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TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury

2012; Elsevier BV; Volume: 53; Issue: 12 Linguagem: Inglês

10.1194/jlr.m031773

ISSN

1539-7262

Autores

Tsutomu Matsubara, Naoki Tanaka, Misako Sato, Dong Wook Kang, Kristopher W. Krausz, Kathleen C. Flanders, Kazuo Ikeda, Hans Luecke, Lalage M. Wakefield, Frank J. Gonzalez,

Tópico(s)

Drug-Induced Hepatotoxicity and Protection

Resumo

Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury. Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury. alkaline phosphatase alanine aminotransferase bile acid cholic acid choline kinase choline phosphotransferase 1 cytochrome P450 farnesoid X receptor lithocholic acid lysophosphatidylcholine lysophosphatidylcholine acyltransferase organic solute transporter phosphate cytidylyltransferase 1 phospholipase D partial least squares pregnane X receptor solute carrier solute carrier organic anion transporter MAD homolog 3 sulfotransferase touro-5β-cholanic acid-3-one taurocholate taurochenodeoxycholate transforming growth factor-β taurohyodeoxycholate taurolithocholate tauromurideoxycholate tumor necrosis factor α tauroursodeoxycholate ultra-performance liquid chromatography coupled with electrospray ionization quadruple time-of-flight mass spectrometry Bile acids (BA) are required for the absorption and excretion of lipophilic metabolites such as cholesterol. 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Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor.J. Biol. Chem. 2002; 277: 2908-2915Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar–14Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor.J. Biol. Chem. 2001; 276: 28857-28865Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). In addition, FXR activation enhances expression of the organic solute transporter α/β (OSTα/β) genes in the liver (15Boyer J.L. Trauner M. Mennone A. Soroka C.J. Cai S.Y. Moustafa T. Zollner G. Lee J.Y. Ballatori N. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290: G1124-G1130Crossref PubMed Scopus (246) Google Scholar) and accelerates BA excretion to the bloodstream. Thus, FXR functions as the chief sensor of intracellular BA levels and contributes to BA homeostasis (16Matsubara, T., Li, F., Gonzalez, F. J., . FXR signaling in the enterohepatic system. Mol. Cell. Endocrinol. Epub ahead of print. May 17, 2012; doi:10.1016/j.mce.2012.05.004Google Scholar). When hepatic BA accumulate in liver, hepatic inflammation is induced by tumor necrosis factor (TNF)α and transforming growth factor (TGF)β. TNFα signaling is known to downregulate SLCO1A1 and SLC10A1 expression in the liver (17Geier A. Dietrich C.G. Voigt S. Ananthanarayanan M. Lammert F. Schmitz A. Trauner M. Wasmuth H.E. Boraschi D. Balasubramaniyan N. et al.Cytokine-dependent regulation of hepatic organic anion transporter gene transactivators in mouse liver.Am. J. Physiol. Gastrointest. Liver Physiol. 2005; 289: G831-G841Crossref PubMed Scopus (95) Google Scholar) and reduce bile canalicular contraction (18Ikeda S. Mitaka T. Harada K. Sato F. Mochizuki Y. Hirata K. Tumor necrosis factor-alpha and interleukin-6 reduce bile canalicular contractions of rat hepatocytes.Surgery. 2003; 133: 101-109Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In addition, TNFα upregulates multidrug resistance-associated protein 3 (MRP3; basolateral bile salt export transporter, also called ABCC3) and can protect from liver injury that results from obstructive cholestasis (19Bohan A. Chen W.S. Denson L.A. Held M.A. Boyer J.L. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis.J. Biol. Chem. 2003; 278: 36688-36698Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Kupffer cell activation mediates induction of hepatic multidrug resistance-associated protein 4 (MRP4; basolateral bile salt export transporter, also called ABCC4) expression (20Campion S.N. Johnson R. Aleksunes L.M. Goedken M.J. van Rooijen N. Scheffer G.L. Cherrington N.J. Manautou J.E. Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G294-G304Crossref PubMed Scopus (54) Google Scholar), which is involved in basolateral BA export. The TGFβ signal regulates expression of CYP7A1 gene (21Li T. Chiang J.Y. A novel role of transforming growth factor beta1 in transcriptional repression of human cholesterol 7alpha-hydroxylase gene.Gastroenterology. 2007; 133: 1660-1669Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the rate-limiting enzyme of the BA synthesis. Proinflammatory cytokines can also influence BA homeostasis. Among the BA, lithocholic acid (LCA) is the most potent chemical causing liver toxicity. LCA levels are elevated in patients with liver disease (22Festi D. Morselli Labate A.M. Roda A. Bazzoli F. Frabboni R. Rucci P. Taroni F. Aldini R. Roda E. Barbara L. Diagnostic effectiveness of serum bile acids in liver diseases as evaluated by multivariate statistical methods.Hepatology. 1983; 3: 707-713Crossref PubMed Scopus (63) Google Scholar) and intrahepatic cholestasis (23Hofmann A.F. Detoxification of lithocholic acid, a toxic bile acid: relevance to drug hepatotoxicity.Drug Metab. Rev. 2004; 36: 703-722Crossref PubMed Scopus (206) Google Scholar). Experimental interventions to protect against LCA toxicity were investigated using animal models, revealing that the nuclear receptor pregnane X receptor (PXR) protects against LCA toxicity through upregulation of cytochrome P450 3A (CYP3A) and sulfotransferase 2A (SULT2A) that metabolize LCA for elimination (24Kitada H. Miyata M. Nakamura T. Tozawa A. Honma W. Shimada M. Nagata K. Sinal C.J. Guo G.L. Gonzalez F.J. et al.Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity.J. Biol. Chem. 2003; 278: 17838-17844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar–26Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.Proc. Natl. Acad. Sci. USA. 2001; 98: 3375-3380Crossref PubMed Scopus (680) Google Scholar). A variety of LCA metabolites were reported to be associated with this protection (27Cho J.Y. Matsubara T. Kang D.W. Ahn S.H. Krausz K.W. Idle J.R. Luecke H. Gonzalez F.J. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.J. Lipid Res. 2010; 51: 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar–31Miyata M. Watase H. Hori W. Shimada M. Nagata K. Gonzalez F.J. Yamazoe Y. Role for enhanced faecal excretion of bile acid in hydroxysteroid sulfotransferase-mediated protection against lithocholic acid-induced liver toxicity.Xenobiotica. 2006; 36: 631-644Crossref PubMed Scopus (21) Google Scholar). Recently, endogenous bile acid metabolism associated with LCA toxicity was investigated (27Cho J.Y. Matsubara T. Kang D.W. Ahn S.H. Krausz K.W. Idle J.R. Luecke H. Gonzalez F.J. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.J. Lipid Res. 2010; 51: 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and LCA exposure was reported to change serum chemistry, such as phospholipids, cholesterol, free fatty acids, and triglycerides (32Miyata M. Nomoto M. Sotodate F. Mizuki T. Hori W. Nagayasu M. Yokokawa S. Ninomiya S. Yamazoe Y. Possible protective role of pregnenolone-16 alpha-carbonitrile in lithocholic acid-induced hepatotoxicity through enhanced hepatic lipogenesis.Eur. J. Pharmacol. 2010; 636: 145-154Crossref PubMed Scopus (13) Google Scholar). Furthermore, a comprehensive view of LCA-induced alterations in endogenous metabolites was investigated by use of metabolomics for the detection and characterization of small organic chemicals in biological matrices. LCA exposure decreased serum lysophosphatidylcholine (LPC) levels, leading to cholestasis (33Matsubara T. Tanaka N. Patterson A.D. Cho J.Y. Krausz K.W. Gonzalez F.J. Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.Hepatology. 2011; 53: 1282-1293Crossref PubMed Scopus (66) Google Scholar). The change in serum LPC was associated with increased serum alkaline phosphatase (ALP), a marker of cholangiocyte injury, and increased hepatic lysophosphatidylcholine acyltransferase (LPCAT)4 expression. In addition, the TGFβ-MAD homolog 3 (SMAD3)-dependent LPCAT4 induction was observed in mouse primary hepatocytes (33Matsubara T. Tanaka N. Patterson A.D. Cho J.Y. Krausz K.W. Gonzalez F.J. Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.Hepatology. 2011; 53: 1282-1293Crossref PubMed Scopus (66) Google Scholar). TGFβ is ubiquitously expressed as latent type, and it is transformed to active type by a variety of agents. Active TGFβ binds to the TGFβ receptors 1 and 2, resulting in enhanced SMAD3 phosphorylation (34Matsuzaki K. Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells.Cell Tissue Res. 2012; 347: 225-243Crossref PubMed Scopus (70) Google Scholar). Phosphorylated SMAD3 can lead to dynamic alterations in gene expression patterns. Thus, although TGFβ-SMAD3 signaling is expected to mediate the metabolic alteration in cholestasis, in vivo studies are required to confirm this hypothesis. In the current study, LCA-induced metabolic alterations in Smad3-null mice compared with wild-type mice was investigated, revealing that TGFβ-SMAD3 signaling is involved in BA and LPC metabolism in LCA-induced liver injury. Bile acids (tauromurideoxycholate and tauro-5β-cholanic acid-3-one) were synthesized as described in the supplementary data. The other BA and fatty acids were purchased from Sigma-Aldrich (St. Louis, MO) or Steraloids (Newport, RI). Lysophosphatidylcholines ware purchased form Avanti Polar Lipids (Alabaster, AL). Male mice (C57BL/6), MAD homolog 3 (Smad3)-null mice (35Yang X. Letterio J.J. Lechleider R.J. Chen L. Hayman R. Gu H. Roberts A.B. Deng C. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar), and background-matched wild-type mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. For the LCA studies, mice were given 0.6% LCA-supplement diet with the AIN93G diet as a control (Dyets, Bethlehem, PA). Three wild-type and three Smad3-null mice were fed the control diet, and five wild-type and four Smad3-null mice were given the LCA diet. All animal studies were carried out in accordance with Institute of Laboratory Animal Resources (ILAR) guidelines and protocols approved by the National Cancer Institute Animal Care and Use Committee. Serum was prepared using Serum Separator Tubes (Becton, Deckinson and Co., Franklin Lakes, NJ). The serum catalytic activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) was measured with ALT and ALP assay kit, respectively (Catachem, Bridgeport, CT). Serum was prepared using Serum Separator Tubes (Becton, Dickinson and Co.). The serum was diluted with 19 vol of 66% acetonitrile and centrifuged twice at 18,000 g for 20 min to remove insoluble materials. UPLC-ESI-QTOFMS was preformed as previously reported (27Cho J.Y. Matsubara T. Kang D.W. Ahn S.H. Krausz K.W. Idle J.R. Luecke H. Gonzalez F.J. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.J. Lipid Res. 2010; 51: 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In brief, the aliquots (5 μl) were injected into a reverse-phase 50 × 2.1 mm ACQUITY 1.7 μm C18 column (Waters, Milford, MA) using an ACQUITY UPLC system (Waters) with a gradient mobile phase comprising 0.1% formic acid and acetonitrile containing 0.1% formic acid. The eluant was introduced by electrospray ionization into the mass spectrometer [Q-TOF Premier (Waters)] operating in negative electrospray ionization mode. The capillary and sampling cone voltages were set to 3000 and 30 V, respectively. The desolvation gas flow was set to 650 l/h, and the temperature was set to 350°C. The cone gas flow was 50 l/h, and the source temperature was 120°C. Data were acquired in centroid mode from m/z 50 to 800 in MS scanning. Tandem MS collision energy was scanned from 5 to 35 V. Data processing and multivariate data analysis were conducted as previously reported (27Cho J.Y. Matsubara T. Kang D.W. Ahn S.H. Krausz K.W. Idle J.R. Luecke H. Gonzalez F.J. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.J. Lipid Res. 2010; 51: 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Partial least squares (PLS) and contribution analyses were performed using SIMCA-P+12 (Umetrics, Kinnelon, NJ). Serum bile acid levels were determined with standard curves using authentic metabolites. Quantification of serum lysophosphatidylcholine was performed according to a previously reported method (33Matsubara T. Tanaka N. Patterson A.D. Cho J.Y. Krausz K.W. Gonzalez F.J. Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.Hepatology. 2011; 53: 1282-1293Crossref PubMed Scopus (66) Google Scholar). RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), and qPCR was performed using cDNA generated from 1 µg total RNA with a SuperScript II Reverse Transcriptase kit and random oligonucleotides (Invitrogen). Primers were designed using qPrimerDepot. All sequences are listed in supplementary Table I. Quantitative PCR reactions were carried out using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7900HT Sequence Detection System. Values were quantified using comparative CT method, and samples were normalized to 18S rRNA. For whole-cell extracts, a culture cell was lysed with sample buffer and subjected to Western blotting. The samples were boiled for 5 min and then separated and transferred to PVDF membranes using standard Western blotting techniques. The membranes were incubated with an antibody against phospho-SMAD3 at a dilution of 1:1,000, ab52903 (Abcam, Cambridge, UK) or SMAD3 at a dilution of 1:1,000 (ab28379, Abcam). The signals were normalized to signals obtained with a GAPDH Ab used at a dilution of 1:10,000 (MAB374, Millipore, Billerica, MA). Small blocks of liver tissue were immediately fixed in 10% neutral formalin and embedded in paraffin. Sections (4 µm thick) were stained with hematoxylin and eosin. At least three discontinuous liver sections were evaluated for each mouse. Immunolocalization of TGFβ1 and phospho-Smad3 were performed as described (36Figueroa J.D. Flanders K.C. Garcia-Closas M. Anderson W.F. Yang X.R. Matsuno R.K. Duggan M.A. Pfeiffer R.M. Ooshima A. Cornelison R. et al.Expression of TGF-beta signaling factors in invasive breast cancers: relationships with age at diagnosis and tumor characteristics.Breast Cancer Res. Treat. 2010; 121: 727-735Crossref PubMed Scopus (48) Google Scholar) with the addition of antigen retrieval in 1 mM EDTA (pH 8) at 95°C for 10 min for sections stained with the pSmad3 antibody. TGFβ1 was detected with the primary antibody LC-1-30-1 at 3 µg/ml, and pSmad3 was detected with a rabbit monoclonal antibody from Epitomics (Cat. No. 1880-1) at a dilution of 1:500. A Smad3-expressing adenovirus with Cre/LoxP system (Ad-L-Smad3) was generated by using the Adenovirus Cre/LoxP-Regulated Expression Vector Set (Takara, Tokyo, Japan). A Cre-expressing adenovirus (Ad-Cre) and a GFP-expressing adenovirus (Ad-L-GFP) with Cre/LoxP system were previously generated (37Saika S. Ikeda K. Yamanaka O. Sato M. Muragaki Y. Ohnishi Y. Ooshima A. Nakajima Y. Namikawa K. Kiyama H. et al.Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial-mesenchymal transition of lens epithelium in mice.Lab. Invest. 2004; 84: 1259-1270Crossref PubMed Scopus (81) Google Scholar). The titer of these adenoviruses was measured by the 50% tissue culture infectious dose method. Primary hepatocytes were prepared as previously reported (33Matsubara T. Tanaka N. Patterson A.D. Cho J.Y. Krausz K.W. Gonzalez F.J. Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.Hepatology. 2011; 53: 1282-1293Crossref PubMed Scopus (66) Google Scholar). After starvation with FBS-negative Williams' Medium E for 2 h, the hepatocytes were exposed to 5 ng/ml of TGFβ (R and D Systems, Minneapolis, MA) for 12 h, and then collected and lysed for gene expression analysis by use of qPCR. HepG2 cells were seeded on a 12-well plate and infected with recombinant adenovirus. Two days later, the HepG2 cells with the starvation were exposed to 5 ng/ml of TGFβ. The cells were subjected to the Western blotting (treatment with TGFβ for 1 h) or the qPCR (treatment with TGFβ for 12 h). Statistical analysis was performed using Prism version 5.0c (GraphPad Software, San Diego, CA). A P-value of less than 0.05 was considered as significant difference. The influence of lithocholic acid (LCA) exposure on TGFβ signaling was investigated using C57BL/6 mice treated with the synthetic AIN93G diet (Cont) and 0.6% LCA-supplemented AIN93G diet (LCA) for 7 days. Hepatic TGFB1, TGFBR1, and TGFBR2 mRNA levels increased after LCA exposure, although TGFBR3 mRNA level did not changed in the livers (Fig. 1). These results suggest that LCA exposure stimulates TGFβ signaling in the livers. TGFβ activates SMAD3 via the TGFβ receptors. Thus, to investigate whether SMAD3 was involved in LCA-induced liver injury, Smad3-null mice were treated with control diet and LCA diet for 6 days. After LCA exposure, the liver mass of Smad3-null mice was smaller than that of LCA-treated wild-type mice (Fig. 2A). In addition, LCA-increased serum ALP activities were significantly attenuated in the Smad3-null mice, although serum ALT activities were not changed (Fig. 2B, C). Furthermore, liver histology showed mild features of inflammatory cell infiltration around the portal vein in Smad3-null mice, which was not observed in similarly treated wild-type mice (Fig. 2D). Immunohistochemistry revealed TGFβ protein around the portal vein with lower expression of the TGFβ in Smad3-null mice compared with wild-type mice (Fig. 2E), suggesting lower TGFβ stimulation of the SMAD3 activation in the liver (supplementary Fig. I). In addition, a dramatic attenuation of the SMAD3 phosphorylation signal was observed in the hepatocyte nuclei of Smad3-null mice (Fig. 2E). These results suggest that TGFβ-SMAD3 signaling is associated with the LCA-induced biliary injury and raise the possibility that TGFβ-SMAD3 signaling alters hepatic metabolism. To examine serum metabolites, PLS and contribution analyses were performed with UPLC-ESI-QTOFMS negative mode data derived from serum of mice fed LCA or control diet. PLS analysis showed a separation between the LCA-treated wild-type (Fig. 3A) and the LCA-treated Smad3-null group that was further examined with a loadings plot (Fig. 3B). Contribution analysis revealed 10 enhanced and 10 attenuated ions as the top-ranking ions giving rise to the separation. Lysophosphatidylcholine (LPC) and fatty acid fragments were determined as raised ions in Smad3-null mice compared with the wild-type mice (Table 1). The most lowered ions were derived from bile salts (Table 2). After LCA feeding, the serum metabolome of Smad3-null mice was much different from that of the wild-type mice in LPC and bile salts.TABLE 1Top ten of serum metabolite ions that were of higher intensity in Smad3-null group with LCA diet than in wild-type group with LCA dietRankMarkRT (min)Found (m/z)CandidateMass Error (ppm)1I5.37568.3585Stearoyl-LPC (18:0 LPC)5.102II5.94279.2310Linoleic acid5.013III4.90566.3436Oleoyl-LPC (18:1 LPC)3.884IV5.78327.2324Docosahexaenoic acid0.005V4.63540.3279Palmitoyl-LPC (16:0 LPC)4.076VI5.23568.3582Stearoyl-LPC (18:0 LPC)5.637VII4.75540.3275Palmitoyl-LPC (16:0 LPC)4.818VIII5.88303.2313Arachidonic acid3.639IX6.42381.1707Not determined10X6.42281.2467Oleic acid4.98The ion ranking, based on the PLS-DA analysis, indicates the highest confidence and greatest contribution to separation between the wild-type and the Smad3-null mice after LCA exposure. RT, retention time. Open table in a new tab TABLE 2Top ten of serum metabolite ions that were lower intensity in Smad3-null group with LCA-diet than in wild-type group with LCA-dietRankMarkRT (min)Found (m/z)CandidateMass Error (ppm)1i3.19498.2868Taurochenodeoxycholate (TCDC)4.212ii3.72482.2913Taurolithocholate (TLC)5.603iii3.72480.2764Tauro-5β-cholanic acid-3-one (T3KL)4.164iv3.17496.2722Not determined5v2.49514.2813Tauro-α/β-muricholateaNo clear separation between tauro-α-muricholate and tauro-β-muricholate. (TαMC/TβMC)4.866vi2.61498.2856Tauromurideoxycholate (TMDC)6.627vii3.09498.2860Not determined8viii2.77498.2852Tauroursodeoxycholate/TaurohyodeoxycholatebNo clear separation between tauroursodeoxycholate and taurohyodeoxycholate. (TUDC/THDC)7.439ix2.83496.2700Not determined10x2.74496.2703Not determinedThe ion ranking, based on the PLS analysis, indicates the highest confidence and greatest contribution to separation between the wild-type and the Smad3-null mice after LCA exposure. RT, retention time.a No clear separation between tauro-α-muricholate and tauro-β-muricholate.b No clear separation between tauroursodeoxycholate and taurohyodeoxycholate. Open table in a new tab The ion ranking, based on the PLS-DA analysis, indicates the highest confidence and greatest contribution to separation between the wild-type and the Smad3-null mice after LCA exposure. RT, retention time. The ion ranking, based on the PLS analysis, indicates the highest confidence and greatest contribution to separation between the wild-type and the Smad3-null mice after LCA exposure. RT, retention time. Quantification of serum bile salts was performed with authentic compounds. All of the tested bile acid levels were lower in Smad3-null mice after LCA exposure than those in the wild-type mice (Fig. 4A). As bile acid metabolites can be produced from LCA (supplementary Fig. II) in the liver, the expression of the major metabolic enzymes CYP3A11 and sulfotransferase 2A (SULT2A) was investigated. CYP3A11 and SULT2A expression was not, however, different between the wild-type and the Smad3-null mice after LCA feeding (Fig. 4B). Thus, hepatic expression of bile acid-related transporter genes was investigated, including mRNAs encoding ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, solute carrier (SLC)10A1, solute carrier organic anion transporter (SLCO)1A1, SLCO1A4, SLCO1B2, organic solute transporter (OST)α, and OSTβ. Differences in expression of the bile salt uptake transporters SLCO1A1, SLCO1A4, and SLCO1B2 (intake to hepatocyte, Fig. 4C), major bile salt exporters ABCC2 and ABCB11 (export to bile duct, Fig. 4D), and bile acid synthesis enzyme CYP7A1 (Fig

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