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Bile acids enterohepatic circulation

Li, H.

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Citation for published version (APA):Li, H. (2005). Bile acids enterohepatic circulation.

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Download date: 12 Mar 2020

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THEE JOUBNAL or BIOLOGICA L CHEMISTRY Vol.. 277, No. 52, Issue of December 27, pp. 60491-60496, 2002 PrintedPrinted in U.S.A.

Regulationn of the Farnesoid X Receptor (FXR) by Bil e Acidd Flux in Rabbits*

Receivedd for publication, September 6, 2002, and in revised form, October 21, 2002 Published,, JBC Papers in Press, October 24, 2002, DOI 10.1074flbc.M209176200

Guorongg Xu$§1, Lu-xin g Pan§, Hai Li§ , Barr y M. Formanlj , Sandra K. Erickson** , Sarahh Sheferf, Jaya Boll ineni§, Ashok K. Batta§, Jenni fer Christie§, Tsu-hong Wang§, Johnn Michel§, Steve Yang§, Richard Tsai§, Lil y Laill , Kohei Shimadall, G. Stephen Tint |§ , andd Gerald Saleni§

FromFrom the ^Medical Service, Veterans Affairs Medical Center, East Orange, New Jersey 07018, thethe ^Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark,Newark, New Jersey 07103, the ^Department of Molecular Medicine, The City of Hope National Medical Center, Duarte,Duarte, California 91010, and the **Department of Medicine, University of California, San Francisco and the VeteransVeterans Affairs Medical Center, San Francisco, California 94121

Wee investigated the roles of hydrophobic deoxycholic acidd (DCA) and hydrophili c ursocholic acid (UCA) in the regulationn of the orphan nuclear farnesoid X receptor (FXR)) in vivo. Rabbits wit h bil e fistul a drainage (remov-all of the endogenous bil e acid pool), rabbit s wit h bil e fistul aa drainage and replacement wit h either DCA or UCA,, and intact rabbit s fed 0.5% cholic acid (CA) (enlargedd endogenous bil e acid pool) were studied. After b i l ee f istul a dra inage, cholesterol 7a-hydroxylase (CYP7A1)) mRNA and activit y levels increased, FXR-mediatedd transcriptio n was decreased, and FXR mRNA andd nuclear protein levels declined. Replacing the en-terohepaticc bil e acid pool wit h DCA restored FXR mRNA andd nuclear protein levels and activated FXR-mediated transcriptio nn as evidenced by the increased expression off i t s target genes, SHP and BSEP, and decreased CYP7A11 mRNA level and activity . Replacing the bil e acidd pool wit h UCA also restored FXR mRNA and nu-clearr protein levels but did not activate FXR-mediated transcription ,, because the SHP mRNA level and CYP7A1 mRNAA level and activit y were unchanged. Feeding CA to intactt rabbit s expanded the bil e acid pool enriched wit h thee FXR high affinit y ligand, DCA. FXR-mediated tran -scriptionn became activated as shown by increased SHP andd BSEP mRNA levels and decreased CYP7A1 mRNA levell and activit y but did not change FXR mRNA or nuclearr protein levels. Thus, both hydrophobic and hy-drophili cc bil e acids are effective in maintainin g FXR mRNAA and nuclear protein levels. However, the activat-ingg ligand (DCA) in the enterohepatic flux is necessary forr FXR-mediated transcriptiona l regulation, which leadss to down-regulation of CYP7A1.

Thee farnesoid X receptor (FXR)1 is an orphan nuclear tran-scriptionn factor that has recently been identified as a negative

** This study was supported by grants from the Department of Vet-eranss Affairs Research Service, Washington, D. C. and National Insti-tutess of Health Grants DK56830, HL18094, DK57636, DK26766, and HD20632.. The costs of publication of this article were defrayed in part byy the payment of page charges. This article must therefore be hereby markedd "advertisement" in accordance with 18 U.S.C. Section 1734 solelyy to indicate this fact

HH To whom correspondence should be addressed: GI Lab (ISA), Vet-eranss Affairs Medical Center, 385 Tremont Ave., East Orange, NJ 07018-1095.. Tel.: 973-676-1000 ext. 1452; Fax: 973-676-2991; E-mail: xugu@umdnj.edu. .

11 The abbreviations used are: FXR, farnesoid X receptor, BSEP, bile saltt export pump; CA, cholic acid (3a,7a,12a-trihydroxy-50-cholanoic

regulatorr of CYP7A1, a gene encoding cholesterol 7a-hydroxylase,, the rate-limiting enzyme in the classic bile acid synthesiss pathway (1-3). Based on in vitro studies in cell culture,, the most effective (high affinity) ligands for FXR activationn are chenodeoxycholic acid (CDCA), deoxycholic acidd (DCA), and lithocholic acid (LA), which are all hydro-phobicc bile acids. In contrast, hydrophilic bile acids such as ursodeoxycholicc acid and muricholic acid are not effective. Wangg et al. (1) reported that in CV-1 cells co-transfected with bilee acid transporters, free cholic acid (CA), and the glycine or taurinee conjugates of CA, DCA, and LA, which now are hy-drophilic,, became strong activators of FXR comparable with freee CDCA

Recently,, other target genes for FXR have been identified thatt are positively regulated, including the bile salt export pumpp (BSEP), which is responsible for the canalicular trans-portt of bile acids (4), and short heterodimer partner (SHP), whichh plays an important role in the feedback regulation of CYP7A11 by bile acids (5,6). FXR/RXR doess not bind directly to thee bile acid response element in the promoter region of human CYP7A1CYP7A1 (7); thus, CYP7A1 transcriptional down-regulation by bilee acids via FXR is indirect via SHP (5,6). The increased SHP iss believed to inactivate liver receptor homolog-1 (LRH-1), an essentiall transcription factor for CYP7A1 expression (8). Re-cently,, Chen et al. (9) suggested that a-fetoprotein transcrip-tionn factor (FTF), a human homolog of mouse LRH-1, was an inhibitorr rather than a transcription factor for CYP7A1. More recently,, studies in SHP knock-out mice (10,11) suggested that CYP7A11 could also be regulated by bile acids through SHP-independentt pathways, because cholic acid feeding to the SHP -I—-I— mice also repressed CYP7A1 expression. Therefore, the mechanismm by which activated FXR down-regulates CYP7A1 hass not been completely elucidated.

Too examine the proposed theories of the role of bile acids in FXRR activation and the regulation of CYP7A1 under in vivo conditions,, this study was carried out in rabbits with depleted bilee acid pool/flux, which was replaced withh either hydrophobic DCAA or hydrophilic ursocholic acid (UCA), and intact rabbits

acid);; CDCA, chenodeoxycholic acid (3a,7a-dihydroxy-5/3-cholanoic acid);; CYP7A1, cholesterol 7a-hydroxylase; DCA, deoxycholic acid (3a,12aa dihydroxy-50-cholanoic acid); FTF, a-fetoprotein transcription factor;; LA, lithocholic acid (3a-hydroxy-50-cholanoic acid); LRH-1, liver receptorr homolog-1; RXR, 9-cis-retinoic acid receptor; SHP, short het-erodimerr partner, UCA, ursocholic acid (3a,7/3,12a-trihydroxy-50-chol-anoicc acid).

Thiss paper is available on line at http://www.jbc.org 50491 1 57 7

504922 Regulation ofFXR by Hepatic Bile Acid Flux

fedd cholic acid. In addition, the effect of these bile acids on FXR transcriptionn was also evaluated.

EXPERIMENTALL PROCEDURES

ExperimentalExperimental Design—Male New Zealand White (NZW) (n = 24) rabbitss weighing 2.5-2.75 kg (Convance, Denver, PA) were used in this study.. Sixteen rabbits were fed regular rabbit chow, and eight rabbits weree fed regular chow containing 0.5% CA (Purina Mills, St Louis, MO) forr 7 days. Bile fistulas were constructed in 12/16 regular chow-fed rabbitss and 4/8 0.5% CA chow-fed rabbits as described previously (12). Bil ee drainage was continued for 7 days to ensure the complete elimina-tionn of bile acids returning to the Uver. The secondary bile acid, DCA, a high-affinityy ligand for FXR, totally disappeared from the hepatic bile afterr 5 days of bile drainage, indicating interruption of the intestinal bilee acid flux through the Uver. Four of the twelve rabbits with 7 days off bile fistula drainage were sacrificed to collect liver specimens, which weree immediately frozen for measurements of FXR, SHP, BSEP, and FTFF (LRH-1) mRNA levels, FXR/RXR and FTF nuclear protein levels, andd CYP7A1 mRNA levels and cholesterol 7a-hydroxylase activity. The sodiumm salts of glyco-DCA and glyco-UCA were dissolved in water. In thee bile acid depleted rabbits, after bile acid synthesis was maximally stimulatedd and bile acid output became constant, glyco-DCA (n = 4) and glyco-UCAA (n = 4) were then infused intraduodenally. The baseline bile acidd flux was determined by measuring the bile acid output for 30 nun immediatelyy after construction of the bile fistula. To ensure sufficient hepaticc bile acid replacement, glyco-DCA or glyco-UCA were infused at aa rate of 60 mg (conjugated bile acid) per hour for 24 h. After completion off the studies, rabbits, including chow-fed controls and 0.5% CA fed, weree sacrificed, and Uver tissues were taken immediately for analysis. Thee animal protocol was approved by the Subcommittee on Animal Studiess at the Veterans Affairs Medical Center, East Orange, NJ and thee Institutional Animal Care and Use Committee at the University of Medicinee and Dentistry-New Jersey Medical School, Newark, NJ.

ElectrophoretieElectrophoretie Mobility Shift Assay—For the preparation of nuclear extracts,, Uver was minced and homogenized with a Wheaton Dounce homogenizerr (pestle B) in lysis buffer containing 20 mM Hepes (pH 7.6), 100 mM NaCl, 1.5 mM MgCLj 0.2 mM EDTA, ImM dithiothreitol, 10 ,ig/ml leupeptin,, 10 jxg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.1%% Triton X-100, and 20% glycerol. Nuclei were separated by centrif-ugationn at 1800 X g for 5 min suspended in the same lysis buffer that noww contained a high salt concentration (500 mM NaCl). The nuclei weree broken with a homogenizer (pestle A). After centrifugation in an Eppendorff centrifuge at 10,000 rpm for 10 min, the supernatant (nu-clearr extract) was collected and divided into aUquots and stored at - 700 °C. Al l procedures were performed at 4 °C.

Thee response element used as an FXR-specific probe was a double-strandedd oligonucleotide containing the sequence 5'-AAGGTCAATGA-CCTTA-3'' and, for the complementary strand, 5-TAAGGTCATTGAC-CTT-3'.. The sequences of the mutant probe were 5'-AAGAACAATGT-TCTTA-3'' and 5f-TAAGAACATTGTTCTT-3'. The response element for FTFF protein analysis contained the sequence 5-GTTCAAGGCCAGTT-ACTACCA-3'' as the top strand and 5-TGGTAGTAACTGGCCTT-GAAC-3'' as the complementary strand. These probes were end-labeled

ll with T4 polynucleotide kinase and 32P]ATP. In the gel shift assay for II the FTF protein, a standard FTF was appUed to identify the FTF

proteinn in the nuclear protein extracts. The standard FTF lysate was synthesizedd in vitro using a TNT T7 Quick Coupled transcription/trans-lationn system from Promega (Madison, WI) with the expression vectors pCMX.hCPFF or pCDM8.hFTF (from Dr. B. Forman and Dr. J. Chiang, respectively). .

Thee binding reaction contained 2 jxg of poly(dI-dC), 20 mM Hepes (pH 7.5),, 1.5 mM MgCL, 1 mM dithiothreitol, 2 mM EDTA, 50 mM KC1, and 3%% glycerol. Unlabeled competitor probes (mutant or normal response element)) were added at 100-fold excess and were pre-incubated with the extractedd nuclear proteins (10 fig) on ice for 30 min before adding the 32P-labeledd probe. After a 1-h incubation with the labeled probe (0.06 pmol,, 25,000 cpm) on ice, the reactions were analyzed by electrophore-siss through an 8% polyacrylamide gel in 0.375X TBE (0.33 mM Tris boratee (pH 8.7) and 1.0 mM EDTA). The gel was dried and subjected to autoradiography. .

RabbitRabbit cDNA Cloning and Sequencing—Total RNA was isolated from frozenn rabbit Uver tissue using single-step RNA isolation method with Trizoll reagent (Invitrogen) as described by Chomozynski and Sacchi (13).. Poly(A)+ RNA was isolated from 2 mg of total RNA by oUgo(dT)-cellulosee using the FastTrack 2.0 mRNA isolation kit (Invitrogen) de-

scribedd by Biesecker et al. (14). When the RNA was used for reverse transcriptionn PCR, total RNA (1 /Ag) was digested with (10 units) RNase-freee DNase I (Stratagene, La Jolla, CA).

Thee following degenerate oligonucleotide primers were used to clone partiall rabbit cDNAs for *XR, SHP, and FTF for FXR, 5MP, and FTF: FXRR primer, 5-TATGAACTCAGGCGWATG-3' and 5-GTGAGTTC-MGTnTCTCC-3';; SHP primer, 5-CTCAGGAACCTGCCRTC-3' and 5'-GYTCCAGGACTTCACACA-3';; and FTF primer, 5'ATGATGAA-GATCTGGAAGAG-3'' and 5ACAAAGGGACTTCTGTCATA-3'.

Forr rabbit cDNA cloning, cDNAs were prepared as described by Ullrichh et al. (15). First-strand cDNA was obtained from 1 fig fig of rabbit Uverr total RNA by reverse transcription and then digested by RNase H too degrade the RNA using a cDNA synthesis kit (Omniscript reverse transcriptionn kit, Qiagen, Valencia, CA). The first-strand cDNA in the mixturee with the sense and antisense degenerate primer ohgonucleo-tidess was subjected to PCR using a HotStarTaq master mix kit (Qiagen, Valencia,, CA) and a PerkinElmer Life Sciences Thermocycler. The PCR wass run for 30 cycles (30 s at 94 °C; 30 s at 50-60 °C for different primers;; and 60 s at 72 °C). An aliquot of the reaction product was electrophoresedd through a 1.5% agarose gel. When only a single band wass visualized by ethidium bromide staining at the expected size, the PCRR product was cloned by a TOPO TA cloning kit for sequencing (Invitrogen).. At least six clones that contained the PCR product from eachh rabbit gene studied were sequenced by the AppUed Biosystems Divisionn automated 3700 DNA analyzer using Big Dye Terminator chemistryy and AmpUTaq-FS DNA Polymerase.

NorthernNorthern Blotting Analyses—For RNA probe synthesis and Northern blott analysis, rabbit RNA probes were synthesized by the Strip-EZ RNA probee synthesis and removal kit (Ambion, Austin, TX) and labeled with [a-32P]UTPP using cloned cDNA as a template. Northern blot hybridiza-tionn was performed as previously described by Thomas (16). Briefly, 10 tagtag of poly(A)+ RNA was electrophoresed on a formaldehyde-agarose (1.0%)) gel and transferred to a nylon membrane (Nytran supercharge nylonn transfer membrane, Schleicher & Schuell). The membrane was bakedd for 1 h at 80 °C and hybridized to a 32P-labeled RNA probe for 166 h at 60 °C. The membrane was washed at 68 °C in O.lx SSC 0.1% SDSS for 30 min. Relative expression levels were quantified using a Phoephorlmagerr (Molecular Dynamics) and standardized against cyclo-philinn controls.

AssaysAssays for Activities ofCYP7Al—Hepatic microsomes were prepared byy differential ultracentrifugation (17). Protein was determined accord-ingg to Lowry et al. (18). CYP7A1 activity was measured in hepatic microsomess by the isotope incorporation method of Shefer et al. (17).

AssayAssay for Bile Acids—Bile acids were analyzed using a capillary gas-liquidd chromatography method as previously described (12).

StatisticalStatistical Method—Data are shown as means S.D. and were comparedd statistically by Student's t test (unpaired). The BMDP sta-tisticall software (BMDP Statistical Software, Los Angeles, CA) was usedd for statistical evaluations.

RESULTS S

BileBile Fistula Drainage—As we have described previously (12), afterr 7 days of bile fistula drainage the secondary bile acid, DCA,, disappeared from the hepatic bile, which indicated that thee enterohepatic bile acid pool/flux was totally depleted and thatt no further intestinal bile acids had returned to the liver. Removall of the bile acid pool/flux increased CYP7A1 mRNA levelss 2.3-fold and activity 5.7-fold (p < 0.001), respectively (Fig.. 1). FXR/RXR nuclear protein levels measured by gel shift assayss (Fig. 2) were reduced 65% (p < 0.01), and FXR mRNA levelss (Figs. 3 and 4) decreased 41% {p < 0.05). The levels of FXRR target genes SHP and BSEP mRNAs decreased 82% (p < 0.01)) and 58% (p < 0.05), respectively (Figs. 5 and 6). However, theree was no change in FTF nuclear protein (Fig. 7) or mRNA levelss (Fig. 8).

ReplacingReplacing the Bile Acid Flux with either DCA or UCA in Bile Acid-depletedAcid-depleted Rabbits—Infusing sodium glyco-DCA intraduo-denallyy for 24 h restored the hepatic bile acid pool/flux to 4988 mg/h, of which 88% was DCA. At baseline, the hepatic bile acidd pool/flux in these rabbits measured within the first half-hourr immediately after bile fistula drainage was 30 10 mg/h withh 85% DCA and 13% CA. After replacement with sodium glyco-UCA,, the bile acid pool/flux was restored to 46 8 mg/h, off which 82% was UCA (38 8 mg/h) with 17% CA and 0.6%

RegulationRegulation ofFXR by Hepatic Bile Acid Flux 50493 3

CYP7A11 activity (pmol/mg/min) )

CYP7A11 mRNA (unit) )

FIG.. 1. Effect of the bile acid pool/flux on CYP7A1 activity and mRNA.. Shown are CYP7A1 activity (open bar) and mRNA (hatched bar)bar) in control rabbits, rabbits with depleted bile acid pool by bile fistula drainagee (BF), rabbits after the depleted bile acid pool was replaced withh DCA (BF+DCA) or UCA (BF+UCA) for 24 h, and rabbits fed 0.5% CAA for 7 days (CA).

Control l BF F DCA A UCA A CA A

FXR/RXR R

FIG.. 2. FXR/RXR nuclear protein measured by electrophoretic mobilityy shift assays. BF, rabbits with depleted bile acid pool by bile fistulaa drainage; BF+DCA and BF+UCA, the depleted bile acid pool wass replaced with DCA or UCA for 24 h; CA, rabbits fed 0.5% CA for 7 days.. To identify FXR/RXR protein, in the fourth and fifth columns from thee left, unlabeled (non-radioactive) competitor probes, mutant (m), and normall (cooO probes were added at 100-fold excess before adding 32P-labeledd probe. The nuclear protein applied in the fourth and fifth columnscolumns from the left was extracted from the same liver specimen as thatt in the third column from the left.

FXRR mRNA

unit t

1.55 r

FXR/RXRR protein uni t t

X X 111 PI

DCA A UCA A

FIG.. 3. Effect of the bile acid pool/flux on FXR mRNA and protein.. Shown are FXR mRNA (open bar) and FXR/RXR nuclear proteinn (hatched bar) values in control rabbits, rabbits with depleted bilee acid pool by bile fistula drainage (BF), rabbits after the depleted bilee acid pool was replaced with DCA (BF+DCA) or UCA (BF+ UCA) for 244 h, and rabbits fed 0.5% CA for 7 days (CA).

DCA.. Replacement with DCA decreased CYP7A1 mRNA 54% (p(p < 0.01) and activity 55% (p < 0.01), whereas replacing the fluxflux wi th UCA had no effect (Fig. 1). FXR mRNA levels and

FXR R

Cyclo --phili n n

CC BF D U CA FIG.. 4. Northern blott ing analysis for FXR mRNA. C, control

rabbits;; BF, rabbits with depleted bile acid pool by bile fistula drainage; DD and U, rabbits after the depleted bile acid pool was replaced with DCAA (D) or UCA (IT); CA, rabbits fed 0.5% CA for 7 days. Cyclophihn servedd as internal standard.

BSEPmRNA A

FIG.. 5. Effect of the bUe acid pool/flux on SHP and BSEP. Changess in FXR activation were indicated by the expression of its targett genes SHP and BSEP. SHP (open bar) and BSEP (hatched bar) mRNAA levels in control rabbits, rabbits with depleted bile acid pool by bilee fistula drainage (BF), rabbits after replacement with DCA (BF+DCA)(BF+DCA) or UCA (BF+UCA) for 24 h, and rabbits fed 0.5% CA for 7 dayss (CA) are shown.

SHP P

BSEP P

Cyclo --phili n n

CC BF D U CA FIG.. 6. Northern blott ing analysis for SHP and BSEP mRNAs.

C,, control rabbits; BF, rabbits with depleted bile acid pool by bile fistula drainage;; D and U, rabbits after replacement with DCA (D) or UCA (U); CA,CA, rabbits fed 0.5% CA. Cyclophihn served as internal standard.

FXR/RXRR nuclear protein increased significantly and recov-eredd to baseline levels after the bile acid flux was restored wi t h ei therr DCA (hydrophobic) or UCA (hydrophilic) for 24 h (Figs. 22 and 3). SHP and BSEP mRNA levels increased 3.7-fold (p < 0.001)) and 2-fold (p < 0.05), respectively, 24 h after replace-mentt wi t h DCA as compared wi th the low levels in rabbi ts wi th

59 9

50494 4 RegulationRegulation ofFXR by Hepatic Bile Acid Flux

Stand d

Cold d

BF F CA A

F T F -- -- FTF

0.6 6

< < 0.. 1* > > O O

0.0 0 0.0 0 0.4 4 1.2 2 1.6 6

SHP P

FIG.. 9. Relationship between SHP and CYP7A1 mRNA expres-sion.. The data plotted in the graph are from five groups of rabbits with 33 rabbits per group. O, controls; , rabbits with bile fistula drainage;rabbitss after the depleted bile acid pool was replaced with DCA; , rabbitss after the depleted bile acid pool was replaced with UCA; and , rabbitss fed 0.5% CA The relationship can be described by the equation: CYP7A11 = 0.180 - 0.181 ln(SHP), where CYP7A1 and SHP represent thee relative units of mRNA abundance, respectively.

FIG.. 7. Hepatic FTF protein measured by electrophoretic mo-bilit yy shift assays. C, control rabbits; BF, rabbits with depleted bile acidd pool by bile fistula drainage; CA, rabbits fed 0.5% CA for 7 days. Standardd (Stand) FTF protein was synthesized using the TNT system. Inn the first line, non-radioactive (Cold) probe was added as a competitor too identify the location of the FTF protein.

Cyclo--phi n n

CC BF D U CA

60 0

FIG.. 8. Norther n blottin g analysis for FTF mRNA C, control rabbits;; BF, rabbits with depleted bile acid pool by bile fistula drainage; DD and U, after replacement with DCA (D) or UCA ([/); CA, rabbits fed 0.5%% CA for 7 days. Cyclophilin served as internal standard.

depletedd bile acid pools (Figs. 5 and 6). In contrast, when the hepat icc bile acid pool/flux was reestabl ished with UCA, there wass no change in SHP mRNA level but a 49% rise in BSEP mRNAA (Figs. 5 and 6).

CholicCholic Acid Feeding—Feeding 0.5% cholic acid to intact rab-bitss for 7 days expanded the bile acid pool size 2-fold from 3066 25 mg to 605 72 mg (p < 0.01), and the hepatic bile acidd flux increased 2.7-fold (83 6 mg/h versus 30 10 mg/h, pp < 0.001) wi t h 89% DCA. The CYP7A1 mRNA level was inhibi tedd 67% (p < 0.001), and activity decreased 72% (7 2 versusversus 25 9 pmol/mg/min, p < 0.05) (Fig. 1). However, the FXR/RXRR nuclear protein and FXR mRNA level (Figs. 2 and 3)

didd not increase above control values. SHP and BSEP mRNAs increasedd 2.6-fold (p < 0.001) and 34% (p < 0.05), respectively (Figs.. 5 and 6), but FTF nuclear protein and mRNA levels remainedd unchanged (Figs. 7 and 8).

RelationshipRelationship between SHP and CYP7A1 mRNA Expression— Thee relationship between SHP and CYP7A1 mRNA expression iss described by the curve shown in Fig. 9. The data plotted are fromm five groups of rabbits (three rabbits in each group) under differentt t reatments, i.e. controls, rabbits after 7 days of bile fistulaa drainage where the bile acid pool was depleted, rabbits afterr the depleted bile acid pool was replaced wi th DCA or UCA forr 24 h, and rabbits with intact bile acid enterohepatic circu-lationn fed 0.5% CA for 7 days. Mathematical analysis showed thatt the relationship between SHP and CYP7A1 mRNA expres-sionn can be described satisfactorily by the equation CYP7A1 = 0.1800 - 0.181 ln(SHP) (r2 = 0.915, p < 0.0001) where CYP7A1 andd SHP represent the relative uni ts of mRNA abundance, respectively. .

DISCUSSION N

Thee leading theories concerning the mechanism of FXR ac-tivationn by bile acids and the role of activated FXR in feedback regulationn of CYP7A1 were based on findings derived from in vitrovitro studies in cultured cells. Therefore, i t became necessary to investigatee these mechanisms in an in vivo whole animal model.. I n this study, we attempted to clarify: 1) the critical role off the FXR-activating ligand (DCA) in the enterohepatic bile acidd flux for FXR activation; 2) whether bile acids play any role i nn the regulation of FXR transcript ion aside from a ligand role; andd 3) whether FXR is the dominant regulator for bile acid synthesiss (CYP7A1) and the canalicular t ransporter (BSEP).

Thiss s tudy demonstrated that in the in vivo bile acid-de-pletedd rabbit model, replacing the enterohepatic flux wi th DCA effectivelyy activated FXR and regulated its downstream target genes.. As we reported previously (19), when the endogenous bilee acid pool consisting of 85% DCA was totally removed by bilee fistula drainage for 7 days, CYP7A1 mRNA and activity levelss increased significantly, because FXR transcript ional ac-tivit yy was muted as indicated by the decreased expression of FXRR target genes. Importantly, the present work shows that afterr restoration of the enterohepatic bile acid pool/flux with DCA,, FXR target gene expression also was restored as indi-catedd by the increase in the expression of SHP and BSEP

RegulationRegulation ofFXR by Hepatic Bile Acid Flux 50495 5

mRNAss and the decrease in CYP7A1 mRNA and activity rela-tivee to rabbits with depleted bile acid pools. When the entero-hepaticc bile acid pool/flux, which contained 88% DCA, was increasedd 2-fold by feeding 0.5% CA to rabbits with an intact enterohepaticc circulation, CYP7A1 mRNA and activity values weree significantly lower than the control values because FXR wass activated as indicated by the increased mRNA levels of SHPSHP and BSEP as compared with controls. Thus, these results consistentlyy demonstrated that DCA was an effective FXR agonistt in vivo.

Thee inverse relationship between SHP and CYP7A1 mRNA expressionn is shown by the curve in Fig. 9 and described by the equationn CYP7A1 « 0.180 - 0.181 ln(SHP). The data in this figuree were plotted from five groups of rabbits with different treatmentss (controls, bile fistula drainage, DCA and UCA re-placement,, and CA feeding). The expression levels of SHP and CYP7A11 in the curve shown in Fig. 9 represent a significant inversee relationship (r2 = 0.915, p < 0.0001). Mathematical analysiss suggests that the effect of SHP on CYP7A1 is inde-pendentt and saturable. When SHP expression is low (limited supplyy of activating ligand for FXR), CYP7A1 is elevated and sensitivee to the increase in SHP expression, although when SHPP expression rises beyond certain high levels, the increase inn SHP mRNA does not result in further significant repression off CYP7A1. Although recent studies in Shp knock-out mice (10, 11)) demonstrated that other pathways were involved in the down-regulationn of CYP7A1, these studies strongly indicated thatt activated FXR repressed CYP7A1 via SHP. The results of thiss study in the rabbit model support the hypothesis that activationn of FXR induces SHP expression that down-regulates CYP7A11 transcription.

However,, when the depleted bile acid pool was restored with UCA,, there was littl e effect on CYP7A1 mRNA and activity or FXRR activation as indicated by the lack of effect on the SHP mRNAA level. However, after UCA replacement, the FXR nu-clearr protein was restored to baseline levels, although down-streamm FXR-dependent transcriptional activity apparently was not.. This mirrors the in vitro studies on the high affinity ligandss for FXR (1), which show that hydrophilic, ursodeoxy-cholicc acid is not an activating ligand of FXR.

Thiss study further demonstrated that the activation of FXR andd its target genes was not solely dependent on changes in the sizee of the bile acid pool and enterohepatic flux per se. Infusing UCAA restored the enterohepatic bile acid pool/flux but did not activatee FXR, because the composition of the bile acid flux/pool wass changed to 82% UCA with only 17% CA and 0.6% DCA. Thee amounts of CA and/or DCA were apparently below the ligand-activatingg threshold for FXR. Therefore, the composi-tionn of the circulating bile acid pool plays a critical role in determiningg the nuclear activation state of FXR. In other words,, a sufficient enterohepatic circulating flux of activating/ highh affinity ligands is required for the activation of FXR and itss subsequent effects on bile acid metabolism. Previously, we notedd that in cholesterol fed rabbits the expanded bile acid pool wass the detennining factor for the regulation of CYP7A1 (12). Noww we better understand that in cholesterol fed rabbits, >85%% of the expanded bile acid pool was the FXR high affinity activatingg ligand, DCA. Not only did the bile acid pool double in sizee with cholesterol feeding, but the availability of a high affinityy ligand supply of DCA also increased, resulting in the activationn of FXR with the subsequent down-regulation of CYP7A1.. It is interesting to note that in rabbits with depleted bilee acid pool, a 24-h infusion of glyco-DCA at the same hepatic bilee acid flux as controls did not completely restore SHP mRNA too the baseline levels seen in the controls. These results indi-catee that factors other than bile acid pool composition and

hepaticc flux might also affect the expression of SHP, and these non-bilee acid factors might also be induced by bile fistula drainage. .

Ann important new observation in this study is that bile acids alsoo regulate FXR mRNA levels. After the enterohepatic bile acidd pool was removed, FXR mRNA and nuclear protein levels decreasedd significantly. When the depleted bile acid flux was restoredd with either DCA or UCA, the FXR mRNA and nuclear proteinn levels recovered to baseline values in controls without bilee fistula. This suggests that bile acids, including those that aree not activating/high affinity ligands of FXR, are necessary forr maintenance of FXR mRNA and nuclear protein levels. As wass observed in CA fed mice (20), FXR mRNA levels were not increased.. This suggests that increasing the bile acid pool be-yondd normal control levels has no effect on FXR mRNA. How-ever,, the bile fistula/bile acid replacement experiments de-scribedd here demonstrate that a minimum level of bile acids in thee enterohepatic circulation is required for the maintenance of FXRR mRNA and nuclear protein levels at baseline values in the intactt controls. The mechanisms of this regulation are still unknown. .

Inn this study, we did not find significant changes in FTF proteinn and mRNA levels in rabbits after removal of the bile acidd pool where FXR was deactivated and CYP7A1 was up-regulatedd or in CA fed rabbits where the bile acid pool doubled, FXRR was activated, and CYP7A1 was down-regulated. There-fore,, changes in the activation of FXR or the regulation of CYP7A11 are not necessarily reflected by changes in the FTF proteinn or mRNA in the in vivo rabbit model.

Althoughh in vitro studies (1-3) showed that the most effec-tivee ligands for FXR are hydrophobic bile acids such as DCA, CDCA,, and LA, whereas hydrophilic bile acids, such as ur-sodeoxycholicc acid and muricholic acids are not effective li -gandss for FXR, the mechanism of how bile acids activate FXR iss not totally understood. However, we should point out that hydrophobicityy is not the sole criterion for determining whetherr a bile acid is an activating ligand for FXR, because hydrophobicityy refers to the water insolubility of the bile acid.. For example, to infuse DCA dissolved in a water solu-tionn into the duodenum, the hydrophobic DCA was conju-gatedd with the amino acid glycine, making i t hydrophilic; but replacingg the bile acid pool with hydrophilic glyco-DCA also activatedd FXR.

Measurementss of FXR activity in this study were important forr understanding the effect of bile acids on the function of FXR inn the regulation of CYP7A1. Presently, there is no method availablee for the direct measurement of FXR activation in vivo. Inn our studies, we measured the changes in the expression of targett genes of FXR as markers for the activation of FXR. SHP mRNAmRNA levels always mirrored the changes in FXR activation. However,, although BSEP mRNA levels were regulated by ac-tivatedtivated FXR, other bile acid-dependent mechanisms also ap-pearr to be involved. UCA did not activate FXR but did produce similarr quantitative biliary bile acid outputs such as DCA with aa 48% increase of BSEP mRNA. This suggested that in addition too FXR, BSEP might also be regulated by the bile acid flux in ann FXR-independent manner.

Inn summary, this study demonstrated in the rabbit model thatt DCA is a potent ligand for FXR, activated FXR mediates negativee regulation of CYP7A1, and bile acids are required to maintainn FXR mRNA and nuclear FXR protein levels. Non-activatingg ligand bile acids can induce FXR mRNA and protein, butt thee FXR protein is not activated and cannot down-regulate CYP7A1.. Therefore, activation of FXR is not solely dependent onn the size of the bile acid pool but, as importantly, on the

504966 Regulation ofFXR by Hepatic Bile Acid Flux

proportionn of activating ligands of FXR in the circulating bile acidd pool/flux mixture.

REFERENCES S 1.. Wang, H., Chen, J., Hollister, K., Sowers, L. C., and Forman B. M. (1999) Mol.

CellCell 3, 543-553 2.. Makishima, M., Okamoto, A, Y., Repa, J. J., Tu, H., Learned, R. M., Luk, A.,

Hull ,, M. V., Lustig, K. D., Mangelsdorf, D. J., and Shan, B. (1999) Science 284,, 1362-1365

3.. Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G., Consler, T. G., Kliewer,, S. A., Stimmel, J. B., Willson, T. M., Zavacki, A. M., Moore, D. D., andd Lehinann, J. M. (1999) Science 284, 1365-1368

4.. Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D.,, and Suchy, F. J. (2001) J. Biol. Chem. 276, 28857-28865

5.. Lu, T. T., Makishima, M., Repa, J. J., Schoonjans, K , Kerr, T. A., Auwerx, J., andd Mangelsdorf, D. J. (2000) Mol. Celt 6, 507-515

6.. Goodwin, B., Jones, S. A., Price, R. R., Watson, M. A., McKee, D. D., Moore, L.. B., Galardi, C, Wilson, J. G., Lewis, M. C, Roth, M. E., Maloney, P. R., Willson,, T. M., and Kliewer, S. A. (2000) Mol. Cell 6, 517-526

7.. Chiang, J. Y. L., Kimmel, R., Weinberger, C, and Stroup, D. (2000) J. Biol. Chem.Chem. 275, 10918-10924

8.. Nitta, M., Ku, S., Brown, C, Okamoto, A. Y., and Shan, B. (1999) Proc. Natl. Acad.Acad. Sci. U.S. A. 96, 6660-6665

9.. Chen, W., Owsley, E., Yang, Y., Stroup, D„ and Chiang, J. Y. L. (2001) J. Lipid Res.Res. 42, 1402-1412

10.. Kerr, T A , Saeku, S., Schneider, M., Schaefer, K., Berdy, S., Redder, T., Shan, B.,, Russell, D. W„ and Schwartz, M. (2002) Dev. Cell 2, 713-720

11.. Wang, L., Lee, Y.-K., Bundman, D., Han, Y., Thevananther, S., Kim, C.-S., Chua,, S. S., Wei, P., Heyman, R. A., Karin, M., and Moore, D. D. (2002) Dev. CellCell 2, 721-731

12.. Xu, G., Salen, G., Shefer, S., Tint, G. S., Nguyen, L. B., Chen, T. S., and Greenblatt,, D. (1999) J. Clin. Invest. 103, 89-95

13.. Chomozynski, R., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 14.. Biesecker, L. G., Gottachalk, L. R., and Emerson, S. G. (1993) Proc. Natl. Acad.

Sci.Sci. U.S.A. 90, 7044-7048 15.. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tarn, A. W., Lee,

J.,, Yarden, Y., Libermann, T. A., Schlessinger, J., Downward, J., Mayes, E.. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984) Nature 309,, 418-425

16.. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-5205 17.. Shefer, S., Salen, G., and Batta, A K. (1986) Methods of Assay: Cholesterol

7a-Hydroxylase7a-Hydroxylase (7a-Monooxygenase) pp. 43-49, CRC Press, Inc., Boca Raton,, FL

18.. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.Chem. 193, 265-275

19.. Xu, G., Pan, L., Erickson, S. K., Forman, B. M., Shneider, B. L., Ananthanarayanan,, M., Li , X., Shefer, S., Balasubramanian, N., Ma, L., Asaoka,, H., Lear, S. R., Nguyen, L. B., Dussault, I., Suchy, F. J., Tint, G.. S., and Salen, G. (2002) J. Lipid Res. 43, 45-50

20.. Sinai, C. J., Tonkin, M., Miyata, M., Ward, J. M., Lambert, G., and Gonzalez, F.. J. (2000) Cell 102, 731-744

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