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Activation of Constitutive Androstane Receptor Prevents Cholesterol Gallstone Formation

  • Shihai Cheng
    Affiliations
    Department of Clinical Pharmacy and Pharmacy Administration, West China School of Pharmacy, Sichuan University, Chengdu

    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Min Zou
    Affiliations
    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Qinhui Liu
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Jiangying Kuang
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Jing Shen
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Shiyun Pu
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Lei Chen
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Hong Li
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Tong Wu
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Rui Li
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Yanping Li
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Wei Jiang
    Affiliations
    Molecular Medicine Research Center, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Zhiyong Zhang
    Correspondence
    Zhiyong Zhang, Pharm.D., Department of Pharmacy, West China Hospital of Sichuan University, No. 88 South Keyuan Street, Chengdu, Sichuan, China.
    Affiliations
    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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  • Jinhan He
    Correspondence
    Address correspondence to Jinhan He, M.D., Ph.D., Department of Pharmacy, State Key Laboratory of Biotherapy and Cancer Center, and Collaborative Innovation Center of Biotherapy, No. 88 South Keyuan Street, Chengdu, Sichuan, China.
    Affiliations
    Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China

    Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China
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Open ArchivePublished:March 07, 2017DOI:https://doi.org/10.1016/j.ajpath.2016.12.013
      Cholesterol gallstone disease (CGD) is one of the most common gastrointestinal diseases. Lithogenic hepatic bile secretion precedes the formation of cholesterol gallstones. Constitutive androstane receptor (CAR), a member of nuclear family, plays an important role in cholesterol and bile acid metabolism. To examine whether activation of CAR can prevent cholesterol gallstone formation, we treated C57BL6/J mice maintained on a lithogenic diet with CAR agonist 1,4-bis-[2-(3, 5-dichlorpyridyloxy)] benzene and performed bile duct cannulation to study the dynamics of biliary lipids. We report that activation of CAR decreases the biliary cholesterol concentration and prevents CGD formation. The lower biliary cholesterol level was largely attributed to suppressed Abcg5 and Abcg8 expression in CAR-activated mice. CAR activation also promoted cholesterol conversion into bile acids by increasing the expression of Cyp7a1, a rate-limiting enzyme in bile acid biosynthesis. Activation of CAR enhanced bile acid re-absorption via increasing the expression of bile acid transporters Asbt and Ostβ in the ileum. The hepatic steatosis was also improved in the liver of CAR-activated mice. Furthermore, activation of CAR protected the mice against the liver X receptor α–sensitized CGD through suppressing the expression of Abcg5/8. Collectively, CAR plays an important role in maintaining the homeostasis of cholesterol, bile acids, and triglycerides levels, and it might be a promising therapeutic target for preventing or treating CGD.
      Cholesterol gallstone disease (CGD) is one of the most common gastroenterologic disorders. The incidence rate is increasing because of improved standards of living, a chronic high-cholesterol diet, and overnutrition. The precipitation of excess cholesterol in the bile as solid crystals is a prerequisite for cholesterol gallstone formation. In the normal physiological state, cholesterol can be solubilized in mixed micelles composed of phospholipids and bile salts, all in dynamic equilibrium. However, under certain pathophysiological conditions, especially the supersaturated bile of cholesterol, the relative excess cholesterol will precipitate as solid crystals, then aggregate, fuse, and eventually form gallstones that cause disease.
      • Claudel T.
      • Zollner G.
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      Role of nuclear receptors for bile acid metabolism, bile secretion, cholestasis, and gallstone disease.
      The main components of bile are cholesterol, phospholipids, and bile acids (BAs), which are controlled by an elaborate network of transporters. Cholesterol exits the body principally by efficient secretion into bile, mediated by the ATP-binding cassette (Abc) transporters Abcg5 and Abcg8.
      • Graf G.A.
      • Yu L.
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      • Gerard R.
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      ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion.
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      • Moschetta A.
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      • Cohen J.C.
      Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion.
      The canalicular efflux of phospholipids depends on Abcb4, also known as multidrug resistance protein 2 (Mdr2).
      • Smit J.J.
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      • van Roon M.A.
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      • Offerhaus G.J.
      • Berns A.J.
      • Borst P.
      Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.
      Abca1 is a basolateral transporter that effluxes both cholesterol and phospholipids. The primary BAs are synthesized from cholesterol in the liver controlled by multiple cytochrome P450 (Cyp) enzymes via the classical pathway (mainly mediated by Cyp7a1 and Cyp8b1) or alternative pathways (mainly controlled by Cyp7b1 and Cyp27a1), which accounts for approximately 75% and 25% of the total primary BAs, respectively.
      • de Aguiar Vallim T.Q.
      • Tarling E.J.
      • Ahn H.
      • Hagey L.R.
      • Romanoski C.E.
      • Lee R.G.
      • Graham M.J.
      • Motohashi H.
      • Yamamoto M.
      • Edwards P.A.
      MAFG is a transcriptional repressor of bile acid synthesis and metabolism.
      Cyp7a1 is the rate-limited enzyme and can be suppressed by the negative feedback regulation of BA synthesis. Cyp7a1 is down-regulated by small heterodimerizing partner (Shp) in the liver and fibroblast growth factor 15 (Fgf15) in the small intestine.
      • Kerr T.A.
      • Saeki S.
      • Schneider M.
      • Schaefer K.
      • Berdy S.
      • Redder T.
      • Shan B.
      • Russell D.W.
      • Schwarz M.
      Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis.
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • Luo G.
      • Jones S.A.
      • Goodwin B.
      • Richardson J.A.
      • Gerard R.D.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      • Inagaki T.
      • Moschetta A.
      • Lee Y.K.
      • Peng L.
      • Zhao G.
      • Downes M.
      • Yu R.T.
      • Shelton J.M.
      • Richardson J.A.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor.
      Fgf15, derived from the small intestine, circulates in the blood to the liver, binds to the Fgf receptor 4 (Fgfr4)/β-Klotho receptor on the surface of hepatocytes to repress the expression of Cyp7a1.
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • Luo G.
      • Jones S.A.
      • Goodwin B.
      • Richardson J.A.
      • Gerard R.D.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      When emptied from the gallbladder to the intestine, >90% of BAs can be reabsorbed by the apical sodium-dependent BA transporter (Asbt) and basolateral organic solute transporter α/β (Ostα/β) in the ileum and then circulates back to the liver through the hepatic uptake transporters sodium (Na+)-taurocholate cotransport protein (Ntcp) and the organic anion transporting polypeptides (Oatps).
      • Dawson P.A.
      • Lan T.
      • Rao A.
      Bile acid transporters.
      Both newly synthesized and reabsorbed BAs are secreted into the bile by the BA export pump (Bsep) and multidrug-resistance protein 2 (Mrp2). Mrp3 and Mrp4 play a role in the sinusoid export of conjugated bile salts and promote their renal secretion.
      • He J.
      • Nishida S.
      • Xu M.
      • Makishima M.
      • Xie W.
      PXR prevents cholesterol gallstone disease by regulating biosynthesis and transport of bile salts.
      Constitutive androstane receptor (CAR), primarily considered a xenobiotic sensor, is predominantly expressed in liver and small intestine.
      • Wei P.
      • Zhang J.
      • Egan-Hafley M.
      • Liang S.
      • Moore D.D.
      The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.
      It mediates specific xenobiotic induction of drug metabolism and plays an important role in stimulating BAs and bilirubin detoxification and elimination.
      • Qatanani M.
      • Moore D.D.
      CAR, the continuously advancing receptor, in drug metabolism and disease.
      CAR activation can increase the elimination of cholesterol by its conversion into BAs and subsequent fecal excretion, with positive consequences on reverse cholesterol transport and whole-body cholesterol homeostasis.
      • Sberna A.L.
      • Assem M.
      • Gautier T.
      • Grober J.
      • Guiu B.
      • Jeannin A.
      • Pais de Barros J.P.
      • Athias A.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation stimulates faecal bile acid excretion and reverse cholesterol transport in mice.
      Despite the role of CAR in the elimination of BAs in liver, a recent study showed that CAR activation still maintains the biliary excretion of BAs.
      • Lickteig A.J.
      • Csanaky I.L.
      • Pratt-Hyatt M.
      • Klaassen C.D.
      Activation of constitutive androstane receptor (CAR) in mice results in maintained biliary excretion of bile acids despite a marked decrease of bile acids in liver.
      In addition, CAR plays an important role in preventing atherosclerosis.
      • Sberna A.L.
      • Assem M.
      • Gautier T.
      • Grober J.
      • Guiu B.
      • Jeannin A.
      • Pais de Barros J.P.
      • Athias A.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation stimulates faecal bile acid excretion and reverse cholesterol transport in mice.
      • Sberna A.L.
      • Assem M.
      • Xiao R.
      • Ayers S.
      • Gautier T.
      • Guiu B.
      • Deckert V.
      • Chevriaux A.
      • Grober J.
      • Le Guern N.
      • Pais de Barros J.P.
      • Moore D.D.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation decreases plasma apolipoprotein B-containing lipoproteins and atherosclerosis in low-density lipoprotein receptor-deficient mice.
      However, the effect of CAR activation on the pathogenesis of CGD remains unclear.
      In this study, we examined activation of the nuclear receptor CAR in preventing cholesterol gallstone formation in mice. We treated C57BL6/J mice with the CAR agonist 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene (TCPOBOP) and simultaneously fed them with a lithogenic diet (LGD). Activation of CAR decreased the biliary cholesterol concentration and prevented CGD formation. The lower biliary cholesterol level was largely attributed to suppressed Abcg5 and Abcg8 expression in CAR-activated mice. CAR activation also promoted cholesterol conversion to BAs by increasing Cyp7a1 expression. Modulation of CAR activity might be a therapeutic target for CGD formation.

      Materials and Methods

       Animals, LGD, and Drug Treatment

      All animal care and experimental procedures complied with the guidelines of the Animal Care and Utilization Committee of the institute and were approved by this committee. Eight-week-old C57BL6/J male mice were purchased from Sichuan University Laboratory Animal center (Chengdu, China), housed with a 12:12-hour light/dark cycle, and permitted ad libitum consumption of water and a standard mouse diet. When necessary, mice received an intraperitoneal injection of vehicle or the CAR ligand TCPOBOP (sc-203291; Santa Cruz Biotechnology, Santa Cruz, CA) 1.0 mg/kg body weight, once per week. The vehicle- or TCPOBOP-treated mice were fed a chow diet (CD) or LGD (Harlan Teklad TD.88051) containing approximately 15.8% fat, 1.25% cholesterol, 0.5% sodium cholate, and 7.5% casein for 2 weeks. The TCPOBOP was injected 1 day before the start of LGD feeding, and the drug treatment was continued until the end of the experiments. After 2 weeks, mice were sacrificed after being deprived of food overnight, and gallbladders were removed by ligation of the cystic duct. Gallbladder bile and contents were gently squeezed into tubes after cholecystectomy and immediately examined under a polarized microscope for cholesterol crystals and gallstones. The remaining bile was stored at −20°C for biochemical analysis. Livers, gallbladders, and small intestines were frozen in liquid nitrogen and stored at −80°C. To demonstrate whether CAR activation can prevent liver X receptor (LXR)α-sensitized gallstone formation, mice were treated with intraperitoneal injection of TCPOBOP (once a week) and daily gavage of 30 mg/kg GW3965, and then fed with LGD for 2 weeks.

       Bile Duct Cannulation and Collection of Hepatic Bile

      Bile duct cannulation was performed as described.
      • Biddinger S.B.
      • Haas J.T.
      • Yu B.B.
      • Bezy O.
      • Jing E.
      • Zhang W.
      • Unterman T.G.
      • Carey M.C.
      • Kahn C.R.
      Hepatic insulin resistance directly promotes formation of cholesterol gallstones.
      Briefly, mice were deprived of food overnight and were anesthetized with isoflurane. The lower end of the common bile duct of mice was ligated, and the common bile duct was cannulated with a PE-10 polyethylene catheter below the entrance of the cystic duct. The cystic duct was then doubly ligated, and a cholecystectomy was performed. Hepatic bile was collected in 0.2-mL tubes by gravity for 1 hour.

       Histologic Evaluation and Biochemical Analysis of Bile, Hepatic Bile, Serum, Urine, and Feces

      Livers and gallbladders were fixed in 10% formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin. Frozen sections (6 μm) were used for Oil Red O staining. Images were captured by a microscope (Nikon, Tokyo, Japan). Biliary, hepatic bile, and serum BA, cholesterol, and triglyceride levels were measured with assay kits from Biosino (Beijing, China). Phospholipids were measured by using a kit from Wako (296-63801; Osaka, Japan). The cholesterol saturation index (CSI) of bile and hepatic bile was calculated according to Carey's critical tables.
      • Carey M.C.
      Critical tables for calculating the cholesterol saturation of native bile.
      To determine urinary and fecal BA excretion, mice were individually housed, and urine and feces were collected. Fecal BAs were extracted as described.
      • Yu C.
      • Wang F.
      • Kan M.
      • Jin C.
      • Jones R.B.
      • Weinstein M.
      • Deng C.X.
      • McKeehan W.L.
      Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4.
      Briefly, 0.5 g of dried feces was extracted in 10 mL of 75% ethanol at about 50°C for 2 hours, followed by centrifugation at 1500 × g for 10 minutes. BAs were then measured enzymatically in the resulting supernatant fluid. The daily feces output (in g/d per 100 g body weight) and fecal BA content (in μmol/d per 100 g body weight) were used to calculate the rate of BA excretion. The total BA pool size was determined as BA content of the small intestine, the gallbladder, the liver, and their contents.
      • Yu C.
      • Wang F.
      • Kan M.
      • Jin C.
      • Jones R.B.
      • Weinstein M.
      • Deng C.X.
      • McKeehan W.L.
      Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4.
      After the mice were weighed, anesthetized, and exsanguinated, the fresh organs were collected, minced together, and extracted with 75% ethanol at about 50°C for 2 hours. The extract was centrifuged, BAs were determined enzymatically, and the pool size was expressed as micromoles of BAs per 100 g of body weight.

       Hepatic Lipid Analysis

      To analyze hepatic triglycerides and cholesterol levels, 150 mg of liver tissue was lysated into 1 mL of phosphate-buffered saline by homogenization. Total lipids were extracted by chloroform/methanol (2:1, v/v)
      • Folch J.
      • Lees M.
      • Sloane Stanley G.H.
      A simple method for the isolation and purification of total lipides from animal tissues.
      and dissolved in 1% Triton-X100 in ethanol. Triglyceride and cholesterol levels were measured by commercial assay kits from Biosino.

       Hepatic Total BA Analysis

      Hepatic total BAs were extracted as described.
      • Ma K.
      • Xiao R.
      • Tseng H.T.
      • Shan L.
      • Fu L.
      • Moore D.D.
      Circadian dysregulation disrupts bile acid homeostasis.
      Briefly, liver tissues were homogenized in 75% ethanol and incubated at 50°C for 2 hours. The extracted supernatant fluids were assayed by using commercial assay kits from Biosino.

       Western Blot Analysis

      Liver and ileum were lyzed in lysis buffer and subjected to Western blot analysis with the following primary antibodies: anti-Abcg5 (dilution 1:1000; sc-25796), anti-Abcg8 (dilution 1:1000; sc-30111), and anti-Fgf15 (dilution 1:1000; sc-27177) from Santa Cruz Biotechnology; anti-Cyp7a1 (dilution 1:1000; ab-65596) from Abcam (Cambridge, MA); anti–β-actin (dilution 1:4000, A-4700) from Sigma-Aldrich (St. Louis, MO). Blots were visualized by LI-COR (Lincoln, NE) Odyssey System. Quantitative determination of band intensity was conducted by Image Studio analysis software version 4.0 (LI-COR).

       Quantitative Real-Time RT-PCR

      Total hepatic and ileal RNA was extracted with TRIzol reagent and reverse-transcribed into complementary DNA (RR037A; TaKaRa, Kyoto, Japan). Quantitative real-time PCR (no. 170-8882AP; Bio-Rad, Hercules, CA) involved the SYBR Green-based assay with the CFX96 Real-Time system.

       Statistical Analysis

      Data are expressed as means ± SEM. One-way analysis of variance (Tukey's test) involved the use of GraphPad Prism version 5.0 (GraphPad Inc., San Diego, CA). P < 0.05 was considered statistically significant.

      Results

       Activation of CAR Prevents CGD in C57BL/6J Mice

      To determine the effect of CAR on lithogenesis, C57BL/6J mice were fed a CD or LGD for 2 weeks with or without the CAR agonist TCPOBOP. With a CD, vehicle- or TCPOBOP-treated mice showed no cholesterol crystals (Supplemental Figure S1A). However, with the LGD, the incidence of gallstones was 94.7% in vehicle-treated mice but decreased to 33.3% in TCPOBOP-treated mice (Figure 1A). The gallbladder in TCPOBOP-treated mice showed transparent bile, almost without any impurities compared with vehicle-treated mice that were filled with gallstones (Figure 1B). Polarizing light microscopy examination of bile revealed aggregated cholesterol crystals in vehicle-treated gallbladders, whereas TCPOBOP-treated mice were generally free of cholesterol precipitates (Figure 1C). Two-week LGD treatment caused inflammation in vehicle-treated mice, including thickening of gallbladder wall and submucosal vasodilatation. TCPOBOP treatment ameliorated these signs of inflammation (Figure 1D).
      Figure thumbnail gr1
      Figure 1Activation of constitutive androstane receptor (CAR) prevents cholesterol gallstone disease (CGD) in C57BL/6J mice. A: Proportion of crystals in male C57BL/6J mice treated with vehicle (VEH) and TC (1.0 mg/kg body weight, once per week). All mice were fed a LGD for 2 weeks. The number of mice is indicated. B: Gross appearance of representative gallbladders. C: Polarizing light microscopy of cholesterol crystals. D: Histologic examination of gallbladder by H&E staining. The white arrow indicates the thickness of the gallbladder wall. The black arrow indicates submucosal vasodilatation. n = 5 per group. Scale bar = 50 μm (D). Original magnification, ×100 (C). H&E, hematoxylin and eosin; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.

       Activation of CAR Alters the Biochemical Composition of the Gallbladder and Hepatic Bile for Antilithogenesis Effects

      The protective effect of CAR activation on CGD prompted us to detect the biliary biochemical composition in the gallbladder. TCPOBOP-treated mice consuming a CD had similar biliary cholesterol, BAs, and phospholipid levels as vehicle-treated mice (Supplemental Figure S1, B–D). However, with 2 weeks of LGD feeding, TCPOBOP-treated mice showed significantly decreased biliary cholesterol concentration (Figure 2A) and slightly decreased phospholipid levels (Figure 2B). The biliary BA concentration was comparable in vehicle- and TCPOBOP-treated mice (Figure 2C). Despite its protective effect against lithogenesis, TCPOBOP treatment did not decrease biliary CSI in this phenotype (Figure 2D).
      Figure thumbnail gr2
      Figure 2Activation of CAR alters the biochemical composition of the gallbladder and hepatic bile for antilithogenesis effects. A–D: Biliary concentrations of cholesterol (A), phospholipids (B), bile acids (C), and cholesterol saturation index (CSI) (D) of gallbladder bile. Male C57BL/6J mice treated with VEH and TC (1.0 mg/kg body weight, once per week). All mice were fed an LGD for 2 weeks. E–I: Hepatic bile flow rate (E) and secretary rate of cholesterol (F), phospholipids (G), bile acids (H), and CSI (I). Bile was collected from the common bile duct of 10-week-old mice. J: Bile acid pool size. Data are expressed as means ± SEM. n = 5 per group (A–D); n = 7 for each group (E–I); n = 5 for each group (J). P < 0.05, ∗∗P < 0.01. BW, body weight; CAR, constitutive androstane receptor; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.
      To further investigate the effect of CAR activation to prevent cholesterol gallstone formation, we performed bile duct cannulation to clarify the dynamics of bile flow and fresh biliary lipid outputs after a LGD. TCPOBOP did not change the bile flow but substantially the decreased biliary cholesterol secretion rate (Figure 2, E and F). The decreased cholesterol secretion rate was accompanied by a mild decrease in phospholipid secretion (Figure 2G) and constant BA output (Figure 2H). As a result, TCPOBOP treatment decreased CSI in hepatic bile flow (Figure 2I). The decreased hepatic biliary CSI provided a pathophysiological explanation for the protective role of CAR activation in the lithogenic phenotype. Consistent with the similar secretion rate of BAs, total pool size of BAs in the mice of the two groups were equally comparable (Figure 2J).

       Activation of CAR Decreases Canalicular Efflux of Cholesterol and Increases Cyp7a1 Expression in Liver

      To gain insights into the molecular mechanisms by which CAR activation prevented cholesterol gallstone formation in C57BL6/J mice, first, we evaluated the expression levels of the genes involved in cholesterol, phospholipid, and BA transport. The mRNA expression of Cyp2b10, a target gene of CAR, was highly induced with both CD and LGD feeding after TCPOBOP treatment, indicating successful CAR activation (Supplemental Figure S2). In the liver, cholesterol is secreted by the canalicular transporters Abcg5 and Abcg8 or is converted into BAs via classic and alternative pathways that generate approximately 75% and 25% of the total primary BAs, respectively.
      • Russell D.W.
      The enzymes, regulation, and genetics of bile acid synthesis.
      Activation of CAR significantly decreased the mRNA and protein expression of Abcg5 and Abcg8 induced by the LGD (Figure 3, A, F and G), which agreed with reduced cholesterol levels in the gallbladder and hepatic bile flow. When profiling cholesterol-metabolizing genes, we found the expression of cholesterol 7a-hydroxylase (Cyp7a1), a key gene involved in the classic BA synthesis pathway, was higher in the liver of TCPOBOP-treated mice (Figure 3B). LGD feeding suppressed Cyp7a1 expression. However, this suppression of Cyp7a1 expression was markedly reversed by CAR activation. Western blot analysis also demonstrated a significant increase in Cyp7a1 protein expression in the liver of TCPOBOP-treated mice (Figure 3, F and G). The expression of other BA synthetic genes such as Cyp8b1, Cyp7b1, and Cyp27a1 were not changed. This finding agreed with a previous report that overexpression of Cyp7a1 prevented CGD.
      • Miyake J.H.
      • Duong-Polk X.T.
      • Taylor J.M.
      • Du E.Z.
      • Castellani L.W.
      • Lusis A.J.
      • Davis R.A.
      Transgenic expression of cholesterol-7-alpha-hydroxylase prevents atherosclerosis in C57BL/6J mice.
      Figure thumbnail gr3
      Figure 3Activation of CAR decreases canalicular efflux of cholesterol and increases Cyp7a1 expression. A and B: Real-time PCR analysis of hepatic mRNA expression of cholesterol canalicular transporters (A) and bile acid synthesis enzyme genes (B). C: Ileal mRNA expression of Shp and Fgf15. D: Hepatic mRNA expression of Fgfr4 and β-klotho. E: Hepatic level of cholesterol. F: Expression of hepatic Abcg5/8, Cyp7a1, and ileal Fgf15 proteins were measured by Western blot analysis. G and H: Quantitative determination of band intensity in panel F. Data are expressed as means ± SEM. n = 5 to 6 for each group. P < 0.05, ∗∗P < 0.01. CAR, constitutive androstane receptor; CD, chow diet; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.
      To understand the mechanism of reversed Cyp7a1 expression with TCPOBOP treatment, we measured the expression of Shp and ileal Fgf15, two farnesoid X receptor (FXR) target genes that play an important role in negative feedback regulation of Cyp7a1 expression.
      • Kerr T.A.
      • Saeki S.
      • Schneider M.
      • Schaefer K.
      • Berdy S.
      • Redder T.
      • Shan B.
      • Russell D.W.
      • Schwarz M.
      Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis.
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • Luo G.
      • Jones S.A.
      • Goodwin B.
      • Richardson J.A.
      • Gerard R.D.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      • Inagaki T.
      • Moschetta A.
      • Lee Y.K.
      • Peng L.
      • Zhao G.
      • Downes M.
      • Yu R.T.
      • Shelton J.M.
      • Richardson J.A.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor.
      In the liver, Shp expression was unchanged (Supplemental Figure S3), suggesting the increased Cyp7a1 expression was not caused by hepatic expression of Shp. The hepatic BA levels were not changed either (Supplemental Figure S4A). In the ileum, the mRNA levels of Shp and Fgf15 were significantly decreased after TCPOBOP treatment (Figure 3C). Fgf15 is secreted from the intestine into the blood circulation, then binds to the hepatic Fgfr4, and suppresses Cyp7a1 expression.
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • Luo G.
      • Jones S.A.
      • Goodwin B.
      • Richardson J.A.
      • Gerard R.D.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      Activation of CAR had little effect on Fgfr4 expression with the basal and LGD conditions (Figure 3D). However, the expression of β-klotho, a coreceptor of Fgfr4, was significantly inhibited in TCPOBOP-treated mice with an LGD (Figure 3D). Consistent with the mRNA level, the protein expression of Fgf15 was also decreased as illustrated by Western blot analysis (Figure 3, F and H). The decreased Fgf15 and β-klotho expression may coordinately relieve the suppression of this signal on Cyp7a1 transcription. Consistent with higher conversion from cholesterol into BAs, lipid analysis revealed that the hepatic cholesterol level was significantly decreased (Figure 3E).

       Activation of CAR Induces the Systematic Expression of Genes Involved in BA Transport

      BAs are controlled by an elaborate network of BA transporters. In the biliary secretion system, TCPOBOP treatment had no effect on the expression of Bsep, regardless of diet, but significantly induced Mrp2 expression with a CD (Figure 4A). For sinusoid export transporters, TCPOBOP induced Mrp3 and Mrp4 mRNA levels 2.4- and 23.9-fold with a CD, respectively. When challenged with an LGD, Mrp3 and Mrp4 levels were increased 1.7- and 13.4-fold, respectively, in TCPOBOP-treated mice. This finding is consistent with the notion that Mrp4 was a target gene of CAR.
      • Assem M.
      • Schuetz E.G.
      • Leggas M.
      • Sun D.
      • Yasuda K.
      • Reid G.
      • Zelcer N.
      • Adachi M.
      • Strom S.
      • Evans R.M.
      • Moore D.D.
      • Borst P.
      • Schuetz J.D.
      Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice.
      With a CD, TCPOBOP significantly up-regulated the expression of Oatp2 and sodium (Na+)-taurocholate cotransport protein, two transporters for hepatic uptake of BAs. However, activation of CAR had no effect on these two genes after an LGD. TCPOBOP treatment robustly inhibited the expression of Oatp1 regardless of the diet. Consistent with the induction of Mrp3/4, the urinary BA outputs (Figure 4B) were drastically increased in LGD-fed TCPOBOP-treated mice; however, the serum BA levels were unchanged (Figure 4C).
      Figure thumbnail gr4
      Figure 4Pharmacologic activation of CAR alters relative mRNA levels of genes involved in bile acid transport. A: Real-time PCR analysis of hepatic expression of genes involved in bile acids transport. B: Urinary level of bile acids. C: Serum level of bile acids. D: Ileal expression of bile acid transporters. E: Protein expression of ileal bile acid transporters was measured by Western blot analysis. F: Quantitative determination of band intensity in E. G: Fecal output of bile acids. Data are expressed as means ± SEM. n = 5 to 6 for each group. P < 0.05, ∗∗P < 0.01. CAR, constitutive androstane receptor; CD, chow diet; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.
      More than 90% of BA is reabsorbed in the ileum; therefore, we also profiled the expression of intestinal genes involved in BA transport. The LGD significantly suppressed the expression of Asbt regardless of drug treatment (Figure 4D). TCPOBOP treatment did not affect Asbt mRNA expression compared with vehicle treatment. TCPOBOP increased Ostβ mRNA expression with a CD but not with a LGD. The LGD markedly decreased the Mrp3 mRNA level in TCPOBOP-treated mice. The mRNA expression of Ostα was unchanged among the four groups of mice. Although no changes were observed in the mRNA expression of these ileal BA transporters, Western blot analysis revealed that TCPOBOP dramatically increased Asbt and Ostβ protein expression in LGD-fed mice (Figure 4, E and F). It indicated that activation of CAR increased BA reabsorption in the ileum of the mice consuming a LGD. Consistent with these facts, we also found the average fecal BA excretion was significantly reduced in TCPOBOP-treated mice at day 9 of the LGD (Figure 4G).

       Activation of CAR Decreases the Expression of Genes Involved in Phospholipid Transport

      Although activation of CAR protected mice against CGD, we observed decreased phospholipid secretion into the gallbladder (Figure 2G). The level of Abcb4, a major phospholipid transporter, was markedly decreased with both a CD and LGD in TCPOBOP-treated animals (Figure 5). The level of Abca1 was slightly increased with a CD but unchanged with an LGD.
      Figure thumbnail gr5
      Figure 5Activation of CAR decreases the expression of phospholipid transporters. Real-time PCR analysis of hepatic mRNA expression of phospholipid transporters. Data are expressed as means ± SEM. n = 5 to 6 for each group P < 0.05, ∗∗P < 0.01. CAR, constitutive androstane receptor; CD, chow diet; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.

       Activation of CAR Decreases the Expression of Genes Involved in Lipogenesis

      CAR is an antiobesity nuclear receptor.
      • Gao J.
      • He J.
      • Zhai Y.
      • Wada T.
      • Xie W.
      The constitutive androstane receptor is an anti-obesity nuclear receptor that improves insulin sensitivity.
      In our study, TCPOBOP treatment significantly decreased the expression of a gene involved in lipogenesis, sterol regulatory element–binding protein (Srebp-1c), regardless of diet. The expression of stearoyl-CoA desaturase-1 (Scd-1) was unregulated with a CD but markedly suppressed with an LGD (Figure 6A). TCPOBOP did not change hepatic tissue architecture in mice maintained on a CD (Figure 6B). With an LGD, the livers of vehicle-treated mice were filled with large lipid droplets by evidence of both macrovesicular and microvesicular steatosis. However, TCPOBOP-treated mice showed less lipid accumulation, which was further illustrated by Oil Red O staining of the liver (Figure 6C). Biochemical analysis revealed that, as expected, LGD feeding increased hepatic triglyceride levels, and TCPOBOP treatment decreased hepatic triglyceride content both with a CD and LGD compared with vehicle-treated mice (Figure 6D). Similarly, serum triglycerides were decreased in TCPOBOP-treated mice, whereas the circulating cholesterol was slightly increased (Supplemental Figure S4B).
      Figure thumbnail gr6
      Figure 6Pharmacologic activation of CAR decreases hepatic steatosis. A: Real-time PCR analysis of hepatic mRNA expression of genes involved in lipogenesis. B: Representative images of H&E-stained sections of livers. Arrows indicate macrovesicular and microvesicular steatosis. C: Oil Red O staining of liver sections. D: Analysis of hepatic triglycerides level. Data are expressed as means ± SEM. n = 5 to 6 for each group (A and D). P < 0.05, ∗∗P < 0.01. Scale bars = 50 μm (B and C). CAR, constitutive androstane receptor; CD, chow diet; H&E, hematoxylin and eosin; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.

       Activation of CAR Prevents the LXRα-Sensitized Gallstone Disease

      It was reported that activation of LXRα sensitized mice to gallbladder cholesterol crystallization by increasing cholesterol transport to the gallbladder.
      • Uppal H.
      • Zhai Y.
      • Gangopadhyay A.
      • Khadem S.
      • Ren S.
      • Moser J.A.
      • Xie W.
      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      We were interested in whether CAR activation can attenuate gallbladder cholesterol crystallization caused by LXRα agonist in vivo. Activation of LXRα sensitized mice to gallstone disease, similar to a previous report.
      • Uppal H.
      • Zhai Y.
      • Gangopadhyay A.
      • Khadem S.
      • Ren S.
      • Moser J.A.
      • Xie W.
      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      However, TCPOBOP treatment attenuated the aggravated gallbladder cholesterol crystallization induced by GW3965 (Figure 7, A and B). The cholesterol transporters Abcg5 and Abcg8 were highly induced by LXRα (Figure 7C). However, this induction was significantly inhibited by CAR activation (Figure 7C).
      Figure thumbnail gr7
      Figure 7Activation of CAR prevents the LXRα-sensitized gallstone disease. A: Gross appearance of representative gallbladders in C57BL/6J mice treated with VEH, TC (1.0 mg/kg body weight, once per week), and/or GW (30 mg/kg body weight, once per day). All mice were fed a LGD for 2 weeks. B: Polarizing light microscopy of cholesterol crystals. C: Real-time PCR analysis of hepatic mRNA expression of cholesterol transporters Abcg5 and Abcg8. Data are expressed as means ± SEM. n = 5 for each group. P < 0.05, ∗∗P < 0.01. Original magnification, ×100 (B). CAR, constitutive androstane receptor; CD, chow diet; GW, GW3965; LGD, lithogenic diet; LXRα, liver X receptor α; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.

      Discussion

      Previous studies have shown that CAR played important roles in reverse cholesterol transport, atherosclerosis, and BA elimination.
      • Sberna A.L.
      • Assem M.
      • Gautier T.
      • Grober J.
      • Guiu B.
      • Jeannin A.
      • Pais de Barros J.P.
      • Athias A.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation stimulates faecal bile acid excretion and reverse cholesterol transport in mice.
      • Sberna A.L.
      • Assem M.
      • Xiao R.
      • Ayers S.
      • Gautier T.
      • Guiu B.
      • Deckert V.
      • Chevriaux A.
      • Grober J.
      • Le Guern N.
      • Pais de Barros J.P.
      • Moore D.D.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation decreases plasma apolipoprotein B-containing lipoproteins and atherosclerosis in low-density lipoprotein receptor-deficient mice.
      • Wagner M.
      • Halilbasic E.
      • Marschall H.U.
      • Zollner G.
      • Fickert P.
      • Langner C.
      • Zatloukal K.
      • Denk H.
      • Trauner M.
      CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice.
      However, the role of CAR in CGD is still unknown. In this study, we showed that activation of CAR prevented cholesterol gallstone formation in mice consuming an LGD. The protective role was associated with the markedly decreased secretion of cholesterol into the gallbladder. Although the CSI of bile in the gallbladder did not change, the prevalence of cholesterol gallstones was markedly decreased in TCPOBOP-treated mice. In the liver, the relative greatly decreased biliary cholesterol level and slightly decreased phospholipid secretion, combined with unchanged BA secretion, contributed to the decreased CSI of hepatic fresh bile, thus being antilithogenic. Activation of CAR also protected mice against LXRα-sensitized gallstone formation. Although we showed that the activation of CAR by TCPOBOP had beneficial effects with the CGD, this study is limited because CAR knockout mice were not used to evaluate whether TCPOBOP effect is CAR dependent. In addition, CAR knockout mice should be used to determine whether ablation of this receptor sensitized mice to gallstone disease in the future.
      Recently, the role of CAR in cholesterol transport was highlighted, especially the role in reverse cholesterol transport and atherosclerosis. Therefore, CAR may play an important role in whole-body cholesterol homeostasis. Indeed, activation of CAR can decrease high-density lipoprotein cholesterol level, the main component in the reverse transport of cholesterol to the liver.
      • Masson D.
      • Qatanani M.
      • Sberna A.L.
      • Xiao R.
      • Pais de Barros J.P.
      • Grober J.
      • Deckert V.
      • Athias A.
      • Gambert P.
      • Lagrost L.
      • Moore D.D.
      • Assem M.
      Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice.
      However, CAR activation can also stimulate reverse cholesterol transport and can protect mice against atherosclerosis along with decreased biliary cholesterol level by repressing the expression of the Abcg5/8 cholesterol transporters.
      • Sberna A.L.
      • Assem M.
      • Gautier T.
      • Grober J.
      • Guiu B.
      • Jeannin A.
      • Pais de Barros J.P.
      • Athias A.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation stimulates faecal bile acid excretion and reverse cholesterol transport in mice.
      In agreement, we found decreased biliary secretion of cholesterol into the gallbladder of TCPOBOP-treated mice as well as the significantly suppressed mRNA and protein levels of Abcg5/8, which are required for essential secretion of cholesterol into bile. In addition, we observed a slight reduction in phospholipid secretion in bile of TCPOBOP-treated mice. This finding could be explained by the decreased biliary export of cholesterol. Abcg5/8 required Abcb4 for efficient secretion of cholesterol from liver into bile.
      • Langheim S.
      • Yu L.
      • von Bergmann K.
      • Lutjohann D.
      • Xu F.
      • Hobbs H.H.
      • Cohen J.C.
      ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile.
      We also found that altered hepatic secretion of biliary lipids is a prerequisite for cholesterol supersaturated bile in the gallbladder and subsequent cholesterol gallstone formation.
      • Wang D.Q.
      • Lammert F.
      • Paigen B.
      • Carey M.C.
      Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology of biliary lipid secretion.
      Therefore, the decreased CSI of the hepatic bile in TCPOBOP-treated mice precedes the effect of CAR on protecting cholesterol gallstone formation, for a pathophysiological explanation of the protective role of CAR activation in the lithogenic phenotype.
      We found that an LGD markedly inhibited the expression of Cyp7a1 (the rate-limiting enzyme of BA biosynthesis), which was consistent with a previous study.
      • Uppal H.
      • Zhai Y.
      • Gangopadhyay A.
      • Khadem S.
      • Ren S.
      • Moser J.A.
      • Xie W.
      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      This finding is due to the presence of BAs in the LGD, which may signal the negative feedback mechanism of regulating BAs.
      • Kim I.
      • Ahn S.H.
      • Inagaki T.
      • Choi M.
      • Ito S.
      • Guo G.L.
      • Kliewer S.A.
      • Gonzalez F.J.
      Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine.
      The expression of Cyp7a1 was significantly higher in TCPOBOP-treated mice than in vehicle-treated mice regardless of diet. Miao et al
      • Miao J.
      • Fang S.
      • Bae Y.
      • Kemper J.K.
      Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha.
      reported that activation of CAR inhibited Cyp7a1 expression, which is opposite to our results. We reasoned this discrepancy may be the result of different mouse strains used. BALB/c mice were used in their study, whereas we used C57BL/6J mice. It is also possible that a different dosage of TCPOBOP might have a different effect on Cyp7a1 expression (TCPOBOP 0.3 mg/kg body weight in their study and 1.0 mg/kg body weight in ours). Cyp7a1 is negatively regulated by Shp signaling in the liver
      • Kir S.
      • Zhang Y.
      • Gerard R.D.
      • Kliewer S.A.
      • Mangelsdorf D.J.
      Nuclear receptors HNF4alpha and LRH-1 cooperate in regulating Cyp7a1 in vivo.
      and Fgf15 from the intestine.
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • Luo G.
      • Jones S.A.
      • Goodwin B.
      • Richardson J.A.
      • Gerard R.D.
      • Repa J.J.
      • Mangelsdorf D.J.
      • Kliewer S.A.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      We found the hepatic expression of Shp did not changed, which agreed with the constant hepatic BA level (Supplemental Figure S4A). However, the ileal expression of Shp and Fgf15 was significantly suppressed by the TCPOBOP treatment. The lower level of Fgf15 could weaken negative feedback and thereby resulted in higher expression of Cyp7a1.
      Previous studies have shown that CAR played an important role in xenobiotic molecular elimination and also controlled the metabolism of endogenous compounds such as BAs and triglycerides.
      • Wagner M.
      • Halilbasic E.
      • Marschall H.U.
      • Zollner G.
      • Fickert P.
      • Langner C.
      • Zatloukal K.
      • Denk H.
      • Trauner M.
      CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice.
      • Guo G.L.
      • Lambert G.
      • Negishi M.
      • Ward J.M.
      • Brewer Jr., H.B.
      • Kliewer S.A.
      • Gonzalez F.J.
      • Sinal C.J.
      Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity.
      • Maglich J.M.
      • Lobe D.C.
      • Moore J.T.
      The nuclear receptor CAR (NR1I3) regulates serum triglyceride levels under conditions of metabolic stress.
      Despite the activation of Cyp7a1, our TCPOBOP-treated mice showed a constant biliary level of BAs, which was due to the unchanged Bsep and Mrp2 expression that mediated canalicular BA efflux. With increased synthesis of BAs in the liver, CAR activation decreased hepatic BA uptake. TCPOBOP treatment robustly suppressed the expression of Oatp1, thereby indicating a decreased hepatic Na+-independent import of BAs. The increased BA synthesis but constant biliary BA level in TCPOBOP-treated mice might be due to the overwhelming up-regulation of Mrp4 (ABCC4), a target gene of CAR, which is responsible for the sinusoidal efflux of BAs from the hepatocytes into the space of Disse.
      • Assem M.
      • Schuetz E.G.
      • Leggas M.
      • Sun D.
      • Yasuda K.
      • Reid G.
      • Zelcer N.
      • Adachi M.
      • Strom S.
      • Evans R.M.
      • Moore D.D.
      • Borst P.
      • Schuetz J.D.
      Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice.
      • Mennone A.
      • Soroka C.J.
      • Cai S.Y.
      • Harry K.
      • Adachi M.
      • Hagey L.
      • Schuetz J.D.
      • Boyer J.L.
      Mrp4-/- mice have an impaired cytoprotective response in obstructive cholestasis.
      When mice were maintained on a western-type diet, Sberna et al
      • Sberna A.L.
      • Assem M.
      • Gautier T.
      • Grober J.
      • Guiu B.
      • Jeannin A.
      • Pais de Barros J.P.
      • Athias A.
      • Lagrost L.
      • Masson D.
      Constitutive androstane receptor activation stimulates faecal bile acid excretion and reverse cholesterol transport in mice.
      found that activation of CAR by TCPOBOP stimulated fecal BA excretion. In our results, TCPOBOP treatment suppressed the fecal excretion of BAs. In their study, CAR activation increased the sinusoidal efflux and enhanced the hepatic BA uptake. In addition, the canalicular membrane secretion of BAs was significantly increased, which is essential to the stimulated fecal excretion of BAs. In our study, we found that the protein levels of ileal BA transporters Asbt and Ostβ were significantly higher in TCPOBOP-treated mice (Figure 4, E and F). The higher expression of Asbt and Ostβ will increase the reabsorption of BA and subsequently decrease fecal BA excretion. We reasoned this discrepancy might be due to the presence of cholate in the diet. The cholate will signal a negative feedback on Asbt, Ostα, and Ostβ expression. However, this negative feedback might be relieved when CAR is activated.
      Several nuclear receptors affect lithogenesis and include FXR, LXR, pregnane X receptor (PXR), and estrogen receptors.
      • He J.
      • Nishida S.
      • Xu M.
      • Makishima M.
      • Xie W.
      PXR prevents cholesterol gallstone disease by regulating biosynthesis and transport of bile salts.
      • Uppal H.
      • Zhai Y.
      • Gangopadhyay A.
      • Khadem S.
      • Ren S.
      • Moser J.A.
      • Xie W.
      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      • Moschetta A.
      • Bookout A.L.
      • Mangelsdorf D.J.
      Prevention of cholesterol gallstone disease by FXR agonists in a mouse model.
      • Wang H.H.
      • Afdhal N.H.
      • Wang D.Q.
      Estrogen receptor alpha, but not beta, plays a major role in 17beta-estradiol-induced murine cholesterol gallstones.
      • de Bari O.
      • Wang T.Y.
      • Liu M.
      • Portincasa P.
      • Wang D.Q.
      Estrogen induces two distinct cholesterol crystallization pathways by activating ERalpha and GPR30 in female mice.
      We found that CAR played an antilithogenic role in lithogenesis similar to FXR and PXR. However, the mechanisms were quite different. CAR activation played a central role in canalicular membrane transport of cholesterol, whereas FXR activation had profound effects on canalicular BA and phospholipid secretion.
      • Moschetta A.
      • Bookout A.L.
      • Mangelsdorf D.J.
      Prevention of cholesterol gallstone disease by FXR agonists in a mouse model.
      In addition, TCPOBOP induced the conversion from cholesterol into BAs, whereas FXR suppressed the expression of Cyp7a1.
      Here, we demonstrated that activation of CAR by TCPOBOP prevents cholesterol gallstone formation via inhibiting cholesterol transport from the liver to the gallbladder. PXR, a sister xenobiotic receptor of CAR, also prevents CGD.
      • He J.
      • Nishida S.
      • Xu M.
      • Makishima M.
      • Xie W.
      PXR prevents cholesterol gallstone disease by regulating biosynthesis and transport of bile salts.
      Like in the case of drug metabolism, the function of these two xenobiotic receptors in CGD is also conserved. CAR and PXR share many of their target genes and indirectly regulated genes. Activation of PXR prevented CGD by decreasing biliary concentrations of cholesterol and increasing the level of bile salts. In contrast to PXR, activation of CAR prevented CGD by decreasing biliary concentrations of cholesterol without changing the level of bile salts. Mechanistically, both receptors decreased cholesterol secretion through inhibiting the expression of Abcg5/8. In contrast to the unchanged biliary BA level in CAR-activated mice, PXR activation increased biliary BAs via induction of Mrp2 without Bsep. In the intestine, both PXR and CAR increased BAs reabsorption by increasing Asbt expression. Thus, both receptors provide a rationale for pharmacologic interference with CGD via these nuclear receptors. The present and previous studies establish a crucial and collective role for PXR and CAR in preventing CGD in vivo.
      • He J.
      • Nishida S.
      • Xu M.
      • Makishima M.
      • Xie W.
      PXR prevents cholesterol gallstone disease by regulating biosynthesis and transport of bile salts.
      We conclude that CAR and PXR mediate overlapping yet distinct defense to prevent CGD. Combined loss of PXR and CAR heightens the sensitivity to toxic BAs in mice.
      • Uppal H.
      • Toma D.
      • Saini S.P.
      • Ren S.
      • Jones T.J.
      • Xie W.
      Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice.
      It is also tempting to speculate that a combined defect in PXR and CAR expression and/or activation may increase and/or decrease susceptibility to CGD and may present a novel research topic.
      LXRα has a lithogenic effect opposite to that of CAR. The lithogenic effect of LXRα was due to the combined effect of increased biliary cholesterol and phospholipids and decreased biliary BAs.
      • Uppal H.
      • Zhai Y.
      • Gangopadhyay A.
      • Khadem S.
      • Ren S.
      • Moser J.A.
      • Xie W.
      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      In our study, we found markedly decreased hepatic secretion of cholesterol, slightly decreased phospholipid secretion, and unchanged BA secretion. Activation of LXRα suppresses CAR-mediated xenobiotic response, and, conversely, activation of CAR inhibits the LXRα-mediated lipogenesis.
      • Xiao L.
      • Xie X.
      • Zhai Y.
      Functional crosstalk of CAR-LXR and ROR-LXR in drug metabolism and lipid metabolism.
      Indeed, we observed that the hepatic triglyceride level was markedly decreased after a 2-week TCPOBOP treatment, probably because of the suppressed expression of Srebp-1c and Scd-1 in the liver. Activation of CAR also protected the mice from LXRα-sensitized gallstone disease by suppressing the expression of Abcg5 and Abcg8 induced by LXRα. Our findings expand the cognitive territory of the checks and balances behind LXRα and CAR.

      Conclusions

      We provide evidence that CAR may be a new factor in preventing cholesterol gallstone formation in a mouse model under lithogenic conditions. Activation of CAR may represent a new approach for treating CGD. Certainly, whether CAR could be a novel target in preventing or treating CGD in humans remains to be further determined.

      Acknowledgments

      S.C., M.Z., and J.H. designed and performed experiments and wrote the manuscript; Q.L., J.K., J.S., S.P., L.C., H.L., T.W., and R.L. helped with experiments; Y.L., W.J., and Z.Z. contributed to the Discussion and reviewed the manuscript; J.H. obtained funding, designed experiments, and wrote the manuscript; S.C., M.Z., and J.H. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

      Supplemental Data

      • Supplemental Figure S1

        Vehicle (VEH)- and TCPOBOP-treated mice are free of cholesterol gallstone disease with a chow diet (CD). A: Polarizing light microscopy of bile in mice fed a CD. B–D: Levels of biliary cholesterol (B), phospholipids (C), and bile acids (D). Data are expressed as means ± SEM. TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene.

      • Supplemental Figure S2

        TCPOBOP efficiently induces the expression of Cyp2b10 in both vehicle (VEH)- and TCPOBOP-treated mice. The mRNA expression of Cyp2b10 was measured by real-time PCR analysis. Data are expressed as means ± SEM. ∗∗P < 0.01. CD, chow diet; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene.

      • Supplemental Figure S3

        TCPOBOP has no effect on the expression of Shp in both CD- and LGD-maintained mice. The mRNA expression of hepatic Shp was measured by real-time PCR analysis. Data are expressed as means ± SEM. CD, chow diet; LGD, lithogenic diet; TC, TCPOBOP or 1,4-bis-[2-(3,5-dichlorpyridyloxy)] benzene; VEH, vehicle.

      • Supplemental Figure S4

        Liver bile acids (BAs), serum triglicerides (TAG), and cholesterol (CHO) levels in lithogenic diet (LGD)-maintained mice. A: Hepatic BA level in LGD-maintained mice. B: Serum concentration of TAGs and CHO. Data are expressed as means ± SEM. P < 0.05. TC, TCPOBOP; VEH, vehicle.

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