxylation. Hepatic TG and cholesterol content directlycorrelated with the amount of FXR antagonistic muricholates andinversely correlated with the amount of FXR agonist TCDCA+TDCAin the liver. Similar decreases in hepatic lipid accretion were observedin Asbt−/− mice versus WT mice with short-term HFD feeding and inthe hepatic BA composition of theAsbt−/−mice, regardless of diet. Expo-

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sure of transfected HepG2 cells to synthetic BA mixtures modeling thehepatic BAcomposition found in ilealASBT-inhibitedmice increased theactivation of an FXR luciferase reporter plasmid, as compared to the he-patic BA composition ofHFDmice. Together, these findings suggest thatinhibition of ileal BA uptake has meaningful biological and gene regula-tory consequences that address multiple features of NASH in mice.

Ileal ASBT inhibition resulted in profound reduction in hepatic lip-ids, improvement in insulin sensitivity, and alterations in the expres-sion of hepatic lipid, inflammatory, and BA regulatory genes. Theunderlying mechanisms may relate to alterations in BA, FXR, or LXRsignaling or other pathways. Recent works by Jiang et al. (8, 18) demon-



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strated that intestinal-specific inactivationor inhibition of FXR-produced metabolicbenefit by reducing the intestinal produc-tion of bioactive ceramides, whichpromotefatty liver and obesity-related metabolicdysfunction in HFD-fed mice. Here, inhi-bitionof ileal FXRactivationwas associatedwith metabolic benefit without a change inileal or hepatic ceramide content, suggest-ing alternative mechanisms leading to im-provements in NASH. These differentialresults may be related to consequences ofspecifically targeting FXR versus BA-mediated signaling. For example, with re-gard to the ileal enterocyte, inhibition orinactivationofASBTblocksBA internaliza-tion and is predicted to causepan-inhibitionof FXR as well as other intercellular BAtargets and signaling pathways. This isin contrast to intestinal-specific in-activation of FXR or administration of in-testinally selective FXR antagonists, both ofwhich would block only the FXR signalingcomponent. A relevant confounder tofocusing on inhibition of intestinal FXRsignaling to improve NASH is a recentwork by Fang et al. (17), which demon-strated improved lipid handling andNASH in mice with a gut-restricted FXRagonist. Thus, with regard to NASH inmice, the beneficial effects associated withmodifying BA signaling in ileal entero-cytes may involve FXR and non-FXR tar-gets as well as ceramide and nonceramidepathways, whose relative contributions re-main to be determined.

Several metabolic pathways are involvedin maintaining hepatic lipid homeostasis,whereby dysregulation of each can resultin accumulation of lipid droplets. It is wellestablished that lipid and TG synthesesare increased in NAFLD (33). In addition,changes in BA composition can elicit majoreffects on regulation ofmetabolic pathwaysin liver (14, 30, 34, 35). Hepatic expressionof genes involved in lipid synthesis andlipid droplet formationwere induced after

Fig. 7. Hepatic accumulationof TGsand cholesterol is reduced inAsbt−/−mice. (A) Bodyweight gain in theindicated ad libitum–fed groups. * indicates value significantly different fromWT. (B) Average caloric intake over
1weekof feeding. (C) Liverweightafter1weekofHFDorchow. (D) Liverweight tobodyweight ratioafter1weekofHFDor chow. (E toH) Hematoxylin and eosin–stained liver sections from (E)WT/chow, (F)WT/HFD 1week,(G) Asbt−/−/chow, and (H) Asbt−/−/HFD 1weekmice. (I) Hepatic TG content. (J) Hepatic total cholesterol con-tent. (K) Hepatic free cholesterol content. (L) Hepatic cholesteryl ester content. The labeling scheme for eachgroup is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate signif-icant differences between groups; individual P values are provided in table S1. Scale bars, 100 mm.



16weeks ofHFD and reduced in the presence of the ASBTi. In contrast,there was little effect on gene expression for b-oxidation or lipoproteinsecretion pathways in this study, suggesting that they did not play ama-jor role in the changes in hepatic TGcontent in thismodel.Note that themodified ALIOS diet used in this study contained a modest amount ofadded [0.2% (w/w)] cholesterol, and cholesterol accumulation is a likely

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contributor to NASH (36–40). Cholesterol accumulation may cause in-creases in hepatic inflammation and oxysterol-mediated alterations ingene expression, mostly through LXRa target genes including Srebp1c.Additional studies are needed to explore the hepatic consequences ofreducing cholesterol and cholesterol-derived metabolites (such as oxy-sterols) and to determine whether this is an important determinant of



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the improvements in response to anASBTi.Thus, themechanismsmediating the effectsof ASBT inhibition may extend beyondFXR-mediated signaling in animal modelsof NAFLD.

Although adding anASBTi for the final4 weeks of the 16-week HFD treatmentaltered the hepatic BA composition, it didnot produce a histological improvement inNASH. The effects were intermediate, withsimilar decreases in hepatic TG and choles-terol accumulation as observed with the16-week treatment, but not some of theother metabolic and transcriptional effects.It is not clear whether this was a result ofan insufficient duration of treatment orwhether the changes in BA metabolismand signaling induced by the ASBTi areinsufficient to affect NASH once it isestablished. Additional studies using lon-ger periods of intervention and othermodels of NASH will be required toanswer these questions.

In summary, treatment of mice with aluminally restricted ileal ASBTi markedlyimproved several facets of NASH and in-sulin resistance by engaging multiplecomponents of BA signaling within thegut-liver axis. The finding that hepaticBA composition became more FXRagonistic in response to inhibition of ilealASBT is likely to be mechanistically im-portant but may explain only a portionof the positive effects of ASBT inhibitionobserved in this mouse NASH model.There is also the question of whether theseBAcompositional changes are relevant forhumans, who largely lack 6-hydroxylatedBAs under most physiological conditions.However, the targeting of ileal BA uptaketo alter liver FXR agonism may alleviatesome of the concerns seen in the recenthuman NASH trial of a potent FXRagonist, OCA, which not only improvedthe histological features ofNASH, but alsoincreased pro-atherogenic serum lipidconcentrations in some subjects (6). Bothsurgical ileal exclusion and interruption ofileal BA uptake by pharmacologicalmeans have been shown to reduce serumlow-density lipoprotein cholesterol byincreasing hepatic cholesterol conversion

Fig. 8. Hepatic BA composition is altered in Asbt−/− mice. (A) Total hepatic BA content and (B) BAcomposition. (C andD) Hydrophobicity index (C) and pie charts (D) for hepatic FXR antagonist/non-agonist
(black) and agonist (white) BA content. (E) Hepatic gene expression in WT and Asbt−/− mice fed a HFD for1 week. Values shown are relative to chow-fed WT mice. (F) Protein expression of full-length (fl) SREBP1increased in HFD-fed WT mice but not in HFD-fed Asbt−/− mice. SREBP1 expression was normalized tothe amount of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and thequantitation is shown below the blot. The labeling scheme for each group is indicated in the embeddedlegend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups;individual P values are provided in table S1.



to BAs (22, 41), so it is intriguing to speculate that inhibition of ASBTmay provide a means to improve features of hepatic lipotoxicity andmetabolic syndrome while alleviating some atherosclerotic risk factors.Mechanistic preclinical studies, in addition to well-controlled humantrials, are needed to determine whether these data from amouse NASHmodel will translate to humans affected by NASH and to verify thelong-term effects of such an intervention.

MATERIALS AND METHODS

See the Supplementary Materials for Materials and Methods.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/357/357ra122/DC1Materials and MethodsFig. S1. Inhibition of ileal BA absorption does not affect intestinal and colonic gene expression,food and water consumption, and hepatic injury markers.Fig. S2. Inhibition of ileal BA absorption improves whole-body insulin tolerance and hepaticcholesterol concentration.Fig. S3. Inhibition of ileal BA absorption does not affect hepatic fibrosis.Fig. S4. Inhibition of ileal BA absorption changed hepatic BA composition and correlated withTG and cholesterol concentrations.Fig. S5. Inhibition of ileal BA absorption changed global hepatic gene expression during 16 weeksof HFD feeding.Fig. S6. Inhibition of ileal BA absorption changed gene expression in the mouse liver.Fig. S7. Inhibition of ileal BA absorption does not affect hepatic and ileal ceramide amount andcomposition.Fig. S8. Inhibition of ileal BA absorption affects hepatic gene expression in WT and Asbt−/−

mice fed with chow or HFD for 1 week.Table S1. P values for all comparisons (provided as an Excel file).Table S2. Individual elements of NAS.Table S3. Composite BA mixtures of six BAs representing the major in vivo hepatic BA speciesin HFD and HFD/ASBTi16w mice.Table S4. Differentially expressed genes and pathway analysis (provided as an Excel file).Table S5. HFD composition and macronutrient information.Table S6. Primer sequences used for real-time PCR analysis.Table S7. Primary data (provided as an Excel file).References (42–53)

13, 2018
REFERENCES AND NOTES
1. R. Loomba, A. J. Sanyal, The global NAFLD epidemic. Nat. Rev. Gastroenterol. Hepatol. 10,686–690 (2013).

2. Z. M. Younossi, A. B. Koenig, D. Abdelatif, Y. Fazel, L. Henry, M. Wymer, Global epidemiol-ogy of nonalcoholic fatty liver disease—Meta-analytic assessment of prevalence, inci-dence, and outcomes. Hepatology 64, 73–84 (2016).

3. A. J. Sanyal, N. Chalasani, K. V. Kowdley, A. McCullough, A. M. Diehl, N. M. Bass,B. A. Neuschwander-Tetri, J. E. Lavine, J. Tonascia, A. Unalp, M. Van Natta, J. Clark, E. M. Brunt,D. E. Kleiner, J. H. Hoofnagle, P. R. Robuck; Nash CRN, Pioglitazone, vitamin E, or placebofor nonalcoholic steatohepatitis. N. Engl. J. Med. 362, 1675–1685 (2010).

4. B. Nie, H. M. Park, M. Kazantzis, M. Lin, A. Henkin, S. Ng, S. Song, Y. Chen, H. Tran, R. Lai, C. Her,J. J. Maher, B. M. Forman, A. Stahl, Specific bile acids inhibit hepatic fatty acid uptake in mice.Hepatology 56, 1300–1310 (2012).

5. J. K. Kim, K. S. Lee, D. K. Lee, S. Y. Lee, H. Y. Chang, J. Choi, J. I. Lee, Omega-3 poly-unsaturated fatty acid and ursodeoxycholic acid have an additive effect in attenuatingdiet-induced nonalcoholic steatohepatitis in mice. Exp. Mol. Med. 46, e127 (2014).

6. B. A. Neuschwander-Tetri, R. Loomba, A. J. Sanyal, J. E. Lavine, M. L. Van Natta, M. F. Abdelmalek,N. Chalasani, S. Dasarathy, A. M. Diehl, B. Hameed, K. V. Kowdley, A. McCullough, N. Terrault,J. M. Clark, J. Tonascia, E. M. Brunt, D. E. Kleiner, E. Doo; NASH Clinical Research Network,Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steato-hepatitis (FLINT): A multicentre, randomised, placebo-controlled trial. Lancet 385, 956–965(2015).

www.ScienceTran

7. M. Mueller, A. Thorell, T. Claudel, P. Jha, H. Koefeler, C. Lackner, B. Hoesel, G. Fauler, T. Stojakovic,C. Einarsson, H.-U. Marschall, M. Trauner, Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity. J. Hepatol. 62,1398–1404 (2015).

8. C. Jiang, C. Xie, F. Li, L. Zhang, R. G. Nichols, K. W. Krausz, J. Cai, Y. Qi, Z.-Z. Fang, S. Takahashi,N. Tanaka, D. Desai, S. G. Amin, I. Albert, A. D. Patterson, F. J. Gonzalez, Intestinal farnesoid Xreceptor signaling promotes nonalcoholic fatty liver disease. J. Clin. Invest. 125, 386–402 (2015).

9. B. Schnabl, D. A. Brenner, Interactions between the intestinal microbiome and liver dis-eases. Gastroenterology 146, 1513–1524 (2014).

10. P. A. Dawson, S. J. Karpen, Intestinal transport and metabolism of bile acids. J. Lipid Res. 56,1085–1099 (2015).

11. J. Y. L. Chiang, Bile acid metabolism and signaling. Compr. Physiol. 3, 1191–1212 (2013).12. J. P. Arab, S. J. Karpen, P. A. Dawson, M. Arrese, M. Trauner, Bile acids and nonalcoholic

fatty liver disease: Molecular insights and therapeutic perspectives. Hepatology 10.1002/hep.28709 (2016).

13. C. Fuchs, T. Claudel, M. Trauner, Bile acid-mediated control of liver triglycerides. Semin.Liver Dis. 33, 330–342 (2013).

14. M. Watanabe, Y. Horai, S. M. Houten, K. Morimoto, T. Sugizaki, E. Arita, C. Mataki, H. Sato,Y. Tanigawara, K. Schoonjans, H. Itoh, J. Auwerx, Lowering bile acid pool size with a syntheticfarnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energyexpenditure. J. Biol. Chem. 286, 26913–26920 (2011).

15. J. Prawitt, M. Abdelkarim, J. H. M. Stroeve, I. Popescu, H. Duez, V. R. Velagapudi, J. Dumont,E. Bouchaert, T. H. van Dijk, A. Lucas, E. Dorchies, M. Daoudi, S. Lestavel, F. J. Gonzalez,M. Oresic, B. Cariou, F. Kuipers, S. Caron, B. Staels, Farnesoid X receptor deficiency improvesglucose homeostasis in mouse models of obesity. Diabetes 60, 1861–1871 (2011).

16. F. Li, C. Jiang, K. W. Krausz, Y. Li, I. Albert, H. Hao, K. M. Fabre, J. B. Mitchell, A. D. Patterson,F. J. Gonzalez, Microbiome remodelling leads to inhibition of intestinal farnesoid X recep-tor signalling and decreased obesity. Nat. Commun. 4, 2384 (2013).

17. S. Fang, J. M. Suh, S. M. Reilly, E. Yu, O. Osborn, D. Lackey, E. Yoshihara, A. Perino, S. Jacinto,Y. Lukasheva, A. R. Atkins, A. Khvat, B. Schnabl, R. T. Yu, D. A. Brenner, S. Coulter, C. Liddle,K. Schoonjans, J. M. Olefsky, A. R. Saltiel, M. Downes, R. M. Evans, Intestinal FXR agonismpromotes adipose tissue browning and reduces obesity and insulin resistance. Nat. Med.21, 159–165 (2015).

18. C. Jiang, C. Xie, Y. Lv, J. Li, K. W. Krausz, J. Shi, C. N. Brocker, D. Desai, S. G. Amin, W. H. Bisson,Y. Liu, O. Gavrilova, A. D. Patterson, F. J. Gonzalez, Intestine-selective farnesoid X receptorinhibition improves obesity-related metabolic dysfunction. Nat. Commun. 6, 10166 (2015).

19. M. Watanabe, K. Morimoto, S. M. Houten, N. Kaneko-Iwasaki, T. Sugizaki, Y. Horai, C. Mataki,H. Sato, K. Murahashi, E. Arita, K. Schoonjans, T. Suzuki, H. Itoh, J. Auwerx, Bile acid bindingresin improves metabolic control through the induction of energy expenditure. PLOS One7, e38286 (2012).

20. K. K. Ryan, V. Tremaroli, C. Clemmensen, P. Kovatcheva-Datchary, A. Myronovych, R. Karns,H. E. Wilson-Pérez, D. A. Sandoval, R. Kohli, F. Backhed, R. J. Seeley, FXR is a moleculartarget for the effects of vertical sleeve gastrectomy. Nature 509, 183–188 (2014).

21. L. H. Tetri, M. Basaranoglu, E. M. Brunt, L. M. Yerian, B. A. Neuschwander-Tetri, SevereNAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructosecorn syrup equivalent. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G987–G995 (2008).

22. B. G. Bhat, S. R. Rapp, J. A. Beaudry, N. Napawan, D. N. Butteiger, K. A. Hall, C. L. Null, Y. Luo,B. T. Keller, Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE–/–

mice by SC-435. J. Lipid Res. 44, 1614–1621 (2003).23. A. G. Miethke, W. Zhang, J. Simmons, A. E. Taylor, T. Shi, S. K. Shanmukhappa, R. Karns, S. White,

A. G. Jegga, C. S. Lages, S. Nkinin, B. T. Keller, K. D. R. Setchell, Pharmacological inhibition ofapical sodium-dependent bile acid transporter changes bile composition and blocks progres-sion of sclerosing cholangitis in multidrug resistance 2 knockout mice. Hepatology 63,512–523 (2016).

24. A. Baghdasaryan, C. D. Fuchs, C. H. Österreicher, U. J. Lemberger, E. Halilbasic, I. Påhlman,H. Graffner, E. Krones, P. Fickert, A. Wahlström, M. Ståhlman, G. Paumgartner, H.-U. Marschall,M. Trauner, Inhibition of intestinal bile acid absorption improves cholestatic liver and bile ductinjury in a mouse model of sclerosing cholangitis. J. Hepatol. 64, 674–681 (2016).

25. C. L. Sears, W. S. Garrett, Microbes, microbiota, and colon cancer. Cell Host Microbe 15, 317–328(2014).

26. J. I. Barrasa, N. Olmo, M. A. Lizarbe, J. Turnay, Bile acids in the colon, from healthy tocytotoxic molecules. Toxicol. In Vitro 27, 964–977 (2013).

27. E. M. Brunt, D. E. Kleiner, L. A. Wilson, P. Belt, B. A. Neuschwander-Tetri; NASH ClinicalResearch Network (CRN), Nonalcoholic fatty liver disease (NAFLD) activity score and thehistopathologic diagnosis in NAFLD: Distinct clinicopathologic meanings. Hepatology 53,810–820 (2011).

28. C. D. Klaassen, J. Y. Cui, Review: Mechanisms of how the intestinal microbiota alters theeffects of drugs and bile acids. Drug Metab. Dispos. 43, 1505–1521 (2015).

29. D. J. Parks, S. G. Blanchard, R. K. Bledsoe, G. Chandra, T. G. Consler, S. A. Kliewer, J. B. Stimmel,T. M. Willson, A. M. Zavacki, D. D. Moore, J. M. Lehmann, Bile acids: Natural ligands for anorphan nuclear receptor. Science 284, 1365–1368 (1999).

slationalMedicine.org 21 September 2016 Vol 8 Issue 357 357ra122 10

http://www.sciencetranslationalmedicine.org/cgi/content/full/8/357/357ra122/DC1




http://stm.sciencem

ag.org/D

ownloaded from

30. S. I. Sayin, A. Wahlstrom, J. Felin, S. Jäntti, H.-U. Marschall, K. Bamberg, B. Angelin, T. Hyötyläinen,M. Orešič, F. Bäckhed, Gut microbiota regulates bile acid metabolism by reducing the levels oftauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab. 17, 225–235 (2013).

31. W. R. Howard, J. A. Pospisil, E. Njolito, D. J. Noonan, Catabolites of cholesterol synthesispathways and forskolin as activators of the farnesoid X-activated nuclear receptor. Toxicol.Appl. Pharmacol. 163, 195–202 (2000).

32. F. G. Schaap, M. Trauner, P. L. M. Jansen, Bile acid receptors as targets for drug develop-ment. Nat. Rev. Gastroenterol. Hepatol. 11, 55–67 (2014).

33. J. A. G. Duarte, F. Carvalho, M. Pearson, J. D. Horton, J. D. Browning, J. G. Jones, S. C. Burgess, Ahigh-fat diet suppresses de novo lipogenesis and desaturation but not elongation and tri-glyceride synthesis in mice. J. Lipid Res. 55, 2541–2553 (2014).

34. R. A. Haeusler, M. Pratt-Hyatt, C. L. Welch, C. D. Klaassen, D. Accili, Impaired generation of12-hydroxylated bile acids links hepatic insulin signaling with dyslipidemia. Cell Metab. 15,65–74 (2012).

35. M. Boesjes, V. W. Bloks, J. Hageman, T. Bos, T. H. van Dijk, R. Havinga, H. Wolters, J. W. Jonker,F. Kuipers, A. K. Groen, Hepatic farnesoid X-receptor isoforms a2 and a4 differentially mod-ulate bile salt and lipoprotein metabolism in mice. PLOS One 9, e115028 (2014).

36. G. C. Farrell, D. van Rooyen, Liver cholesterol: Is it playing possum in NASH? Am. J. Physiol.Gastrointest. Liver Physiol. 303, G9–G11 (2012).

37. C. Savard, E. V. Tartaglione, R. Kuver, W. G. Haigh, G. C. Farrell, S. Subramanian, A. Chait,M. M. Yeh, L. S. Quinn, G. N. Ioannou, Synergistic interaction of dietary cholesterol anddietary fat in inducing experimental steatohepatitis. Hepatology 57, 81–92 (2013).

38. D. M. Van Rooyen, G. C. Farrell, SREBP-2: A link between insulin resistance, hepatic cho-lesterol, and inflammation in NASH. J. Gastroenterol. Hepatol. 26, 789–792 (2011).

39. D. M. Van Rooyen, L. T. Gan, M. M. Yeh, W. G. Haigh, C. Z. Larter, G. Ioannou, N. C. Teoh,G. C. Farrell, Pharmacological cholesterol lowering reverses fibrotic NASH in obese,diabetic mice with metabolic syndrome. J. Hepatol. 59, 144–152 (2013).

40. D. M. Van Rooyen, C. Z. Larter, W. G. Haigh, M. M. Yeh, G. Ioannou, R. Kuver, S. P. Lee, N. C. Teoh,G. C. Farrell, Hepatic free cholesterol accumulates in obese, diabetic mice and causes non-alcoholic steatohepatitis. Gastroenterology 141, 1393–1403 1403.e1-5 (2011).

41. K. L. West, T. L. Zern, D. N. Butteiger, B. T. Keller, M. L. Fernandez, SC-435, an ileal apicalsodium co-dependent bile acid transporter (ASBT) inhibitor lowers plasma cholesterol andreduces atherosclerosis in guinea pigs. Atherosclerosis 171, 201–210 (2003).

42. P. A. Dawson, J. Haywood, A. L. Craddock, M. Wilson, M. Tietjen, K. Kluckman, N. Maeda,J. S. Parks, Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cyclingof bile acids in mice. J. Biol. Chem. 278, 33920–33927 (2003).

43. J. E. Mells, P. P. Fu, P. Kumar, T. Smith, S. J. Karpen, F. A. Anania, Saturated fat and choles-terol are critical to inducing murine metabolic syndrome with robust nonalcoholic stea-tohepatitis. J. Nutr. Biochem. 26, 285–292 (2015).

44. M. B. Tollefson, S. A. Kolodziej, T. R. Fletcher, W. F. Vernier, J. A. Beaudry, B. T. Keller, D. B. Reitz,A novel class of apical sodium co-dependent bile acid transporter inhibitors: The 1,2-benzothiazepines. Bioorg. Med. Chem. Lett. 13, 3727–3730 (2003).

45. D. E. Kleiner, E. M. Brunt, M. Van Natta, C. Behling, M. J. Contos, O. W. Cummings, L. D. Ferrell,Y.-C. Liu, M. S. Torbenson, A. Unalp-Arida, M. Yeh, A. J. McCullough, A. J. Sanyal; NonalcoholicSteatohepatitis Clinical Research Network, Design and validation of a histological scoring sys-tem for nonalcoholic fatty liver disease. Hepatology 41, 1313–1321 (2005).

46. G. K. Reddy, C. S. Enwemeka, A simplified method for the analysis of hydroxyproline inbiological tissues. Clin. Biochem. 29, 225–229 (1996).

47. M. Hagio, M. Matsumoto, M. Fukushima, H. Hara, S. Ishizuka, Improved analysis of bile acidsin tissues and intestinal contents of rats using LC/ESI-MS. J. Lipid Res. 50, 173–180 (2009).

www.ScienceTran

48. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Ultrafast and memory-efficient alignmentof short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

49. R. C. Gentleman, V. J. Carey, D. M. Bates, B. Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier,Y. Ge, J. Gentry, K. Hornik, T. Hothorn, W. Huber, S. Iacus, R. Irizarry, F. Leisch, C. Li, M. Maechler,A. J. Rossini, G. Sawitzki, C. Smith, G. Smyth, L. Tierney, J. Y. H. Yang, J. Zhang, Bioconductor:Open software development for computational biology and bioinformatics. Genome Biol. 5,R80 (2004).

50. H. Wu, C. Wang, Z. Wu, A new shrinkage estimator for dispersion improves differentialexpression detection in RNA-seq data. Biostatistics 14, 232–243 (2013).

51. D. W. Huang, B. T. Sherman, R. A. Lempicki, Systematic and integrative analysis of largegene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

52. A. Kosters, D. D. White, H. Sun, S. Thevananther, S. J. Karpen, Redundant roles for cJun-N-terminal kinase 1 and 2 in interleukin-1b-mediated reduction and modification of murinehepatic nuclear retinoid X receptor a. J. Hepatol. 51, 898–908 (2009).

53. A. Kosters, F. Tian, Y. J. Wan, S. J. Karpen, Gene-specific alterations of hepatic gene expres-sion by ligand activation or hepatocyte-selective inhibition of retinoid X receptor-asignalling during inflammation. Liver Int. 32, 321–330 (2012).

Acknowledgments: We thank Lumena/Shire for the SC-435; D. Shayakhmetov and N. C. Di Paolo(Emory University School of Medicine) for assistance and use of their microscope; the YerkesNonhuman Primate Genomics Core (Emory University School of Medicine) for performingRNA-seq analysis; the Children’s Healthcare of Atlanta Pathology Services for processing his-tology tissues; T. Osborne and P. Phelan (Sanford Burnham Prebys Medical Discovery Institute)for assistance with SREBP1 immunoblotting. Funding: This study was supported by the NIH[DK56239 (S.J.K.); DK047987 (P.A.D.); ES023485, AG038746, HL113451, ES019776, ES025632,EY022618, HD075784, HL095479, HL086773, and HL125042 (D.P.J.)]. J.E.M. was supported byan NIH Fellowship in Research and Science Teaching (FIRST) Institutional Research andAcademic Career Development Award (K12 GM000680). S.J.K. and P.A.D. were also supportedby the Center for Transplantation and Immune-mediated Disorders and the Children’s Health-care of Atlanta. Author contributions: A.R., A.K., J.E.M., W.Z., K.D.R.S., T.X., B.T.K., H.Y., G.M.W.,A.M.A., S.B., D.P.J., and H.W. performed the experiments and collected and analyzed the data;S.J.K. and P.A.D. managed and designed the study and conceived the experiments; A.K., A.R.,A.M.A., P.A.D. and S.J.K. wrote the manuscript. Competing interests: B.T.K. was an employeeof Lumena Pharmaceuticals, which is now part of Shire Pharmaceuticals. P.A.D. had previ-ously served as a consultant for Lumena Pharmaceuticals. The other authors declare thatthey have no competing interests. Data and materials availability: Asbt−/− mice are avail-able for purchase through the Jackson Laboratory. SC-435 was made available as a researchgift from Lumena/Shire Pharmaceuticals. RNA-seq data are available through Gene ExpressionOmnibus (GSE84994).

Submitted 15 February 2016Accepted 2 August 2016Published 21 September 201610.1126/scitranslmed.aaf4823

Citation: A. Rao, A. Kosters, J. E. Mells, W. Zhang, K. D. R. Setchell, A. M. Amanso, G. M. Wynn,T. Xu, B. T. Keller, H. Yin, S. Banton, D. P. Jones, H. Wu, P. A. Dawson, S. J. Karpen, Inhibition ofileal bile acid uptake protects against nonalcoholic fatty liver disease in high-fat diet–fed mice.Sci. Transl. Med. 8, 357ra122 (2016).

slationalMedicine.org 21 September 2016 Vol 8 Issue 357 357ra122 11


fed mice−high-fat diet Inhibition of ileal bile acid uptake protects against nonalcoholic fatty liver disease in

KarpenWynn, Tianlei Xu, Brad T. Keller, Hong Yin, Sophia Banton, Dean P. Jones, Hao Wu, Paul A. Dawson and Saul J. Anuradha Rao, Astrid Kosters, Jamie E. Mells, Wujuan Zhang, Kenneth D. R. Setchell, Angelica M. Amanso, Grace M.

DOI: 10.1126/scitranslmed.aaf4823, 357ra122357ra122.8Sci Transl Med

may be useful for treating multiple components of the metabolic syndrome.the treatment improved glucose tolerance and cholesterol concentrations in the treated animals, suggesting that it thereby reduces the severity of fatty liver disease in a mouse model. In addition to its beneficial effects on the liver,can be given by mouth and is not systemically absorbed, but inhibits bile acid absorption from the intestine and

have now identified a drug thatet al.understood, it is known that bile acids play key roles in lipid metabolism. Rao common, and there is no specific treatment available. Although the pathogenesis of this disorder is not yet fully

Nonalcoholic fatty liver disease, which is associated with the metabolic syndrome, is becoming increasinglyBlocking bile acids to protect the liver

ARTICLE TOOLS http://stm.sciencemag.org/content/8/357/357ra122

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2016/09/19/8.357.357ra122.DC1

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http://stm.sciencemag.org/content/scitransmed/9/412/eaan8732.fullhttp://stm.sciencemag.org/content/scitransmed/9/407/eaad4000.fullhttp://stke.sciencemag.org/content/sigtrans/10/491/eaal3336.fullhttp://stm.sciencemag.org/content/scitransmed/9/396/eaal3694.fullhttp://stm.sciencemag.org/content/scitransmed/8/323/323rv1.fullhttp://stm.sciencemag.org/content/scitransmed/8/352/352ra108.fullhttp://stm.sciencemag.org/content/scitransmed/8/337/337ra63.full

REFERENCES

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