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Originally published In Press as doi:10.1194/jlr.M700330-JLR200 on August 24, 2007

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Journal of Lipid Research, Vol. 48, 2664-2672, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine

Insook Kim*, Sung-Hoon Ahn*, Takeshi Inagaki{dagger}, Mihwa Choi{dagger}, Shinji Ito*, Grace L. Guo1,*, Steven A. Kliewer{dagger} and Frank J. Gonzalez2,*

* Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
{dagger} University of Texas Southwestern Medical Center, Dallas, TX 75390

Published, JLR Papers in Press, August 24, 2007.

1 Present address of G. L. Guo: University of Kansas Medical Center, Kansas City, KS 66160. Back

2 To whom correspondence should be addressed. e-mail: fjgonz{at}helix.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acid concentrations are controlled by a feedback regulatory pathway whereby activation of the farnesoid X receptor (FXR) represses transcription of both the CYP7A1 gene, encoding the rate-limiting enzyme in the classic bile acid synthesis pathway, and the CYP8B1 gene, required for synthesis of cholic acid. The tissue-specific roles of FXR were examined using liver- and intestine-specific FXR-null models. FXR deficiency in either liver (Fxr{Delta}L) or intestine (Fxr{Delta}IE) increased bile acid pool size. Treatment with the FXR-selective agonist GW4064 significantly repressed CYP7A1 in Fxr{Delta}L mice but not Fxr{Delta}IE mice, demonstrating that activation of FXR in intestine but not liver is required for short-term repression of CYP7A1 in liver. This intestinal-specific effect of FXR is likely mediated through induction of the hormone FGF15, which suppresses CYP7A1. In comparison to CYP7A1, FXR-mediated repression of CYP8B1 was more dependent on the presence of FXR in liver and less dependent on its presence in intestine. Consistent with these findings, recombinant FGF15 repressed CYP7A1 mRNA levels without affecting CYP8B1 expression. These data provide evidence that FXR-mediated repression of bile acid synthesis requires the complementary actions of FXR in both liver and intestine and reveal mechanistic differences in feedback repression of CYP7A1 and CYP8B1.

Supplementary key words FXR • FGF15 • CYP7A1 • CYP8B1 • liver-specific FXR null mice • intestine-specific FXR null mice

Abbreviations: BW, body weight; CA, cholic acid; DCA, deoxycholic acid; FXR, farnesoid X receptor; LC-MS/MS, liquid chromatography tandem mass spectrometry; MCA, murocholic acid; RT-qPCR, real-time quantitative PCR; TCA, taurocholic acid; TDCA, taurodeoxycholic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acids are amphiphilic end products of cholesterol catabolism that facilitate lipid absorption in the intestine. Bile acids are synthesized in the liver, released into the proximal intestine, and reabsorbed in the distal intestine (1). Release of bile from the gallbladder to the proximal intestine is induced by cholecystokinin, a hormone secreted from the duodenum in response to food intake (2). Conversely, gallbladder filling is regulated by the hormone FGF15, which is secreted from the ileum in response to bile acids (3). Disruption of bile acid homeostasis can cause cholestasis, diarrhea, and lipid malabsorption (4–7) and can affect lipid homeostasis by changing lipid absorption and altering the expression of genes that control lipid metabolism (8–11). In addition, owing to their cytotoxic, detergent-like properties, chronic exposure to elevated bile acids causes spontaneous liver tumor development in Fxr-null mice (12, 13). Bile acids also modulate liver regeneration (14) and energy expenditure (15).

The farnesoid X receptor (FXR) is a major bile acid sensor that protects the liver from bile acid toxicity by regulating the transcription of genes involved in bile acid homeostasis. Recently, the physiological ramifications of FXR have been expanded to include broader roles in glucose and lipid homeostasis (16–19). FXR is expressed in liver, intestine, kidney, and adrenal. Among the genes regulated by FXR are those encoding enzymes involved in bile acid synthesis, such as CYP7A1 and CYP8B1, and transport, such as BSEP and OST{alpha}/ß (20–25). The global Fxr-null mouse model has been a valuable model for investigating the roles of FXR in vivo (26). FXR-deficient mice fail to adapt to dietary cholic acid overload and exhibit dyslipidemia with elevated serum cholesterol and triglyceride concentrations (26, 27). The recent finding that FXR regulates transcription of the gene encoding the hormone FGF15/19 in intestine suggested a role for FXR in the cross-talk between intestine and liver. FGF15/19 cooperates with the orphan nuclear receptor SHP, whose expression is also regulated by FXR, to repress CYP7A1 and bile acid synthesis in liver (20, 28, 29). However, direct evaluation of the relative importance of FXR in liver and intestine in regulating bile acid homeostasis has not been possible owing to the lack of tissue-specific Fxr-null mice. In this report, we provide the first characterization and comparison of liver- and intestine-specific Fxr-null mice. Our results demonstrate that FXR in intestine is crucial for feedback regulation of bile acid synthesis in liver. Furthermore, they reveal unexpected differences in the mechanisms whereby FXR represses CYP7A1 and CYP8B1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of liver-specific and intestine-specific Fxr-null mice
Generation of global Fxr-null mice (26) and FGF15-null mice (29) was previously described, and the background matched wild-type animals used as controls. To generate liver-specific Fxr-null mice (Fxr{Delta}L) and intestine-specific Fxr-null mice (Fxr{Delta}IE), homozygous Fxr-floxed mice (Fxrfl/fl) (26) were crossed with mice harboring the cre-recombinase under the control of the albumin promoter [Alb-Cre mice, from Derek LeRoith (30)] and villin promoter [Villin-Cre mice, from Deborah Gumucio (31)], respectively. Animals were maintained on a C57B/6;129 mixed background. For genotyping, 50 ng of tail DNA was amplified in a 10 µl final reaction mixture containing 1.5 mM MgCl2, 0.25 mM deoxynucleotide triphosphates, 2.5% Me2SO, 0.25 U taq-polymerase, 0.2 µM FXR-geno-F (5'-atagacaaccccagtgaccc-3'), and 0.2 µM FXR-geno-R (5'-tctaaaggatagccgaatct-3'). Cycling conditions were 94°C for 3 min and then 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by a 10 min extension at 72°C. These primers yield products of 300 bp and 380 bp for the wild-type and the Fxrfl/fl alleles, respectively. Genotyping for cre-transgene was performed as previously described using microsomal epoxide hydrolase as an internal control (32). Disruption of Fxr was quantified by real-time quantitative PCR (RT-qPCR) using primers designed against the deleted exon: forward 5'-cacagcgatcgtcatcctctct-3' and reverse 5'-tctcaggctggtacatcttgca-3'. Mice were maintained under a standard 12 h light/12 h dark cycle with water and chow provided ad libitum. Animal handling was in accordance with animal study protocols approved by the National Cancer Institute Animal Care and Use Committee.

Animal treatment
All the experiments were performed with age-matched 8 to 10 week-old male mice, and Fxrfl/fl littermates without albumin-cre recombinase transgene or villin-cre recombinase transgene were used as controls to Fxr{Delta}L mice and Fxr{Delta}IE mice, respectively.

GW4064 treatment Mice received a first dose of GW4064 (100 mg/kg in 1% Tween 80/1% methylcellulose) or vehicle by oral gavage followed by a second dose 12 h later and were killed 2 h later for tissue collection. Tissues were flash-frozen in liquid nitrogen and stored at –80°C until RNA was prepared. Small and large intestines were removed and flushed with ice cold phosphate-buffered saline. Small intestine was divided into three equal lengths designated duodenum (proximal), jejunum (medial), and ileum (distal). The three segments were cut open longitudinally, and the mucosa was gently scraped and flash-frozen in liquid nitrogen.

Recombinant FGF15 treatment Wild-type mice (C57B6, male) were treated with saline control or 100 µg/kg recombinant FGF15 by tail vein injection and euthanized for tissue collection 1 h later. Recombinant FGF15 was expressed by infection of human embryonic retinoblast 911 cells with an FGF15-expressing adenovirus and purified from the media as previously described (29).

Serum chemistry
Serum was prepared by centrifugation at 8,000 rpm for 10 min in a serum separator, and serum triglyceride and total cholesterol were analyzed by using colorimetric assay kits, Infinity triglyceride assay reagent and cholesterol liquid stable reagents (Thermo Electronics, Inc.; Melbourne, Australia), respectively. Total bile acids in serum were analyzed by colorimetric enzyme assay using a bile acid kit (Trinity Biotech; St. Louis, MO).

Gene expression analysis
Northern blot analysis Total RNA was extracted using Trizol® (Invitrogen; Carlsbad, CA) following the manufacturer's protocol. RNA (10 µg) was separated on 1% formaldehyde-agarose gels and transferred to GeneScreen Plus membranes (Dupont; Wilmington, DE) overnight. The blots were hybridized at 62°C in PerfectHyb plus solution (Sigma) with random primer-labeled cDNA probes and exposed to a phosphorimager screen cassette and visualized using the Storm 860 PhosphorImager system (Amersham Biosciences).

RT-qPCR qPCR was performed using cDNA generated from 1 µg total mRNA with the SuperScript III Reverse Transcriptase kit (Invitrogen). Primers were designed for qPCR using the Primer Express software (Applied Biosystems; Foster City, CA) based on GenBank sequence data (CYP7A1: forward 5'-agcaactaaacaacctgccagtacta-3', reverse 5'-gtccggatattcaaggatgca-3'; FGF15: forward 5'-gaggaccaaaacgaacgaaatt-3', reverse 5'-acgtccttgatggcaatcg-3'; SHP: forward 5'-cgatcctcttcaacccagatg-3', reverse 5'-agggctccaagacttcacaca-3'; BSEP: forward 5'-acagaagcaaagggtagccatc-3', reverse 5'- ccatttgtgatttacaaacattcca-3'; IBABP: forward 5'-ggtcttccaggagacgtgat-3', reverse 5'-acattctttgccaatggtga-3'; paraoxonase 1: forward 5'-tgggtctgtcgtggtccaat-3', reverse 5'-atcaagggaaatgccaatgc-3'; CYP8B1: forward 5'-acgcttcctctatcgcctgaa-3', reverse 5'-gtg cctcagacgcagaggat-3'; OST{alpha}: forward 5'-atgcatctgggtgaacagaa-3', reverse 5'-gagtagggaggtgagcaagc-3'; OSTß: forward 5'-gaccacagtgcagagaaagc-3', reverse 5'-cttgtcatgaccaccaggac-3'; and ß-actin: forward 5'-tattggcaacgagcggttcc-3', reverse 5'-ggcatagaggtctttacggatgtc-3'). RT-qPCR reactions contained 25 ng of cDNA, 150 nM of each primer, and 5 µl of SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 10 µl. All reactions were performed in triplicate on an Applied Biosystems Prism 7900HT sequence detection system, and relative mRNA levels were calculated by the comparative threshold cycle method using ß-actin as the internal control.

Liquid chromatography tandem mass spectrometry analysis of conjugated bile acids in the bile acid pool and free bile acids in fecal extracts
To determine the bile acid pool size, liver, gallbladder, and small intestine with its contents were removed from 4 h-fasted mice, weighed and freeze-dried overnight. Fecal excretion of bile acid was determined in stools collected over 24 h in metabolic cages. Collected stools were air-dried over 48 h. Dried bile acid pool and stool samples were powdered; bile acids were extracted in 5 ml of 70% ethanol at 55°C for 4 h, and bile salts species were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) performed on a PESCIEX API200 ESI triple-quadrupole mass spectrometer (PerkinElmer Life Sciences) controlled by Analyst software, as described previously (33). Briefly, 200 µl of acetonitrile and an internal standard, 50 µl of dehydrocholic acid (20 µM in methanol), were added to 100 µl alcoholic extract of bile acids from bile acid pool or stools, and the mixture was centrifuged at 14,000 g for 5 min. Bile acids were further solid-phase extracted and eluted by 100% methanol using Sepak®, and then the organic solvent was evaporated by Speed-Vac (Thermo Savant, Waltham, MA). The residue was reconstituted in 60 µl of 0.1% formic acid in 50% methanol. Final extract (10 µl) was injected to LC-MS/MS and separated on the mobile phase of 0.1% formic acid in acetonitrile-water-ethanol (25:50:20).

Statistical analysis
Data are presented as mean ± standard deviation unless described otherwise, and statistical significance was analyzed by Student's t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tissue-specific deletion of FXR in liver or intestinal epithelium
Liver-specific or intestinal epithelium-specific disruption of Fxr expression was examined by Northern blot analysis (Fig. 1A , B). In Fxr{Delta}L mice harboring the homozygous floxed Fxr allele with the albumin-cre recombinase transgene, FXR mRNA was undetectable in liver but expression of FXR mRNA in ileal mucosa was comparable to that in Fxrfl/fl mice. On the other hand, in Fxr{Delta}IE mice harboring the homozygous floxed Fxr allele and the villin-cre recombinase transgene, the expression of FXR in the epithelium of the intestinal tract (including duodenum, jejunum, ileum, and colon) was significantly lower than in Fxrfl/fl mice, and its expression in liver and kidney was comparable to that of Fxrfl/fl mice (Fig. 1C). The Fxr{Delta}L or Fxr{Delta}IE mice are viable and display no overt abnormalities.


Figure 1
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  		Fig. 1. Tissue-specific disrupition of farnesoid X receptor (FXR) expression in liver-specific and intestinal-specific FXR-null mouse. Total RNA from (A) livers or (B) ileal mucosa of liver-specific and intestine-specific FXR-null mice and littermate controls was extracted and subjected to Northern blot analysis. C: Real-time quantitative PCR (RT-qPCR) analysis of FXR expression in the Fxr{Delta}IE mouse model. L, liver; K, kidney; D, duodenum; J, jejunum; I, ileum; C, colon; F/F, homozygous floxed FXR littermate controls; {Delta}L, Fxrfl/fl, albumin-cre+; {Delta}IE, Fxrfl/fl, villin-cre+; n = 3. Error bars indicate ± SD. * P < 0.05.

 
The whole-body Fxr-null mice exhibit dyslipidemia with elevated triglyceride, cholesterol, and bile acid levels (Fig. 2A ) (26). In contrast, total serum bile acid levels in Fxr{Delta}L and Fxr{Delta}IE mice were not significantly different compared with controls (Fig. 2A). As in the case of the Fxr-null mice, serum triglyceride and cholesterol levels of Fxr{Delta}L were significantly elevated compared with littermate controls, whereas serum lipid levels of Fxr{Delta}IE were not significantly different from littermate controls (Fig. 2B, C). These data indicate that the presence of FXR in either liver or intestine is sufficient to maintain normal serum bile acid concentrations but that FXR in liver is required for controlling serum triglyceride and cholesterol levels.


Figure 2
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  		Fig. 2. Serum chemistry of Fxr-null, Fxr{Delta}L, and Fxr{Delta}IE mice. Serum samples from 4 h-fasted animals were analyzed for (A) total bile acids, (B) cholesterol, and (C) triglyceride. BA, bile acids; CHOL, cholesterol; TG, triglycerides; F/F: homozygous floxed FXR littermate controls; {Delta}L, Fxrfl/fl, albumin-cre+; {Delta}IE, Fxrfl/fl, villin-cre+; n = 5–6. Error bars indicate ± SD. * P < 0.05.

 
Bile acid composition in the bile acid pool and feces
It was previously shown that the total bile acid pool size is increased in Fxr-null mice (12, 34). To investigate the relative contribution of FXR in liver and intestine to maintaining the bile acid pool size, alcohol extracts of total bile acids were prepared from liver, gallbladder, and small intestine and analyzed for both total bile acid levels and bile acid species by LC-MS/MS. Consistent with previous reports, the total bile acid pool size was increased in Fxr-null mice (Fig. 3A ). A significant increase in the bile acid pool size was also observed in Fxr{Delta}L mice. Although a trend toward increased total bile acid pool size was seen in Fxr{Delta}IE mice (Fig. 3A), this difference did not achieve statistical significance. The magnitude of the increase was greater in the Fxr-null mice than in either the Fxr{Delta}L or Fxr{Delta}IE mice, suggesting that FXR in both liver and intestine contributes to controlling the overall bile acid pool size.


Figure 3
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  		Fig. 3. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of conjugated bile acids in bile acid pool. Total bile acids were extracted from freeze-dried bile acid pool composed of liver, gallbladder, and small intestine with its contents and analyzed for conjugated bile acids as described in MATERIALS AND METHODS. A: Total bile acid pool (% control). Individual bile acids: global Fxr-null (n = 4–5) (B); liver-specific FXR-null ({Delta}L) (n = 4–5) (C); and intestine-specific Fxr-null ({Delta}IE) (n = 9–10) (D); BW, body weight; F/F, homozygous floxed FXR littermate controls; TCA, taurocholic acid; TßMCA, tauro-ß-muricholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; GCA, glycocholic acid.

 
To determine the contribution of different pathways to the increased bile acid pool size, individual bile acid species were analyzed in more detail. In our initial analysis of free-form bile acids, including cholic acid (CA), murocholic acid ({alpha}MCA), ßMCA, and deoxycholic acid (DCA), the combined free bile acid level was less than 10% of the bile acid pool, and no significant differences between controls and Fxr-null models were observed. Briefly, the combined free bile acid level in the bile acid pool was 2.6 ± 0.9 and 3.8 ± 0.9 µmol/100 g body weight (BW) in wild-type and Fxr-null mice, respectively; 3.9 ± 1.4 and 4.5 ± 2.0 µmol/100 g BW in controls and Fxr{Delta}L mice, respectively; and 2.3 ± 1.6 and 2.6 ± 1.3 µmol/100 g BW in controls and Fxr{Delta}IE mice, respectively. Because most bile acids are present in their conjugated forms (1), only the conjugated bile acids were analyzed. There was a significant increase in taurocholic acid (TCA), the most abundant bile acid, in Fxr-null, Fxr{Delta}L, and Fxr{Delta}IE groups compared with their controls (Fig. 3B–D). TCA levels increased 2.6-, 1.4-, and 1.2-fold in Fxr-null, Fxr{Delta}L, and Fxr{Delta}IE mice, respectively, compared with control mice. The relative magnitude of these changes is in good agreement with the overall changes in the bile acid pool size (Fig. 3A). The cholate derivative, taurodeoxycholic acid (TDCA), was also increased in all three null groups (Fig. 3B–D). In contrast, the second most abundant species, tauro-ß-muricholic acid (TßMCA), was not significantly different in Fxr-null and Fxr{Delta}L mice and was slightly decreased in Fxr{Delta}IE mice compared with their controls. No significant difference was detected in taurochenodeoxycholic acid (TCDCA) levels in all three null groups (Fig. 3B–D). These results indicate that the increase in bile acid pool size in the Fxr-deficient models is due specifically to an increase in cholate and its derivatives.

During enterohepatic circulation, a small fraction of bile acids are lost in the stool, whereas the vast majority of bile acids are reabsorbed in the ileum (1). Bile acid composition was analyzed in fecal extracts to determine whether the increased bile acid pool size was reflected in fecal bile acid excretion (Fig. 4A –C). As expected, the fecal bile acids were almost exclusively unconjugated, owing to the actions of gut microflora (data not shown). Interestingly, DCA was significantly increased in fecal extracts from Fxr{Delta}IE mice but not from Fxr-null or Fxr{Delta}L mice. Although there was a trend toward increased fecal CA in Fxr-null, Fxr{Delta}l, and Fxr{Delta}IE mice compared with their controls, there were no other significant changes in fecal bile acid concentrations.


Figure 4
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  		Fig. 4. LC-MS/MS analysis of free bile acids in fecal extracts. Total bile acids were extracted from air-dried feces collected over 24 h and analyzed for free bile acids. Fecal extracts of Fxr-null (n = 4) (A), liver-specific Fxr-null ({Delta}L) (n = 5) (B), and intestine-specific Fxr-null ({Delta}IE) (n = 8) (C). DCA, deoxycholic acid; MCA, murocholic acid; CA, cholic acid; WT, wild-type; F/F, homozygous floxed FXR littermate controls.

 
Gene expression analysis
To investigate the mechanistic basis for the increased bile acid pool size in Fxr{Delta}L and Fxr{Delta}IE mice, gene expression was analyzed by qPCR using total RNA from liver and ileal mucosa of Fxr{Delta}L and Fxr{Delta}IE mice. Similar to previous findings in Fxr-null mice, the basal level of CYP7A1 mRNA expression was significantly increased in Fxr{Delta}L mouse livers (Fig. 5 ). Surprisingly, no change in basal CYP7A1 levels was seen in Fxr{Delta}IE mice (Fig. 6 ). There was no significant change in basal CYP8B1 mRNA levels in either the Fxr{Delta}L or Fxr{Delta}IE mice (Figs. 5, 6). There was a trend toward decreased basal SHP expression in liver but not ileum of Fxr{Delta}L mice; conversely, there was a trend toward decreased SHP expression in ileum but not liver of Fxr{Delta}IE mice. IBABP mRNA levels were significantly lower in ileum of Fxr{Delta}IE mice (Fig. 6).


Figure 5
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  		Fig. 5. Gene expression induced by GW4064 treatment in the liver-specific Fxr-null mice. Real-time quantitative PCR (RT-qPCR) analysis was performed on cDNA prepared from total RNA of liver or ileal mucosa of Fxrfl/fl littermate controls (F/F) or liver-specific Fxr-null ({Delta}L) treated with vehicle (open bars) or 100 mg/kg GW4064 (closed bars) by oral gavage as described in MATERIALS AND METHODS. CYP7A1, CYP8B1, BSEP, and PON1 expression was analyzed in liver. Data were presented as a relative value to the expression level of control treated with vehicle (n = 4–5; * P < 0.05 versus control treatment group). SHP, small heteromer partner; FGF15, fibroblast growth factor 15; BSEP, bile salts export pump; PON1, paraoxonase1. Error bars indicate ± SD.

 

Figure 6
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  		Fig. 6. Gene expression by GW4064 treatment in the intestine-specific FXR-null mice. RT-qPCR analysis was performed on total RNA from livers or ileal mucosa of Fxrfl/fl littermate controls (F/F) or intestine-specific Fxr-null ({Delta}IE) mice treated with vehicle (open bar) or GW4064 (closed bar) as described in MATERIALS AND METHODS. IBABP, OST{alpha}, and OSTß expression was analyzed in ileal mucosa. CYP7A1, CYP8B1, BSEP, and PON1 expression was analyzed in liver. Data are presented as a relative value to the expression level of control treated with vehicle (n = 4–5; * P < 0.05 versus control treatment group). Error bars indicate ± SD.

 
To further examine the role of FXR in regulating gene expression in liver and intestine, Fxr{Delta}L, Fxr{Delta}IE, and control mice were administered either the FXR-selective synthetic agonist GW4064 or vehicle. As expected, GW4064 induced SHP and BSEP mRNA expression in livers of control mice but not Fxr{Delta}L mice (Fig. 5) and induced IBABP, OST{alpha}, and OSTß in ileum of control mice but not Fxr{Delta}IE mice (Fig. 6). Interestingly, in Fxr{Delta}L mouse livers, CYP7A1 mRNA expression was markedly repressed by GW4064. In contrast, GW4064 did not significantly repress CYP7A1 in Fxr{Delta}IE mice, whereas BSEP expression was induced consistently in Fxr{Delta}IE mice (Fig. 6). Taken together, these data show that FXR in intestine but not liver is required for acute repression of CYP7A1.

Unexpectedly, CYP8B1 showed a completely different pattern of regulation compared with CYP7A1. Although GW4064 treatment repressed CYP8B1 expression >5-fold in livers of control mice, repression was reduced to <2-fold in Fxr{Delta}L mice (Fig. 5). In marked contrast to CYP7A1, deletion of FXR in intestine had only a modest effect on GW4064-mediated repression of CYP8B1 (Fig. 6). These data demonstrate that CYP7A1 and CYP8B1 are differentially regulated by FXR, with CYP7A1 repression more dependent on FXR in intestine and CYP8B1 repression more dependent on FXR in liver.

The demonstration that intestinal FXR is required for efficient repression of CYP7A1 is consistent with the FXR-FGF15 enterohepatic signaling cascade playing a prominent role in CYP7A1 regulation. FGF15 was not induced by GW4064 in ileum of Fxr{Delta}IE mice but was induced in ileum of control and Fxr{Delta}L mice (Figs. 5, 6). The potential role of the FXR-FGF15 signaling cascade in regulating gene expression was further investigated by analyzing the hepatic expression of paraoxonase 1 (PON1), which is regulated by FGF15 (35, 36). PON1 mRNA levels were significantly reduced by GW4064 in control and Fxr{Delta}L mouse liver but not in liver of Fxr{Delta}IE mice (Figs. 5, 6). These data provide additional evidence that FGF15 originating from the small intestine contributes to the regulation of hepatic genes in response to GW4064 in FXR-deficient liver.

Differential effects of FGF15 on CYP7A1 and CYP8B1
To test directly whether FGF15 has differential effects on CYP7A1 and CYP8B1, wild-type mice were injected with recombinant FGF15 or saline control. The regulation of CYP7A1 by FGF15 and in Fgf15-null mice was consistent with previous observations (29). Notably, FGF15 efficiently repressed CYP7A1 expression but did not significantly affect CYP8B1 mRNA levels (Fig. 7A ). To complement these studies, the effect of GW4064 administration on CYP7A1 and CYP8B1 expression was evaluated in wild-type and Fgf15-null mice. Whereas basal CYP7A1 mRNA levels were increased 3.5-fold in Fgf15-null mice, CYP8B1 mRNA levels were elevated a more modest 1.7-fold (Fig. 7B). As expected, GW4064 treatment repressed both CYP7A1 and CYP8B1 expression to a comparable degree in wild-type mice. However, in Fgf15-null mice, GW4064 repressed CYP8B1 expression but did not cause a significant decrease in CYP7A1 mRNA levels. Taken together, these data demonstrate that feedback repression of CYP7A1 is more strongly regulated by the FXR-FGF15 signaling cascade than is CYP8B1.


Figure 7
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  		Fig. 7. Hepatic gene expression by recombinant FGF15 treatment in wild-type mice and GW4064 treatment in the Fgf15-null mice. RT-qPCR analysis was performed on total RNA from livers of wild-type mice injected with recombinant FGF15 or saline and euthanized 1 h later (A–C) (n = 3), and Fgf15-null and wild-type control mice treated with vehicle (open bars) or GW4064 (closed bars) and euthanized 14 h later (D–F) (n = 4–5). Mean ± SEM; * P < 0.05 versus control treatment group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The regulation of bile acid homeostasis by FXR is an area of intense interest. In liver, FXR regulates a program of genes including CYP7A1, CYP8B1, SHP, and BSEP that impact bile acid pool size and composition. Likewise, in intestine, FXR regulates genes involved in bile acid homeostasis, including IBABP, ASBT, OST{alpha}/ß, and FGF15 (20–25). Although the whole-body knockout of FXR in mice causes marked changes in bile acid homeostasis (26, 27), its relative importance in liver and intestine was not known. In the current study, this question was systematically investigated using liver- and intestine-specific FXR-null mice.

These studies reveal that FXR in both liver and intestine makes important contributions to the regulation of bile acid homeostasis. Although the elimination of FXR in either liver or intestine caused increases in the bile acid pool size, including TCA and TDCA, these increases were not as pronounced as those seen in the Fxr-null mice. Moreover, whole-body deletion of FXR caused a significant increase in serum bile acid concentrations (26) that was not seen in either the Fxr{Delta}L or Fxr{Delta}IE mice. Taken together, these data demonstrate additive effects of FXR in liver and intestine under normal dietary conditions.

Notably, fecal DCA excretion, which is usually proportional to cholate pool size, was increased only in Fxr{Delta}IE but not in Fxr-null and Fxr{Delta}L mice. CA excretion tended to be higher in Fxr-null and Fxr{Delta}L mice compared with controls but did not achieve statistical significance. Despite increased bile acid synthesis in Fxr-null mice, bile acid release to small intestine was not increased but was decreased (26) or comparable (27). The findings that fecal bile acid excretion is not altered in either Fxr-null or Fxr{Delta}L mice and is increased in Fxr{Delta}IE mice suggest that the increased bile acid pool size in Fxr{Delta}IE mice is probably due to deregulation of bile acid synthesis rather than to an increase in bile acid reabsorption. Nevertheless, we cannot exclude the possibility of the contribution of deregulation of the bile salts transporter OST{alpha}/ß to the increased fecal bile acid excretion in Fxr{Delta}IE mice. The effect of the selective FXR agonist GW4064 in liver- and intestine-specific FXR-null mice was also compared. As expected, SHP and BSEP were induced by GW4064 in the livers of Fxr{Delta}IE but not Fxr{Delta}L mice. Conversely, IBABP, SHP, and FGF15 were induced by GW4064 in small intestine of Fxr{Delta}L but not Fxr{Delta}IE mice. It was previously shown that FXR represses CYP7A1 through the coordinate induction of FGF15 in intestine (29) and SHP in liver (20, 21). FGF15 and SHP then act cooperatively to repress CYP7A1 transcription through a mechanism that is not yet understood. Although mice completely devoid of either FGF15 or SHP have markedly elevated basal CYP7A1 expression (29, 37), the relative contribution of intestinal and liver FXR to the repression of CYP7A1 was not known. The loss of significant repression of CYP7A1 in Fxr{Delta}IE mice treated with GW4064 provides the first direct evidence that FXR in the gut regulates bile acid synthesis in the liver. The coincident loss of FGF15 induction suggests that this hormone is responsible for signaling from intestine to liver. Consistent with this possibility, FXR-mediated repression of PON1, which is also suppressed by FGF15 (35, 36), was lost in Fxr{Delta}IE but not Fxr{Delta}L mice. Notably, FXR-mediated repression of CYP7A1 was largely intact in the Fxr{Delta}L mice despite the lack of hepatic SHP induction. These data demonstrate that induction of SHP above basal levels is not essential for FXR-mediated repression of CYP7A1. They further suggest that most of the acute effect of FXR on CYP7A1 transcription is mediated by induction of FGF15 in intestine.

There are several caveats that must be kept in mind when interpreting the CYP7A1 data from these studies. First, if intestinal FGF15 regulates CYP7A1, why do the Fxr{Delta}L but not the Fxr{Delta}IE mice have increased basal CYP7A1 mRNA levels? The regulation of CYP7a1 via intestinal FXR-FGF15 is presumably very cyclical, such that under normal physiological conditions, the bile acid flux to the distal small intestine at the end of the enterohepatic circulation would activate FXR in the ileum and then send signals to the liver for CYP7a1 repression. Thus, it is possible not to see a basal level induction of CYP7a1 in the liver of Fxr{Delta}IE mice, especially when liver FXR and other transcription factors are intact. Nonetheless, deregulation of CYP7a1 in Fxr{Delta}IE mice, which we demonstrated using a synthetic FXR agonist, may contribute to a trend toward increasing bile acid pool size. In addition, it is important to note that unlike the Fgf15-null mice, the Fxr{Delta}IE mice still express FGF15, albeit at reduced levels, in the ileum. This residual FGF15 may be adequate to constrain CYP7A1 transcription in the context of an intact FXR response in liver. On the other hand, as with all gene knockout mice, it is also possible that the Fxr{Delta}IE mice have undergone a long-term, compensatory response that constrains CYP7A1 expression even in the absence of the normal contribution of intestinal FXR to the regulation of bile acid synthesis. A related point is why the intestine-specific FXR-null mice show a trend toward an increased bile acid pool size even if they do not have increased basal CYP7A1 expression. One possibility is that the alternative pathway for bile acid synthesis is upregulated in the intestine FXR-null mice. However, according to the bile acid species analysis, a trend toward increasing bile acid pool size in Fxr{Delta}IE mice is attributed mainly to an increase in cholate pool (taurocholate); thus, we hypothesized that an increase in bile acid pool size is due more to the classic (neutral) pathway than to the alternative pathway. Moreover, we did not observe an increase in muricholates or chenodeoxycholate, which are the main products of the acidic pathway in Fxr{Delta}IE mice, compared with controls. At this point, the molecular basis for this trend is not known. A final caveat relates to the FXR-selective agonist, GW4064, which has poor bioavailability. Although the presence of GW4064 in liver is evidenced by the induction of BSEP in controls and Fxr{Delta}IE mice, we cannot exclude the possibility of more-pronounced effects on FXR activity in intestine than liver due to the poor bioavailability of GW4064. This relative lack of efficacy in liver could potentially lead to an underestimation of the importance of FXR-mediated induction of SHP and possibly other genes in liver in the feedback regulation of CYP7A1.

FXR also plays an important role in the feedback repression of CYP8B1. An unexpected outcome of our studies was the differential regulation of CYP7A1 and CYP8B1 by FXR. Whereas FXR-induced repression of CYP7A1 was more strongly affected by disruption of FXR in intestine, repression of CYP8B1 was more sensitive to loss of FXR in liver. One interpretation of these data is that CYP7A1 is regulated more strongly by the FXR-FGF15 pathway in intestine and CYP8B1 is more sensitive to repression via FXR activation in liver. In support of this hypothesis, CYP7A1 was efficiently repressed by injection of FGF15, whereas CYP8B1 was not. Moreover, CYP8B1 was repressed by FXR activation in Fgf15-null mice, whereas CYP7A1 was not. Finally, whereas basal expression of CYP7A1 increased 3-fold in Fgf15-null mice, CYP8B1 mRNA levels increased less than 2-fold. Regarding this latter point, it must be noted that CYP8B1 mRNA levels were significantly increased in the Fgf15-null mice, indicating that CYP8B1 expression is affected by FGF15, at least in the long term.

It was previously shown that treatment of humans with the bile acid sequestrant cholestyramine, which strongly reduces circulating levels of FGF19 (the human ortholog of FGF15), induced CYP7A1 expression but not CYP8B1 (38, 39). Furthermore, repression of CYP7A1 by dietary bile acids was attenuated in livers of mice lacking ßKlotho, an essential component of the FGF15 receptor complex, whereas CYP8B1 repression was not (40). Taken together with data in this study, these results demonstrate that the CYP7A1 gene is more responsive than CYP8B1 to repression by FGF15/19 emanating from the intestine. The molecular basis for this differential regulation and its physiological relevance remain to be determined.

In summary, liver-specific and intestine-specific Fxr-null mice were developed and used to demonstrate differential but complementary roles for FXR in liver and intestine in regulating bile acid homeostasis. This study provides the first direct evidence that FXR in intestine controls bile acid synthesis in liver and supports a previous hypothesis for intestine-produced FGF15 to regulate liver CYP7A1. Moreover, prominent differences were demonstrated in the mechanisms underlying feedback repression of CYP7A1 and CYP8B1. The tissue-specific Fxr-null mice will be powerful models for further investigation of the tissue-specific roles of FXR in bile acid metabolism and other physiologic and pathophysiologic processes.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health National Cancer Institute Intramural Research Program, Grant DK-067158 (I.K., S-H.A., and S.I.), and the Robert A. Welch Foundation (S.A.K.). The authors thank Derek LeRoith and Deborah L. Gumucio for providing the albumin- and villin-cre transgenic mouse lines, respectively, and Robert Gerard for help in preparing recombinant FGF15 protein.

Manuscript received July 20, 2007 and in revised form August 22, 2007.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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