Materials and Methods

Compounds.

1α(OH)D₃ was kindly provided by Dr. Yoji Tachibana (Nisshin Flour Milling Co., Saitama, Japan). CA, DCA, CDCA, LCA, hyodeoxycholic acid (HDCA), and ursodeoxycholic acid (UDCA) were purchased from Sigma-Aldrich (St. Louis, MO), and α-muricholic acid (MCA), β-MCA, and ω-MCA were from Steraloids (Newport, RI). [1-¹⁴C]Glycocholic acid (50 mCi/mmol) was purchased from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK).

Animals and Treatment.

C57BL/6J male mice (5–6 weeks of age; Tokyo Laboratory Animals Science Co., Tokyo, Japan) were housed under controlled temperature (23 ± 1°C) and humidity (45–65%) and a standard 12-h light/dark cycle. Before feeding of chow supplemented with bile acid, mice were fed standard rodent chow (Lab Animal Diet MF; Oriental Yeast Co., Tokyo, Japan). For bile acid supplementation, standard chow was finely powdered and mixed thoroughly with CA, CDCA, DCA, or LCA at 0.4% (w/w) composition. In initial experiments, mice were fed powdered standard or 0.4% bile acid-supplemented chow for 8 days (Fig. 1A). On days 6, 7, and 8, mice were orally administered 1α(OH)D₃ dissolved in corn oil (0.2 ml) at a dose of 2.5 nmol/mouse/day (n = 5–8). At day 9, mice were anesthetized with ether, and blood was collected by cardiac puncture with a heparinized syringe. Tissue samples were collected, frozen on dry ice immediately after removal and weighing, and then stored at −80°C. In the second experiment, mice were fed chow or 0.4% CDCA-supplemented chow from day 1 until day 5, fasted from day 6, and administered 2.5 nmol/mouse 1α(OH)D₃ via gavage on days 6 and 7 (Fig. 1B). Forty-eight-hour urine samples (on days 7–8) were collected in glass metabolic bowls. The experimental protocol adhered to the Guidelines for Animal Experiments of the Nihon University School of Medicine and was approved by the Ethics Review Committee for Animal Experimentation of Nihon University School of Medicine.

Extraction and Derivatization of Bile Acids.

For extraction of bile acids, liver samples (approximately 0.4 g) were homogenized in 6 ml of 80% (v/v) ethanol with a Polytron homogenizer (Kinematica AG, Littau-Lucerne, Switzerland) on ice and centrifuged at 9400g for 10 min. Precipitates were rehomogenized with 6 ml of 80% (v/v) ethanol with a Sonifier (Branson Ultrasonics Corporation, Danbury, CT) and then centrifuged. Extraction with ethanol was repeated three times, and combined supernatant from liver homogenate was evaporated to dryness. [1-¹⁴C]Glycocholic acid was added to a liver sample, and the extraction yield was measured by liquid scintillation counting. The recovery yield of [1-¹⁴C]glycocholic acid was 94% (mean from triplicate assay). Plasma bile acids were extracted by solid-phase extraction using a C18 Bond Elute column (Varian, Inc., Palo Alto, CA) as reported previously (Setchell and Worthington, 1982). Plasma samples (0.4 ml) were mixed with 1 ml of 0.5 mol/l triethylammonium sulfate, pH 7.5, and heated at 65°C for 15 min to liberate the protein-bound bile acids. After chilling, the mixture was applied to a C18 column, rinsed briefly, eluted with 4 ml of ethanol, and evaporated to dryness. Using a C18 Bond Elute column, quantitative yield is successfully attained for both nonpolar, such as lithocholic acid, and polar bile acids, including conjugated bile acids (Setchell and Worthington, 1982). We estimated the recovery yield of plasma bile acids from a C18 Bond Elute column using [1-¹⁴C]glycocholic acid. More than 98% of [1-¹⁴C]glycocholic acid was obtained in the ethanol solution by liquid scintillation counting.

Extracted liver and plasma bile acids were subjected to gas chromatography-mass spectrometry (GC-MS) after derivatization. After the addition of an internal standard, bile acids were treated with 0.6 ml of acetone-methanol-6 M HCl (36:4:0.4 by volume) at 37°C for 14 h to remove the conjugated sulfonyl group (Parmentier and Eyssen, 1975) and then with 15% (w/v) NaOH at 120°C for 2 h in an autoclave to deconjugate the taurine or glycine moiety (Keller and Jahreis, 2004). Samples were then extracted with 2 ml of hexane twice to remove cholesterol and then acidified to approximately pH 1 with 2 M HCl. Deconjugated bile acids were extracted with 2 ml of diethyl ether three times and converted to methyl esters with trimethylsilyldiazomethane (GL Sciences, Inc. Japan, Tokyo, Japan) and to trimethylsilyl derivatives with N,O-bis(trimethylsilyl)trifluoroacetamide plus trimethylchlorosilane (Thermo Fisher Scientific, Waltham, MA).

GC-MS Analysis of Bile Acids.

We used a Shimadzu GCMS-QP5050A GC-MS analyzer, equipped with an AOC-20i autoinjector and a GCMS Solution data system (Shimadzu Corporation, Kyoto, Japan). Gas chromatographic separation was carried out with a separation column of HP-5 (cross-linked 5% Ph-Me silicon, 0.32 mm internal diameter, 0.15 μm film thickness, and 25 m in length; Agilent Technologies, Santa Clara, CA). The injection temperature was 150°C, and the column temperature was programmed at 150°C for 5 min and 10°C/min to 260°C and held for 30 min. Ionizing energy was set at 70 eV, ionizing trap current at 60 μA, and ion detector gain at 1.1 kV. Highly purified helium gas was used as the carrier gas at a flow rate of 1.0 ml/min. Split ratio was 1:10, and sampling rate was 0.5 s. The bile acids were analyzed as methylester-trimethylsilyl derivatives (Setchell et al., 1983). An appropriate fragment ion in the high mass region was selected for mass fragmentography. The fragment ions (m/z) and relative intensities (percentage) were as follows: LCA (target ion, m/z 372, 100%; reference ions, m/z 215, 149% and m/z 257, 51%), DCA (target ion, m/z 255,100%; reference ions, m/z 208, 20% and m/z 370, 13%), CDCA (target ion, m/z 370, 100%; reference ions, m/z 255, 31% and m/z 355, 25%), CA (target ion, m/z 253,100%; reference ions, m/z 368, 60% and m/z 458, 35%), HDCA (target ion, m/z 370, 100%; reference ions, m/z 255, 72% and m/z 355, 32%), UDCA (target ion, m/z 460, 100%; reference ions, m/z 370, 42% and m/z 255, 41%), α-MCA (target ion, m/z 458, 100%; reference ions, m/z 443, 25% and m/z 195, 28%), β-MCA (target ion, m/z 195, 100%; reference ions, m/z 285, 68% and m/z 458, 9%), and ω-MCA (target ion, m/z 195, 100%; reference ions, m/z 285, 58% and m/z 369, 29%). Total bile acid concentrations were calculated from summation of individual bile acid concentrations.

Concentrations of Plasma Calcium, Aminotransferases, and Urinary Total Bile Acid.

Plasma total calcium levels, alanine and aspartate aminotransferases, urinary total bile acid concentrations, and urinary and plasma creatinine concentrations were measured using Calcium C-Testwako, Transaminase CII-Testwako, Total bile acid-Testwako, and Creatinine-Testwako kits (Wako Pure Chemicals Industries, Osaka, Japan), respectively. Urinary bile acid concentrations were normalized with creatinine levels.

Immunoblotting.

Kidney membrane was prepared as reported previously (Zollner et al., 2006b). Proteins in the membrane fraction were resolved by 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), and probed with a monoclonal antibody against Mrp4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or an anti-lamin B antibody (Santa Cruz Biotechnology, Inc.) to confirm the equal protein amount in the lanes. Although Mrp4 has a predicted molecular mass of 150 kDa, it has a number of potential transmembrane regions and glycosylation and phosphorylation sites, which may affect the migration of the protein (Zollner et al., 2006b). Homogenized protein was quantified with a BCA protein assay kit (Thermo Fisher Scientific).

Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction.

Total RNAs from samples were prepared by the acid guanidine thiocyanate-phenol/chloroform method, and cDNAs were synthesized using the ImProm-II Reverse Transcription system (Promega, Madison, WI) (Ogura et al., 2009). Real-time polymerase chain reaction was performed on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (Applied Biosystems). Primers for Mrp3 (GenBank accession number NM_029600) were 5′-GCC AAC TTC CTC CGA AAC TA-3′ and 5′-CTT GCG GAC CTC GTA GAT GG-3′, and others have been reported previously (Ishizawa et al., 2008; Ogura et al., 2009). Relative mRNA levels were calculated by the comparative threshold cycle method using glyceraldehyde-3-phosphate dehydrogenase as the internal control (Choi et al., 2006).

Statistical Analysis.

Data are presented as means ± S.D., and statistical differences were determined by analysis of variance.

Results

Modulation of Bile Acid Compositions in Liver and Plasma of Mice Fed Bile Acid-Supplemented Chow by 1α(OH)D₃.

We fed mice with chow supplemented with 0.4% bile acid for 5 days and examined the effects of bile acid supplements on food intake and liver toxicity in mice. Feeding with CA, DCA, and LCA slightly decreased the food intake but did not induce a significant increase in plasma aminotransferase levels (Table 1 ). CDCA feeding did not change food intake or plasma aminotransferase levels. Thus, 0.4% bile acid supplements have no or only modest toxic effects in mice. To examine the effects of VDR activation on bile acid metabolism, we fed mice with control chow or chow supplemented with 0.4% bile acid and then administered 1α(OH)D₃ via gavage as shown in Fig. 1A. 1α(OH)D₃ is rapidly converted to 1,25(OH)₂D₃ after injection and is more effective than 1,25(OH)₂D₃ in prolonging survival time of mice inoculated with leukemia cells (Honma et al., 1983). Treatment of mice with 1α(OH)D₃ for 3 days increased plasma calcium levels to 18.5 mg/dl from 10.3 mg/dl of control mice, consistent with effective VDR activation (Table 2 ). We examined plasma and liver bile acid composition with GC-MS after deconjugation. In mice fed control chow diet, CA (22%) and ω-MCA (56%) were the major hepatic bile acids (Fig. 2 A). CA and β-MCA have been reported to be the major hepatic bile acids in mice (Stedman et al., 2004; Zollner et al., 2006b). Because ω-MCA is a secondary bile acid converted from β-MCA by intestinal bacteria (Eyssen et al., 1983), microflora may influence the concentration of ω-MCA in the bile acid pool. 1α(OH)D₃ treatment decreased the minor bile acid components (LCA, DCA, HDCA, UDCA, and CDCA) but did not change total bile acid, CA, or ω-MCA concentrations. CA, HDCA, UDCA, and DCA were detected in plasma, and 1α(OH)D₃ decreased plasma total bile acid and DCA concentrations (Fig. 2B).

We examined the effects of 1α(OH)D₃ on bile acid metabolism by feeding mice 0.4% bile acid-supplemented chow as shown in Fig. 1A. First, we fed mice chow supplemented with primary bile acids. CDCA feeding increased the total bile acid concentrations in liver and plasma by 2.8- and 2.4-fold, respectively, compared with those in mice fed control chow (Figs. 2 and 3 ). The major bile acid components in the liver of CDCA-fed mice were α-MCA (35%), UDCA (21%), ω-MCA (17%), and CDCA (17%) (Fig. 3A). CDCA is converted to α-MCA and β-MCA in the liver and to UDCA by microflora (Botham and Boyd, 1983; Fromm et al., 1983). 1α(OH)D₃ treatment decreased total bile acid concentration, LCA, DCA, HDCA, UDCA, CDCA, β-MCA, and ω-MCA in the liver. CDCA feeding increased plasma CDCA and α-MCA (Fig. 3B). Because of the high variation for other bile acids, only the 1α(OH)D₃-dependent decrease in DCA was statistically significant. CA feeding increased liver total bile acids 2.2-fold compared with those in mice fed control chow and 89% of the liver bile acids were CA (Fig. 4 A). Plasma total bile acids were increased in mice fed CA 11-fold compared with mice fed control chow (Figs. 2 and 4), and CA and DCA were the major pool components (Fig. 4B). 1α(OH)D₃ treatment decreased plasma total bile acids and CA but did not reduce hepatic bile acids.

Next, we examined the effects of 1α(OH)D₃ on mice fed chow supplemented with secondary bile acids. DCA feeding increased total liver bile acids 4.3-fold compared with mice fed control chow (Figs. 2 and 5 ). The liver of these mice contained CA (72%) and DCA (27%), and 1α(OH)D₃ treatment effectively decreased hepatic total bile acids, DCA, and CA (Fig. 5A). Total plasma bile acids were increased 18-fold compared with those in mice fed control chow, and 1α(OH)D₃ treatment decreased plasma total bile acids (Fig. 5B). Although the effect of 1α(OH)D₃ on plasma DCA was not significant (p = 0.06), the 1α(OH)D₃-induced decrease in the total bile acid concentration in mice fed DCA could be due to enhanced metabolism or elimination of DCA. LCA feeding did not increase the total bile acid concentration of liver or plasma (Figs. 2 and 6 ). In the liver of mice fed LCA the major bile acids were HDCA (24%) and α-MCA (32%) (Fig. 6A). LCA is metabolized to HDCA by CYP3A enzymes, which are induced by LCA-responsive nuclear receptors, such as pregnane X receptor and VDR (Xie et al., 2001; Makishima et al., 2002). 1α(OH)D₃ treatment decreased LCA and DCA, but had no effect on total bile acids, HDCA, and α-MCA in the liver of LCA-fed mice. 1α(OH)D₃ did not alter plasma bile acid levels (Fig. 6B). Therefore, 1α(OH)D₃ administration enhances the metabolism of bile acids, such as CDCA and DCA.

Bile Acid Metabolism Is Enhanced by 1α(OH)D₃ Treatment in Fasted Mice Prefed CDCA-Supplemented Chow.

1α(OH)D₃ treatment induces hypercalcemia and weight loss in mice (Ishizawa et al., 2008). To rule out the possibility that hypercalcemia-associated decreased food intake affects liver and plasma bile acid concentrations, we fed mice chow supplemented with or without CDCA for 5 days and then administered 1α(OH)D₃ under fasting conditions (Fig. 1B). Body weight did not differ between vehicle-treated mice and 1α(OH)D₃-treated mice (Table 3 ). In mice fed normal chow, 2-day fasting decreased liver bile acid concentrations (Figs. 2 and 7 ). The major bile acids were CA and ω-MCA, similar to the pool composition in mice fed control chow ad libitum. It is interesting to note that the total bile acid concentration in the liver of mice prefed CDCA-supplemented chow decreased to control levels after 2 days of fasting, and 1α(OH)D₃ administration further decreased the total hepatic bile acids (Fig. 7, A and B). CDCA prefeeding decreased CA and ω-MCA and increased UDCA, CDCA, and α-MCA in the liver, and 1α(OH)D₃ administration decreased bile acids, such as CA and UDCA (Fig. 7B). Although not statistically significant because of the high variation, 1α(OH)D₃ probably decreased CDCA and α-MCA. This result indicates that the finding of decreased hepatic bile acids by 1α(OH)D₃ is not due to a secondary effect of decreased food intake. 1α(OH)D₃ treatment also probably decreased plasma DCA, CA, and UDCA in mice prefed CDCA, although these effects were not statistically significant (Fig. 7C). We examined total bile acid concentration in urine and found that 1α(OH)D₃ administration increased urinary excretion of bile acids, especially in mice prefed CDCA-supplemented chow (Fig. 7D). These findings suggest that VDR activation stimulates the excretion of bile acids via urine.

1α(OH)D₃ Treatment Induces Expression of Bile Acid Transporters.

We examined mRNA expression of genes involved in bile acid metabolism in mice treated with 1α(OH)D₃. In the liver, bile acids are synthesized from cholesterol by enzymes, such as cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) and are catabolized by detoxifying enzymes, such as CYP3A (Xie and Evans, 2001; Russell, 2003). Sodium taurocholate-cotransporting polypeptide (Ntcp) and organic anion-transporting polypeptides (Oatps) are involved in bile acid uptake at the basolateral membrane of hepatocytes, and bile acids are excreted by the canalicular bile salt export pump (Bsep) and Mrp2 (Zollner et al., 2006a). At hepatocyte basolateral membrane, Mrp3, Mrp4, and the organic solute transporter α/β (Ostα/β) play a role in alternative expression of bile acids into the system circulation. 1α(OH)D₃ treatment increased liver mRNA expression of Cyp7a1 and Ostα, although it was not effective on expression of Cyp24a1, a VDR target gene involved in vitamin D inactivation (Fig. 8 A). Expression of enzymes (Cyp8b1 and Cyp3a11) and transporters (Ntcp, Oapt1a1, Oatp1a4, Oatp1b2, Bsep, Mrp2, Mrp3, Mrp4, and Ostβ) was not significantly changed (data not shown). Because VDR does not induce transcription of a target gene in hepatocytes because of low expression of VDR (Gascon-Barré et al., 2003), the effect of 1α(OH)D₃ treatment on Cyp7a1 and Ostα expression may be through indirect mechanisms. As reported previously (Ishizawa et al., 2008; Ogura et al., 2009), 1α(OH)D₃ induced mRNA expression of Cyp24a1 in the kidney and small intestine (Fig. 8, B and C), indicating that 1α(OH)D₃ treatment effectively activates VDR in these tissues. The bile acid transporters Mrp2, Mrp4, and Ostα/β are expressed in renal tubular cells and are thought to be involved in urinary bile acid excretion (Zollner et al., 2006a). 1α(OH)D₃ increased renal mRNA expression of Mrp2, Mrp3, and Mrp4 (Fig. 8B), but not that of Ostα or Ostβ (data not shown). Asbt, Mrp3, and Ostα/β are suggested to be involved in bile acid transport in enterocytes (Zollner et al., 2006a). Treatment of mice with 1α(OH)D₃ increased intestinal mRNA expression of Asbt and Mrp4 (Fig. 8C), but not that of Mrp2, Mrp3, Ostα, or Ostβ (data not shown).

Because 1α(OH)D₃ administration increased urinary excretion of bile acids (Fig. 7D) and renal mRNA expression of bile acid transporters (Fig. 8B), we examined protein expression of renal bile acid transporters. Immunoblotting analysis showed increased renal expression of Mrp4 protein in mice treated with 1α(OH)D₃ (Fig. 8D). These findings indicate that VDR activation stimulates bile acid excretion through increased bile acid transporter expression in kidney.

Discussion

In this study, we report that 1α(OH)D₃ treatment enhances bile acid metabolism in vivo. A bile acid-supplemented diet increased liver bile acid concentrations, indicating that bile acids absorbed from the intestine accumulate in the liver. In the liver of mice fed CDCA, DCA, and LCA, the major bile acids detected were α-MCA (Fig. 3A), CA (Fig. 5A), and α-MCA (Fig. 6A), respectively. Alternatively, CA was the major bile acid in the liver of mice fed CA (Fig. 4A). These findings indicate that supplemented CDCA, DCA, and LCA are metabolized effectively but that CA is resistant to metabolism. CA and β-MCA have been reported to be major hepatic bile acids in mice (Stedman et al., 2004; Zollner et al., 2006b), and α-MCA is thought to be a precursor of β-MCA (Cherayil et al., 1963) (Fig. 9 ). Hepatic bile acids from the enterohepatic circulation may be subjected to conversion to primary bile acids, CA and α-MCA, although a detailed mechanism of bile acid metabolism in rodents remains to be elucidated. 1α(OH)D₃ treatment decreased several bile acid components in the liver of mice fed normal chow (Fig. 2A), and this effect was observed more clearly in mice fed CDCA (Fig. 3A). Similar effects of 1α(OH)D₃ on hepatic bile acid compositions were observed in mice administered 1α(OH)D₃ under fasting condition (Fig. 7B). Compared with the decrease in hepatic bile acids, such as CDCA, UDCA, and ω-MCA, in 1α(OH)D₃-treated mice, we did not detect increased bile acid components. These findings suggest that 1α(OH)D₃ treatment stimulates bile acid metabolism, particularly enhancing transport for excretion. Bile acids are excreted from hepatocytes into bile ducts by Bsep and Mrp2 (Zollner et al., 2006a) (Fig. 9). At the hepatocyte basolateral membrane, Mrp3, Mrp4, and Ostα/β play a role in the alternative excretion of bile acids into the systemic circulation. 1α(OH)D₃ treatment increased mRNA expression of Cyp7a1 and Ostα in the liver (Fig. 8A). Cyp7a1 catalyzes the rate-limiting step of the classic bile acid synthesis pathway and is negatively regulated by the bile acid receptor FXR (Makishima, 2005). The induction of Cyp7a1 may be due to a decrease in FXR-activating bile acids by excretion from hepatocytes. The bile acid transporters Mrp2, Mrp4, and Ostα/β are thought to be involved in urinary bile acid excretion (Zollner et al., 2006a; Alrefai and Gill, 2007). Although the role of Mrp3 in renal bile acid transport has not been elucidated, Mrp3 is a direct target gene of VDR (McCarthy et al., 2005). 1α(OH)D₃ treatment increased mRNA expression of kidney Mrp2, Mrp3, and Mrp4 (Fig. 8B). We also observed increased protein expression of kidney Mrp4 (Fig. 8D) and increased urinary excretion of bile acids in mice treated with 1α(OH)D₃ (Fig. 7D). These findings suggest that 1α(OH)D₃ treatment decreases hepatic bile acids by inducing bile acid transporters for urinary excretion (Fig. 9).

In DCA-fed mice, 1α(OH)D₃ treatment decreased hepatic DCA effectively but was less effective in altering CA concentration (Fig. 5A). 1α(OH)D₃ treatment was not effective in reducing accumulated CA in the liver of mice fed CA (Fig. 4A). VDR-induced mechanisms may be ineffective in elimination of accumulated CA in the liver. In contrast to hepatic CA, the plasma CA concentration was decreased after 1α(OH)D₃ treatment in mice fed CA (Fig. 4B). Plasma bile acid levels were also increased in mice fed DCA (Fig. 5B), but not in mice fed CDCA (Fig. 3B) or LCA (Fig. 6B). Increased plasma CA and DCA may be regulated by VDR-induced excretion into urine. Although LCA is an endogenous ligand for VDR (Makishima et al., 2002), 1α(OH)D₃ administration was not effective in decreasing bile acid concentrations in mice fed LCA (Fig. 6A). Although hepatic LCA and DCA were decreased by 1α(OH)D₃ treatment, accumulated HDCA and α-MCA were not affected. HDCA and α-MCA may be products of LCA metabolism by LCA-activated VDR, and VDR-induced mechanisms may not be effective in the further elimination of HDCA and α-MCA.

Bile acids are essential detergents that are required for the ingestion and intestinal absorption of hydrophobic nutrients, including vitamin D (Hofmann, 1999). VDR has dual functions as an endocrine receptor for 1,25(OH)₂D₃ and as a metabolic sensor for secondary bile acids, such as lithocholic acid (Makishima et al., 2002). Although several genes involved in bile acid metabolism, such as CYP3A4 and MRP3, have been reported to be VDR target genes (McCarthy et al., 2005; Matsubara et al., 2008), the in vivo role of VDR as a bile acid sensor may be limited. Whereas administration of high concentrations of LCA restored serum calcium levels to the normal range in vitamin D-deficient rats by increasing VDR target gene expression and bone calcium mobilization, it was not effective in rats with normal vitamin D levels (Nehring et al., 2007). These findings indicate that LCA can substitute for vitamin D in calcium homeostasis only in vitamin D-deficient rats. We demonstrated in this study that pharmacological doses of vitamin D enhance bile acid metabolism. 1,25(OH)₂D₃ increases plasma bile clearance of vitamin D₃ and 1,25(OH)₂D₃ (Gascon-Barré and Gamache, 1991). Whereas hepatocytes express low levels of VDR, biliary epithelial cells express functional VDR (Gascon-Barré et al., 2003). VDR activation in biliary epithelial cells may influence biliary excretion of bile acids as well as that of vitamin D compounds. In addition, the extracellular calcium-sensing receptor is expressed in hepatocytes and its activation enhances bile flow (Canaff et al., 2001). Increased blood calcium by vitamin D administration may also influence bile acid elimination. 1α(OH)D₃ administration is not effective in altering bile acids accumulated in bile duct-ligated mice (Ogura et al., 2009), possibly because of artificial obstruction of bile flow. Approximately 20 enzymes have been shown to be involved in bile acid synthesis in the liver (Russell, 2003). The mechanisms of catabolism and transport of bile acids in the liver and kidney remain to be elucidated. The investigation of vitamin D-regulated bile acid metabolism will be helpful for understanding bile acid metabolism, especially elimination, and in the prevention and treatment of bile acid-associated diseases.