Removal of the bile acid pool upregulates cholesterol 7α-hydroxylase by deactivating FXR in rabbits
2002; Elsevier BV; Volume: 43; Issue: 1 Linguagem: Inglês
10.1016/s0022-2275(20)30185-1
ISSN1539-7262
AutoresGuorong Xu, Luxing Pan, Sandra K. Erickson, Barry M. Forman, Benjamin L. Shneider, Meenakshisundaram Ananthanarayanan, Xiaogui Li, Sarah Shefer, Niranjan Balasubramanian, Lin Ma, Hitoshi Asaoka, Steven R. Lear, Lien B. Nguyen, Isabelle Dussault, Frederick J. Suchy, G. Stephen Tint, Gerald Salen,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoWe investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7α-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12α-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels. —Xu, G., L-x. Pan, S. K. Erickson, B. M. Forman, B. L. Shneider, M. Ananthanarayanan, X. Li, S. Shefer, N. Balasubramanian, L. Ma, H. Asaoka, S. R. Lear, L. B. Nguyen, I. Dussault, F. J. Suchy, G. S. Tint, and G. Salen. Removal of the bile acid pool upregulates cholesterol 7α-hydroxylase by deactivating FXR in rabbits. J. Lipid Res. 2002. 43: 45–50. We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7α-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12α-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels. —Xu, G., L-x. Pan, S. K. Erickson, B. M. Forman, B. L. Shneider, M. Ananthanarayanan, X. Li, S. Shefer, N. Balasubramanian, L. Ma, H. Asaoka, S. R. Lear, L. B. Nguyen, I. Dussault, F. J. Suchy, G. S. Tint, and G. Salen. Removal of the bile acid pool upregulates cholesterol 7α-hydroxylase by deactivating FXR in rabbits. J. Lipid Res. 2002. 43: 45–50. Farnesoid X receptor (FXR), one of the members of the orphan nuclear receptor family expressed predominantly in the liver, kidney, intestine, and adrenal gland (1Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C. Identification of a nuclear receptor that is activated by farnesol metabolite.Cell. 1995; 81: 687-693Google Scholar), has been identified as a ligand-activated negative regulator for the transcription of cholesterol 7α-hydroxylase (CYP7A1) (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar, 3Parks 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. Lehmann J.M. Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284: 1365-1368Google Scholar, 4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3: 543-553Google Scholar), the rate-controlling enzyme for classic bile acid synthesis. The most potent in vitro ligands for FXR are the hydrophobic bile acids: chenodeoxycholic, deoxycholic, and lithocholic acids (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar, 3Parks 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. Lehmann J.M. Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284: 1365-1368Google Scholar, 4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3: 543-553Google Scholar). Wang et al. (4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3: 543-553Google Scholar) reported that after cotransfection with the bile acid transporters, ileal apical sodium-dependent bile acid transporter or sodium/taurocholate-cotransporting polypeptide (NTCP), free cholic acid, and all conjugated bile acids became as powerful ligands as free chenodeoxycholic acid for FXR. Furthermore, to respond to bile acids (ligands), FXR must heterodimerize with the retinoid X receptor (RXR) (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar, 4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3: 543-553Google Scholar). Makishima et al. (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar) reported that when associated with its ligand, FXR suppressed transcription of the gene encoding CYP7A1, but coordinately activated the gene encoding ileal bile acid-binding protein (IBABP), also known as ileal lipid-binding protein. Although the specific function of IBABP in bile acid metabolism has not been ascertained, it has been established as a target gene for activated FXR in the ileum (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar, 5Grober J. Zaghini I. Fujii H. Jones S.A. Kliewer S.A. Willson T.M. Ono T. Besnard P. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene.J. Biol. Chem. 1999; 274: 29749-29754Google Scholar, 6Laffitte B.A. Kast H.R. Nguyen C.M. Zavachi A.M. Moore D.D. Edwards P.A. Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor.J. Biol. Chem. 2000; 275: 10638-10647Google Scholar). Bile salt export pump (BSEP) (7Nathanson M.H. Boyer J.L. Mechanisms and regulation of bile secretion.Hepatology. 1991; 14: 551-566Google Scholar) is an ATP-binding cassette-type membrane transporter that is located in canalicular microvilli of hepatocytes and is responsible for excretion of conjugated bile salts into canalicular bile (8Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J. Biol. Chem. 1998; 273: 10046-10050Google Scholar). Ananthanarayanan et al. (9Ananthanarayanan M. Balasubramanian N. Mangelsdorf D. Suchy F.J. Molecular cloning and functional analysis of the promoter for bile salt export pump (BSEP) from human and mouse genes: evidence for transactivation of the human promoter by farnesoid-X-receptor/retinoid-X-receptor (FXR/RXR).Gastroenterology. 2000; 118: A2Google Scholar) reported that in transfected HepG2 cells, BSEP promoter activity was induced by activated FXR and suggested that BSEP also is a target gene for FXR in the liver. The full-length cDNAs of rabbit and mouse hepatic BSEP have been cloned by Ananthanarayanan et al. (9Ananthanarayanan M. Balasubramanian N. Mangelsdorf D. Suchy F.J. Molecular cloning and functional analysis of the promoter for bile salt export pump (BSEP) from human and mouse genes: evidence for transactivation of the human promoter by farnesoid-X-receptor/retinoid-X-receptor (FXR/RXR).Gastroenterology. 2000; 118: A2Google Scholar) (GenBank accession no. AF249879) and Green, Hoda, and Ward (10Green R.M. Hoda F. Ward K.L. Molecular cloning and characterization of the murine bile salt export pump.Gene. 2000; 241: 117-123Google Scholar), respectively. NTCP is the major bile acid transporter located in the sinusoidal membrane of hepatocytes. It recovers bile acids from the portal circulation by an active process (11Ananthanarayanan M. Ng O.C. Boyer J.L. Suchy F.J. Characterization of cloned Na-bile acid transporter using peptide and fusion protein antibodies.Am. J. Physiol. 1994; 267: G637-G643Google Scholar). It is suggested that transcription of NTCP is negatively regulated by FXR (12Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-1477Google Scholar, 13Denson L.J. Sturm E. Echevarria W. Zimmerman T.L. Makishima M. Mangelsdorf D.J. Karpen S.J. Bile acid-mediated feedback inhibition of the NTCP gene promoter occurs via a novel mechanism involving bile acid receptor (FXR) activation of a transcriptional repressor.Hepatology. 2000; 32: 297AGoogle Scholar). Sinal et al. (12Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-1477Google Scholar) demonstrated that in FXR−/− (FXR null) mice, the inhibitory effect of bile acids on CYP7A1 did not occur, supporting a regulatory role for FXR in the transcription of CYP7A1. In addition, their work confirmed that bile acids positively regulated transcription of Bsep, and sterol 12α-hydroxylase (Cyp8b) in the liver and Ibabp in the ileum in an FXR-dependent manner and that FXR was involved in negative control of Ntcp expression. In this study, we investigated the role of FXR in the regulation of CYP7A1 in vivo and established the importance of the bile acid pool in both FXR activation and control of hepatic mRNA levels. A bile fistula model was utilized in rabbits, in which CYP7A1 responds oppositely to dietary cholesterol as seen in rats (14Xu G. Shneider B.L. Shefer S. Nguyen L.B. Batta A.K. Tint G.S. Arrese M. Thevananther S. Ma L. Stengelin S. Kramer W. Greenblatt D. Pcolinsky M. Salen G. Ileal bile acid transport regulates bile acid pool, synthesis and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.J. Lipid Res. 2000; 41: 298-304Google Scholar) and mice (15Peet D.J. Truley S.D. Ma W. Janowski B.A. Lobaccaro J-M.A. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXRα.Cell. 1998; 93: 693-704Google Scholar). Abundance of hepatic BSEP, sterol 12α-hydroxylase (CYP8B), and NTCP mRNAs and intestinal IBABP protein were measured and served as surrogate markers for FXR activation. This in vivo model enabled us to test FXR function when the bile acid (ligand) flux through the liver was interrupted and to examine the mechanism(s) for upregulation of CYP7A1 in vivo. We observed that in rabbits with a depleted bile acid pool, FXR was deactivated as indicated by reduced FXR protein and the responses of its target genes. These results suggested that upregulation of CYP7A1 was due to removal of the inhibitory control from FXR. In addition, in the absence of the enterohepatic bile acid pool, FXR mRNA levels were decreased substantially, suggesting that FXR transcription was downregulated in response to a diminished ligand supply. Male New Zealand White (NZW) (n = 12) and male Watanabe heritable hyperlipidemic (WHHL) (n = 12) rabbits weighing 2.5–2.75 kg (Convance, Denver, PA) were used in this study. All rabbits were fed regular rabbit chow (Purina Mills, St. Louis, MO). Bile fistulas were constructed in half of the rabbits (6 of 12) as described previously (16Xu G. Salen G. Shefer S. Tint G.S. Nguyen L.B. Chen T.S. Greenblatt D. Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis.J. Clin. Invest. 1999; 103: 89-95Google Scholar). Bile drainage was continued for 7 days to ensure the elimination of bile acids returning to the liver. After 3 days of bile drainage, cholic acid synthesis was maximally stimulated (17Xu G. Salen G. Batta A.K. Shefer S. Nguyen L.B. Niemann W. Chen T.S. Arora-Mirchandani R. Ness G.C. Tint G.S. Glycocholic acid and glycodeoxycholic acid but not glycoursocholic acid inhibit bile acid synthesis in the rabbit.Gastroenterology. 1992; 102: 1717-1723Google Scholar), whereas deoxycholic acid, an FXR high affinity ligand, totally disappeared from the bile after 5 days of bile drainage (16Xu G. Salen G. Shefer S. Tint G.S. Nguyen L.B. Chen T.S. Greenblatt D. Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis.J. Clin. Invest. 1999; 103: 89-95Google Scholar), indicating complete interruption of the bile acid flux through the liver. The animals were then killed. The livers were removed and portions were immediately frozen for measurements of FXR, BSEP, CYP8B, and NTCP mRNA levels, FXR protein levels, CYP7A1 activity and mRNA levels, and cholesterol 27-hydroxylase (CYP27) activity. The mucosa from the terminal ileum was harvested for measurements of IBABP protein. The animal protocol was approved by the Subcommittee on Animal Studies at the Veterans Affairs Medical Center (East Orange, NJ) and by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry-New Jersey Medical School (Newark, NJ). Electrophoretic mobility shift assay for FXR protein. To prepare nuclear extracts, liver tissue from control and bile fistula rabbits was minced and homogenized with a Wheaton (Millville, NJ) Dounce homogenizer (pestle B) in lysis buffer containing 20 mM HEPES (pH 7.6), 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, leupeptin (10 μg/ml), aprotinin (10 μg/ml), 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 20% glycerol. Nuclei were separated by centrifugation at 1,800 g for 5 min and suspended in the same kind of lysis buffer but now containing a high salt concentration (500 mM NaCl). The nuclei were broken with a homogenizer (Dounce, pestle A). After centrifugation in an Eppendorf centrifuge (Brinkmann Instruments, Westbury, NY) at 9,300 g for 10 min, the supernatant (nuclear extract) was collected and divided into aliquots and stored at −70°C. All the procedures were performed at 4°C. The response element that was used as a specific FXR protein-binding probe was a double-stranded oligonucleotide containing the sequence 5′-AAGGTCAATGACCTTA-3′ and complementary strand 5′-TAAGGTCATTGACCTT-3′. The oligonucleotide sequences containing a mutation substitution of binding site for FXR were 5′-AAGAACAATGTTCTTA-3′ and 5′-TAAGAACA TTGTTCTT-3′. These probes were end labeled by T4 polynucleotide kinase and [γ-32P]ATP. Binding reaction mixture contained 2 μg of poly(dI-dC), 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 1 mM DTT, 2 mM EDTA, 50 mM KCl, and 3% glycerol. Competitor [unlabeled (cold) or mutated probe] was added in a 100-fold excess and was preincubated with the extracted nuclear proteins (10 μg) on ice for 30 min before adding labeled probe. After another 1 h of incubation with labeled probe (0.06 pmol, 25,000 cpm) on ice, the reactions were analyzed by low ionic strength system electrophoresis on an 8% polyacrylamide gel in 0.375× TBE [0.33 mM Tris borate (pH 8.7)-1.0 mM EDTA]. The gel was then dried and subjected to autoradiography. Western blot analysis (for IBABP). Homogenates of ileal mucosa were prepared as previously described (18Arrese M. Trauner M. Sacchiero R.J. Crossman M.W. Shneider B.L. Neither intestinal sequestration of bile acids nor common bile duct ligation modulates the expression and function of the rat ileal bile acid transporter.Hepatology. 1998; 28: 1081-1087Google Scholar). Protein concentrations were determined by the Bradford method (19Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Google Scholar) with bovine serum albumin as reference standard. Western blotting for IBABP was performed with 10 μg of homogenate. Proteins were separated in a 15% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto nitrocellulose membranes. The blots were blocked overnight at 4°C with Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk and then incubated for 2 h at room temperature with rabbit anti-mouse ileal lipid-binding protein (also called IBABP) polyclonal antibody (a gift from M. W. Crossman, Washington University, St. Louis, MO). Immune complexes were detected with 125I-labeled protein A. Immunoreactive bands were detected in the linear range of response with a PhosphorImager screen and quantified by using a PhosphorImager and Imagequant software (Molecular Dynamics, Sunnyvale, CA). Northern blotting analyses. Total RNA from samples of frozen liver was isolated by acid guanidinium thiocyanate-phenolchloroform extraction (20Chomozynski R. Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Google Scholar). The total RNA pellet was dissolved in 100 μl of diethylpyrocarbonate-treated water. Poly(A)+ was isolated by oligo(dT)-cellulose chromatography (21Aviv N. Ledder P. Purification of biologically active globin mRNA by chromatography on oligothymidylic acid-cellulose.Proc. Natl. Acad. Sci. USA. 1972; 69: 1408-1412Google Scholar). The relative levels of FXR, CYP7A1, BSEP, CYP8B, and NTCP mRNAs were quantitated by Northern blotting analysis as previously described by Ness, Keller, and Pendelton (22Ness G.C. Keller R.K. Pendelton L.C. Feedback regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by dietary cholesterol is not due to altered mRNA levels.J. Biol. Chem. 1991; 266: 14854-14857Google Scholar), except that glyceraldehyde-3-phosphate dehydrogenase served as the internal reference standard. The cDNA for rat CYP7A1 was a gift from J. Y. L. Chiang (Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, OH), that for FXR was from R. Evans and B. M. Forman (Department of Molecular Medicine, City of Hope National Medical Center, Duarte, CA), CYP8B was from I. Björkhem (Karolinska Institute, Stockholm, Sweden), and the cDNAs for BSEP and NTCP were from M. Ananthanarayanan (Department of Pediatrics, Mt. Sinai School of Medicine, New York, NY). Assays for activities of CYP7A1 and CYP27. Hepatic microsomes and mitochondria were prepared by differential ultracentrifugation (23Shefer S. Salen G. Batta A.K. Methods of assay.in: Fears R. Sabine J.R. Cholesterol 7α-Hydroxylase (7α-Monooxygenase). CRC Press, Boca Raton, FL1986: 43-49Google Scholar), and protein was determined according to Lowry et al. (24Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). CYP7A1 activity was measured in hepatic microsomes by the isotope incorporation method of Shefer, Salen, and Batta (23Shefer S. Salen G. Batta A.K. Methods of assay.in: Fears R. Sabine J.R. Cholesterol 7α-Hydroxylase (7α-Monooxygenase). CRC Press, Boca Raton, FL1986: 43-49Google Scholar). The isotope incorporation method for measurement of mitochondrial CYP27 activity was as previously described by Shefer et al. (25Shefer S. Kren B.T. Salen G. Steer C.J. Nguyen L.B. Chen T.S. Tint G.S. Batta A.K. Regulation of bile acid synthesis by deoxycholic acid in the rat: different effects on cholesterol 7α-hydroxylase and sterol 27-hydroxylase.Hepatology. 1995; 22: 1215-1221Google Scholar) Assay for bile acids. Bile acids were analyzed by capillary gas–liquid chromatography as previously described (16Xu G. Salen G. Shefer S. Tint G.S. Nguyen L.B. Chen T.S. Greenblatt D. Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis.J. Clin. Invest. 1999; 103: 89-95Google Scholar). Data are shown as means ± SD and were compared statistically by Student's t-test (unpaired). BMDP Statistical Software (Los Angeles, CA) was used for statistical evaluations. The percentage of glycine-conjugated deoxycholic acid in the bile acid pool was measured in bile collected during the first 30 min after insertion of the bile fistula and comprised 85 ± 7% in NZW and 83 ± 9% in WHHL rabbits.Relatively small amounts of cholic acid (14–16%) and traces of chenodeoxycholic acid (1%) were also detected in these bile samples. However, after 5 days of bile drainage, glycocholic acid became the predominant bile acid (99%) in the bile and deoxycholic acid, which was formed by bacterial 7α-dehydroxylation of cholic acid in the intestine, was no longer detected, demonstrating complete interruption of the enterohepatic circulation of bile acids. After 7 days of bile drainage, CYP7A1 activity increased 3.3 times (P < 0.001) from 30 ± 5 to 98 ± 28 pmol/mg per min in NZW rabbits and 5.3 times (P < 0.0001) from 15 ± 3 to 79 ± 20 pmol/mg per min in WHHL rabbits (Fig. 1A). CYP7A1 mRNA levels also rose 3.2 times from 29 ± 15 to 94 ± 20 units (P < 0.001) in NZW rabbits and four times from 4 ± 2 to 16 ± 6 units (P < 0.01) in WHHL rabbits (Fig. 1B). CYP27 activity that reflected alternative bile acid synthesis did not change after removal of the bile acid pool in either NZW (29.0 ± 5.5 vs. 24.0 ± 9.9 pmol/mg per min) or WHHL rabbits (23.0 ± 4.5 vs. 33.8 ± 9.8 pmol/mg per min). After depletion of the bile acid pool, FXR mRNA levels were barely detected (Fig. 2) and decreased 74% (from 100 ± 38 to 26 ± 4 units, P < 0.05) in NZW rabbits and 56% (100 ± 31 to 44 ± 13 units, P < 0.05) in WHHL rabbits (Fig. 3).Fig. 3.Densitometric measurements of FXR mRNA after removal of the bile acid pool. FXR mRNA levels declined significantly in both NZW and WHHL rabbits after bile fistula drainage (BF).View Large Image Figure ViewerDownload (PPT) Figure 4 shows FXR protein as determined by electrophoretic mobility shift assays (EMSA) in control rabbits (C) and rabbits after bile fistula bile drainage. The specificity of the binding complex was evaluated by adding anexcess of either unlabeled FXR response element (cold probe) or mutated FXR response element (M-probe) as competitor. When excess unlabeled (cold) probe was added, the specific binding of the labeled probe to FXR protein was limited and not visualized (Fig. 4, lanes 1, 4, 7, and 10). Adding an excess of the unlabeled mutated response element (M-probe) did not interfere with the specific binding of the labeled probe to FXR protein (Fig. 4, lanes 2, 5, 8, and 11). In Fig. 4, abundant specific binding of the labeled probe to FXR was noted in controls (Fig. 4, lanes 3 and 6) but barely visualized in rabbits after bile fistula (Fig. 4, lanes 9 and 12). Densitometric measurements revealed that FXR protein decreased 47% (P < 0.01), from a baseline of 52.1 ± 8.9 units (n = 4) to 27.7 ± 8.4 units (n = 4) in NZW rabbits after bile drainage for 7 days. Thus, removal of the bile acid pool produced a significant decrease of FXR protein. The activation status of FXR was evaluated by determining changes in the expression of key target genes for FXR in control and bile fistula rabbits. IBABP served as a marker of FXR activation in the ileum, and was determined by Western blotting analysis. Figure 5 shows that in controls (lanes 1, 3, 5, 7, and 9), the mass of IBABP is higher in intensity than in rabbits after bile fistula (lanes 2, 4, 6, 8, and 10). The protein levels decreased 57% (1.00 ±0.12 to 0.43 ± 0.07 unit, P < 0.001) in NZW rabbits and 56% (3.42 ± 0.71 to 1.49 ± 0.73 units, P < 0.01) in WHHL rabbits (Fig. 6) after bile fistula. Similarly, the mRNA levels of BSEP, which serves as a marker of FXR activation in the liver, declined 58% (from 45.4 ± 6.4 to 19.1 ± 4.0 units, P < 0.001) in NZW rabbits, and 25% (from 40.0 ± 4.2 to 30.1 ± 3.8 units, P < 0.05) in WHHL rabbits (Fig. 7). In contrast, mRNA levels of hepatic CYP8B and NTCP, which are negatively regulated by FXR, increased more than 2-fold (from 0.089 ± 0.013 to 0.200 ± 0.030 unit, P < 0.01) and more than three times (from 0.06 ± 0.02 to 0.19 ± 0.05 unit, P < 0.01), respectively, in NZW rabbits after bile acid depletion (Fig. 8). Northern blots for these target genes of FXR in control and bile fistula NZW rabbits are shown in Fig. 9.Fig. 6.Densitometric measurements of IBABP after removal of the bile acid pool. IBABP levels in the ileum decreased significantly in NZW and WHHL rabbits after bile fistula drainage (BF).View Large Image Figure ViewerDownload (PPT)Fig. 7.Densitometric measurements of BSEP mRNA levels after removal of the bile acid pool. BSEP mRNA levels decreased significantly in NZW and WHHL rabbits after bile fistula drainage (BF).View Large Image Figure ViewerDownload (PPT)Fig. 8.mRNA levels of NTCP and sterol 12α-hydroxlase (CYP8B) in New Zealand White rabbits increased (P < 0.01) after bile fistula drainage (BF).View Large Image Figure ViewerDownload (PPT)Fig. 9.Representative Northern blots for mRNA levels of CYP7A1, BSEP, sterol 12α-hydroxylase (CYP8B), and NTCP in control and bile acid-depleted NZW rabbits (BF). The expression of mRNA for CYP7A1, CYP8B, and NTCP, which are negatively regulated by FXR, increased whereas BSEP mRNA, which is positively regulated by FXR, decreased. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.View Large Image Figure ViewerDownload (PPT) This study was designed to evaluate the role of the orphan nuclear receptor, FXR, in the regulation of CYP7A1in an in vivo whole rabbit model. Specifically, our aim was to elucidate the effect of the absent ligand flux (bile acids) returning to the liver on FXR as reflected by activated FXR protein and changes in the transcription of both positively and negatively regulated FXR target genes. We demonstrated that removal of the bile acid pool diminished the expression of BSEP and IBABP, which coincided with the upregulation of CYP7A1, NTCP, and CYP8B. Most importantly, measurements of FXR protein by EMSA showed a significant reduction (−47%) after bile drainage. Because FXR must first form a heterodimer with RXR (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Google Scholar, 4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3: 543-553Google Scholar) before it can be activated by ligands (bile acids) and bind to the appropriate response element, the FXR protein measured by EMSA was actually in the form of FXR/RXR. These results demonstrated the inverse relationship between CYP7A1 and FXR in the liver such that removal of the bile acid (ligands) pool deactivated FXR and released CYP7A1 transcription from the inhibitory control of FXR. A second major finding was that FXR mRNA levels diminished markedly after the bile acid ligand supply for FXR was eliminated, consistent with the aforementioned finding that FXR protein levels measured by EMSA decreased significantly in rabbits after bile acid depletion.Taken together, these results suggest that the hepatic bile acid flux not only supplies ligands for the activation of FXR but also may be involved in the transcription of FXR. In contrast to studies of the FXR gene knockout mouse model (12Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-1477Google Scholar), we studied the role of FXR in the regulation of CYP7A1 and other key target genes in an in vivo whole rabbit model with normal genes, where FXR was deactivated by the total elimination of the ligand supply. Moreover, bile acid metabolism in rabbits is different from rodents, where deoxycholic acid, a hydrophobic bile acid, predominates (85%) in the enterohepatic pool. Thus, our study of rabbits can be viewed as complementary to the report of Sinal et al. (12Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-1477Google Scholar) on FXR null mice. Until the present, no method was available for the direct measurement of hepatic FXR activity in an in vivo animal model. Thus, in this study, we used IBABP protein levels in the ileum and BSEP, CYP8B, and NTCP mRNA levels in the liver to serve as surrogate markers that would indicate the activation status of FXR in the ileum and the liver, respectively. After 7 days of bile drainage, the bile acid flux returning to the liver was eliminated as evidenced by complete disappearance of deoxycholic acid from hepatic bile. Significant reductions of IBABP protein and BSEP mRNA levels coupled with increased CYP8B and NTCP mRNA levels occurred and strongly indicated that FXR was deactivated by the depletion of the bile acid flux. It was noteworthy that although IBABP is located in the ileum and BSEP, CYP8B, NTCP, and CYP7A1 are expressed in the liver, absence of the circulating bile acid pool from the gut to the liver produced the expected changes in these FXR target genes. Under these conditions, the activation status of FXR in the ileum reflected by IBABP mirrored FXR activation in the liver. We also used WHHL rabbits in these experiments. In WHHL rabbits, where LDL receptors are deficient and baseline CYP7A1 activity and mRNA levels are lower than NZW rabbits (26Xu G. Salen G. Shefer S. Ness G.C. Chen T.S. Zhao Z. Tint G.S. Unexpected inhibition of cholesterol 7α-hydroxylase by cholesterol in New Zealand White and Watanabe heritable hyperlipidemic rabbits.J. Clin. Invest. 1995; 95: 1497-1504Google Scholar), removal of bile acid pool also deactivated FXR and downregulated transcription. It was also noteworthy that CYP27 activity was not affected by depletion of the bile acid pool, and by inference, was not altered by changes in FXR activation or transcription. Thus, in rabbits, alternative bile acid synthesis initiated by CYP27 does not appear to be regulated by FXR. This finding agrees with the negative results for CYP27 transcription in FXR null mice reported by Sinal et al. (12Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-1477Google Scholar). In summary, FXR protein was measured by EMSA and activation status of FXR was assessed by changes in mRNA levels of key FXR target genes BSEP, CYP8B, and NTCP in the liver and by protein levels of IBABP in the ileum. In an in vivo whole rabbit model, removal of the bile acid pool diminished and deactivated FXR protein. Thus, eliminating the hepatic bile acid flux not only resulted in upregulation of CYP7A1 by removing the inhibitory effect of FXR, but also decreased mRNA levels of FXR, suggesting the possibility that the ligand flux might also be involved in the control of FXR transcription. This study was supported by VA Research Service and US Public Health Service Grants HL18094, DK57636, DK26756, DK54165, and HD20632, and by American Heart Association Grant 9850180T.
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