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• W1987127854 abstract "Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that is highly expressed in liver and catalyzes intermembrane transfer of phosphatidylcholines in vitro. To explore a role for PC-TP in the hepatocellular trafficking of biliary phosphatidylcholines, we characterized biliary lipid secretion using Pctp−/− and wild-type littermate control mice with C57BL/6J and FVB/NJ genetic backgrounds, which express PC-TP at relatively high and low levels in liver, respectively. Eight-week-old male Pctp−/− and wild-type mice were fed a chow diet or a lithogenic diet, which served to upregulate biliary lipid secretion. In chow-fed mice, the absence of PC-TP did not reduce biliary phospholipid secretion or alter the phospholipid composition of biles. However, the responses in secretion of biliary phospholipids, cholesterol, and bile salts to the lithogenic diet were impaired in Pctp−/− mice from both genetic backgrounds. Alterations in biliary lipid secretion could not be attributed to transcriptional regulation of the expression of canalicular membrane lipid transporters, but possibly to a defect in their trafficking to the canalicular membrane.These findings support a role for PC-TP in the response of biliary lipid secretion to a lithogenic diet, but not specifically in the hepatocellular transport and secretion of phosphatidylcholines. Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that is highly expressed in liver and catalyzes intermembrane transfer of phosphatidylcholines in vitro. To explore a role for PC-TP in the hepatocellular trafficking of biliary phosphatidylcholines, we characterized biliary lipid secretion using Pctp−/− and wild-type littermate control mice with C57BL/6J and FVB/NJ genetic backgrounds, which express PC-TP at relatively high and low levels in liver, respectively. Eight-week-old male Pctp−/− and wild-type mice were fed a chow diet or a lithogenic diet, which served to upregulate biliary lipid secretion. In chow-fed mice, the absence of PC-TP did not reduce biliary phospholipid secretion or alter the phospholipid composition of biles. However, the responses in secretion of biliary phospholipids, cholesterol, and bile salts to the lithogenic diet were impaired in Pctp−/− mice from both genetic backgrounds. Alterations in biliary lipid secretion could not be attributed to transcriptional regulation of the expression of canalicular membrane lipid transporters, but possibly to a defect in their trafficking to the canalicular membrane. These findings support a role for PC-TP in the response of biliary lipid secretion to a lithogenic diet, but not specifically in the hepatocellular transport and secretion of phosphatidylcholines. During bile formation, a transhepatocellular flux of bile salts promotes the secretion of unilamellar vesicles composed of unesterified cholesterol and phospholipid molecules (1Cohen D.E. Hepatocellular transport and secretion of biliary lipids.Curr. Opin. Lipidol. 1999; 235: 111-120Google Scholar). Hepatocellular membranes, from which biliary phospholipids are derived, are composed of heterogeneous mixtures of phospholipid classes (2White D.A. The phospholipid composition of mammalian tissues.in: Ansel G.B. Hawthorne J.N. Dawson R.M.C. Form and Function of Phospholipids. Elsevier, Amsterdam1973: 441-482Google Scholar). By contrast, phosphatidylcholines constitute 95% of biliary phospholipids (3Hay D.W. Carey M.C. Chemical species of lipids in bile.Hepatology. 1990; 12: 6-16Google Scholar). The molecular mechanism(s) by which phosphatidylcholines are selected from hepatocellular membranes for biliary secretion remains poorly understood. Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein and a member of the steroidogenic acute regulatory (StAR)-related transfer (START) domain-containing superfamily of proteins (4Ponting C.P. Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins.Trends Biochem. Sci. 1999; 24: 130-132Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 5Tsujishita Y. Hurley J.H. Structure and lipid transport mechanism of a StAR-related domain.Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (450) Google Scholar). In vitro, PC-TP [recently designated StarD2 (6Soccio R.E. Breslow J.L. StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.J. Biol. Chem. 2003; 278: 22183-22186Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar)] catalyzes the intermembrane transfer of phosphatidylcholines, but no other phospholipid class (7Wirtz K.W.A. Phospholipid transfer proteins.Annu. Rev. Biochem. 1991; 60: 73-99Crossref PubMed Scopus (380) Google Scholar). Based on its exquisite substrate specificity (7Wirtz K.W.A. Phospholipid transfer proteins.Annu. Rev. Biochem. 1991; 60: 73-99Crossref PubMed Scopus (380) Google Scholar, 8Roderick S.L. Chan W.W. Agate D.S. Olsen L.R. Vetting M.W. Rajashankar K.R. Cohen D.E. Structure of human phosphatidylcholine transfer protein in complex with its ligand.Nat. Struct. Biol. 2002; 9: 507-511PubMed Google Scholar), enrichment in liver (7Wirtz K.W.A. Phospholipid transfer proteins.Annu. Rev. Biochem. 1991; 60: 73-99Crossref PubMed Scopus (380) Google Scholar, 9Cohen D.E. Green R.M. Wu M.K. Beier D.R. Cloning, gene structure, tissue-specific expression and chromosomal localization of human phosphatidylcholine transfer protein.Biochim. Biophys. Acta. 1999; 1447: 265-270Crossref PubMed Scopus (27) Google Scholar, 10Geijtenbeek T.B.H. Smith A.J. Borst P. Wirtz K.W.A. cDNA cloning and tissue specific expression of the phosphatidylcholine transfer protein gene.Biochem. J. 1996; 316: 49-55Crossref PubMed Scopus (21) Google Scholar), and the observation that activity in vitro is stimulated by bile salts (11Cohen D.E. Leonard M.R. Carey M.C. In vitro evidence that phospholipid secretion into bile may be coordinated intracellularly by the combined actions of bile salts and the specific phosphatidylcholine transfer protein of liver.Biochemistry. 1994; 33: 9975-9980Crossref PubMed Scopus (79) Google Scholar), it has been postulated that PC-TP might function to deliver phosphatidylcholines from their principal site of synthesis in the endoplasmic reticulum to the canalicular membrane for secretion into bile (1Cohen D.E. Hepatocellular transport and secretion of biliary lipids.Curr. Opin. Lipidol. 1999; 235: 111-120Google Scholar, 12Smit J.J.M. Schinkel A.H. Oude Elferink R.P.J. Groen A.K. Wagenaar E. Deemter L. van Mol C.A.A.M. Ottenhoff R. van der Lugt N.M.T. van Roon M.A. van der Valk M.A. Offerhaus G.J.A. Berns A.J.M. Borst P. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1336) Google Scholar, 13LaMorte W.W. Booker M.L. Kay S. Determinants of the selection of phosphatidylcholine molecular species for secretion into bile.Hepatology. 1998; 28: 631-637Crossref PubMed Scopus (14) Google Scholar). Using mice with homozygous disruption of the Pctp gene (Pctp−/−), van Helvoort et al. (14van Helvoort A. Brouwer A. de Ottenhoff R. Brouwers J.F. Wijnholds J. Beijnen J.H. Rijneveld A. van der Poll T. van der Valk M.A. Majoor D. Voorhout W. Wirtz K.W. Elferink R.P. Borst P. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces.Proc. Natl. Acad. Sci. USA. 1999; 96: 11501-11506Crossref PubMed Scopus (65) Google Scholar) tested a role for PC-TP in biliary lipid secretion by mice. They observed that biliary phosphatidylcholine secretion occurred normally in Pctp−/− mice under basal conditions and when biliary secretion rates were driven to higher rates by intravenous infusion of a hydrophilic bile salt (14van Helvoort A. Brouwer A. de Ottenhoff R. Brouwers J.F. Wijnholds J. Beijnen J.H. Rijneveld A. van der Poll T. van der Valk M.A. Majoor D. Voorhout W. Wirtz K.W. Elferink R.P. Borst P. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces.Proc. Natl. Acad. Sci. USA. 1999; 96: 11501-11506Crossref PubMed Scopus (65) Google Scholar). Although this finding excluded PC-TP as an exclusive mechanism for hepatocellular selection and transport of biliary phosphatidylcholines, it did not eliminate the possibility that another pathway was able to compensate under these specific experimental conditions. Of the 15 mammalian START domain proteins, several [i.e., StAR (StarD1), MLN64 (StarD3), StarD4, and StarD5] appear to function in the maintenance of cholesterol homeostasis (4Ponting C.P. Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins.Trends Biochem. Sci. 1999; 24: 130-132Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 16Soccio R.E. Adams R.M. Romanowski M.J. Sehayek E. Burley S.K. Breslow J.L. The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6.Proc. Natl. Acad. Sci. USA. 2002; 99: 6943-6948Crossref PubMed Scopus (154) Google Scholar). Considering that secretion of biliary phospholipids is also upregulated in mice by dietary cholesterol supplementation (17Wang 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.J. Lipid Res. 1999; 40: 2066-2079Abstract Full Text Full Text PDF PubMed Google Scholar), we sought to determine whether Pctp−/− mice would respond normally to a dietary cholesterol challenge. Using Pctp−/− mice bred onto two distinct genetic backgrounds, we provide evidence for impaired biliary lipid secretion in Pctp−/− mice in response to a lithogenic diet containing sufficient cholesterol content to promote the formation of cholesterol gallstones. However, these data do not support a specific role for PC-TP in the hepatocellular selection of phosphatidylcholines for secretion into bile. Pctp−/− mice (14van Helvoort A. Brouwer A. de Ottenhoff R. Brouwers J.F. Wijnholds J. Beijnen J.H. Rijneveld A. van der Poll T. van der Valk M.A. Majoor D. Voorhout W. Wirtz K.W. Elferink R.P. Borst P. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces.Proc. Natl. Acad. Sci. USA. 1999; 96: 11501-11506Crossref PubMed Scopus (65) Google Scholar) were obtained as a generous gift from Drs. Ardy van Helvoort and Piet Borst (The Netherlands Cancer Institute, Amsterdam). These were supplied on a pure 129/Ola genetic background and on a mixed FVB/NJ and 129/Ola genetic background that was obtained by backcrossing 129/Ola Pctp−/− mice for three generations to FVB/NJ mice. In their original characterization of Pctp−/− mice, van Helvoort et al. (14van Helvoort A. Brouwer A. de Ottenhoff R. Brouwers J.F. Wijnholds J. Beijnen J.H. Rijneveld A. van der Poll T. van der Valk M.A. Majoor D. Voorhout W. Wirtz K.W. Elferink R.P. Borst P. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces.Proc. Natl. Acad. Sci. USA. 1999; 96: 11501-11506Crossref PubMed Scopus (65) Google Scholar) reported the unexpected finding that PC-TP expression in livers of wild-type littermate controls of mixed FVB/NJ-129/Ola genetic backgrounds was downregulated at 2 weeks of age and undetectable in 12 week old mice. To determine whether this phenomenon was strain specific, preliminary experiments examined hepatic expression of PC-TP by Western blot analysis in livers of 11-week-old wild-type mice of C57BL/6J, 129/Ola, and FVB/NJ genetic backgrounds that were purchased from the Jackson Laboratory (Bar Harbor, ME). The highest levels of PC-TP expression were observed in C57BL/6J mice, with relatively low levels in FVB/NJ mice. Adult 129/Ola mice expressed intermediate levels of PC-TP in liver. For the current experiments, we prepared Pctp−/− mice on genetic backgrounds with high (C57BL/6J) and low (FVB/NJ) expression levels. FVB/NJ and 129/Ola mixed genetic backgrounds were backcrossed three additional generations to FVB/NJ mice to achieve mice that were six generations backcrossed to FVB/NJ. Pctp−/− mice on the pure 129/Ola genetic background were backcrossed eight generations to C57BL/6J mice to obtain Pctp−/− mice on a predominantly C57BL/6J genetic background. Mice were genotyped as described by van Helvoort et al. (14van Helvoort A. Brouwer A. de Ottenhoff R. Brouwers J.F. Wijnholds J. Beijnen J.H. Rijneveld A. van der Poll T. van der Valk M.A. Majoor D. Voorhout W. Wirtz K.W. Elferink R.P. Borst P. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces.Proc. Natl. Acad. Sci. USA. 1999; 96: 11501-11506Crossref PubMed Scopus (65) Google Scholar). Male 6–8-week-old Pctp−/− and littermate control mice were fed either a lithogenic diet consisting of 15% fat, 1.25% cholesterol, and 0.5% sodium cholate (Harlan Teklad, Madison, WI) or a chow diet consisting of 4% fat and <0.02% cholesterol (LabDiet 5001; PMI Nutrition International, Inc., Brentwood, MO) for specified periods of time. Mice were fasted for 3 h and then anesthetized with intraperitoneal injections of 87 mg/kg body weight ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 13 mg/kg body weight xylazine (Lloyd Laboratories, Shenandoah, IA). Surgery commenced at 9 AM with a midline abdominal incision. The gallbladder was first inspected for the presence of gallstones. Using a PE-10 polyethylene catheter (Becton Dickinson, Sparks, MD), the gallbladder was then cannulated immediately after distal ligation of the common bile duct (18Hyogo H. Roy S. Paigen B. Cohen D.E. Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts.J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Bile samples were collected by diverting bile flow to an Eppendorf collection tube. Concentrated gallbladder bile (the first 10–20 μl of bile collected) was immediately analyzed by light microscopy for the presence of liquid crystals or solid cholesterol crystals. Thereafter, flow rates of hepatic bile were measured by assuming a density of 1 g/ml and weighing collection tubes every 10 min. A collection period of 1 h was required to collect sufficient volumes of bile to measure biliary lipid concentrations and compositions for each sample (see below). Samples were frozen at −80°C before analysis. Livers were then excised, rinsed with 0.15 M NaCl to remove blood, snap frozen in liquid nitrogen, and stored at −80°C. These procedures were conducted with the approval of the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. Biliary phospholipid concentrations were determined using an inorganic phosphorus procedure (18Hyogo H. Roy S. Paigen B. Cohen D.E. Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts.J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Phospholipid classes were determined commercially by Lipomics Technologies, Inc. (West Sacramento, CA) (19Watkins S.M. Reifsnyder P.R. Pan H.J. German J.B. Leiter E.H. Lipid metabolome-wide effects of the PPARgamma agonist rosiglitazone.J. Lipid Res. 2002; 43: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Briefly, equal volumes of bile samples were pooled (n = 5 mice/group) and then phospholipids were extracted into chloroform. Phospholipid classes were separated by thin-layer chromatography, after which individual phospholipid classes were scraped, extracted from silica, and quantified according to phosphorus contents. Fatty acid compositions were determined by gas chromatography. Cholesterol concentrations were measured enzymatically (Sigma, St. Louis, MO) (18Hyogo H. Roy S. Paigen B. Cohen D.E. Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts.J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Bile salt concentrations and molecular species were determined by HPLC (18Hyogo H. Roy S. Paigen B. Cohen D.E. Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts.J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The hydrophobic index of bile salts, a concentration-weighted average hydrophobicity of a mixture of bile salts, was determined according to the method of Heuman (20Heuman D.M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions.J. Lipid Res. 1989; 30: 719-730Abstract Full Text PDF PubMed Google Scholar). Expression of PC-TP in liver was determined by Western blot analysis. Liver cytosol was prepared by homogenizing 300 mg of tissue in ice-cold buffer containing 0.25 M sucrose, 10 mM Tris, pH 7.35, 1 mM EDTA, 5 mM DTT, and Complete™ protease inhibitor (Roche, Indianapolis, IN). Samples were then centrifuged at 100,000 g for 1 h at 4°C. Equal amounts of cytosolic proteins (75 μg) were subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and probed using a polyclonal antibody to PC-TP (21Shoda J. Oda K. Suzuki H. Sugiyama Y. Ito K. Cohen D.E. Feng L. Kamiya J. Nimura Y. Miyazaki H. Kano M. Matsuzaki Y. Tanaka N. Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolism in the liver of patients with intrahepatic calculi.Hepatology. 2001; 33: 1194-1205Crossref PubMed Scopus (63) Google Scholar). Visualization was by enhanced chemiluminescence using goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA). mRNA transcripts encoding canalicular membrane transporters were quantified by Northern blot analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA (20 μg) was electrophoresed through agarose gels and transferred to nylon membranes according to standard procedures. Mouse cDNAs were kindly provided by Dr. Richard Green (Northwestern University, Chicago, IL) [ATP binding cassette protein 4 (Abcb4) and Abcb11] and Dr. David Silver (Columbia University, New York, NY) (Abcg5), and a cDNA encoding mouse β-actin was purchased from Stratagene (La Jolla, CA). cDNAs were radiolabeled with [α-32P]dCTP (Perkin-Elmer Life Sciences, Torrance, CA) using a random primer kit (Invitrogen). After hybridization, blots were subjected to autoradiography and quantified by densitometry using a FluorChem 8900 Imaging system (Alpha Innotech Corp., San Leandro, CA). mRNA expression levels were normalized to β-actin expression. Morphology of bile canaliculi was assessed by both light and electron microscopy. Livers were harvested from 6–8-week-old male chow-fed mice immediately after cervical dislocation. Liver slices (2–3 mm thick) were fixed by immersion in a cold mixture of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 3 h with shaking (22Novikoff P.M. Yam A. Stem cells and rat liver carcinogenesis: contributions of confocal and electron microscopy.J. Histochem. Cytochem. 1998; 46: 613-626Crossref PubMed Scopus (22) Google Scholar). After fixation, slices were rinsed with 7.5% sucrose and stored at 4°C in 2.3 M sucrose for cryoprotection before sectioning. Livers were sectioned (10–20 μm) with a Sartorius freezing microtome (Leitz, Gottingen, Germany). To visualize bile canaliculi, sections were incubated for ATPase activity in buffer containing 1 mM adenosine triphosphate sodium salt, 80 mM Tris maleate, pH 7.2, 10 mM MgSO4 and 0.12% lead nitrate at 37°C for 45 min, followed by visualization in diluted ammonium sulfide and mounting in glycerogel (22Novikoff P.M. Yam A. Stem cells and rat liver carcinogenesis: contributions of confocal and electron microscopy.J. Histochem. Cytochem. 1998; 46: 613-626Crossref PubMed Scopus (22) Google Scholar). Mounted liver sections were examined and photographed with a Zeiss Ultraport II (Carl Zeiss, Thornwood, NY). For electron microscopy, sections were postfixed in 4% osmium tetroxide, pH 7.4, for 1 h, embedded in Epon (Polyscience, Warren, PA), and sectioned (1 μm) using an LKB ultramicrotome II (LKB, Stockholm, Sweden). Slides were examined with a Phillips EM 300 equipped with a goniometer stage (Phillips Electronic Optics, Millersville, MD). Data are expressed as mean ± SD. Student's t-test assuming equal variance was used to determine statistical significance between experimental groups. Differences were considered significant for a two-tailed P < 0.05. Figure 1compares the expression of PC-TP in livers of 8-week-old wild-type littermate control and Pctp−/− C57BL/6J and FVB/NJ mice. Compared with wild-type FVB/NJ mice, wild-type C57BL/6J mice expressed high levels of PC-TP. Expression of PC-TP was absent in Pctp−/− mice of both genetic backgrounds. Susceptibility to cholesterol gallstones upon challenge with a lithogenic diet is a readily detectable phenotype that differs dramatically among inbred strains of mice (23Khanuja B. Cheah Y.C. Hunt M. Nishina P.M. Wang D. Q-H. Chen H.W. Billheimer J.T. Carey M.C. Paigen B. Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice.Proc. Natl. Acad. Sci. USA. 1995; 92: 7729-7733Crossref PubMed Scopus (191) Google Scholar) and in part reflects the biliary response to dietary cholesterol. We examined gallbladders and gallbladder biles to determine whether cholesterol gallstone formation was accelerated in Pctp−/− mice. Mice were fed the lithogenic diet for periods ranging up to 4 weeks. Abundant liquid and solid cholesterol crystals, which precede the formation of macroscopic cholesterol gallstones (24Wang D. Q-H. Paigen B. Carey M.C. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile.J. Lipid Res. 1997; 38: 1395-1411Abstract Full Text PDF PubMed Google Scholar), were detected by 2 days for FVB/NJ mice and by 9 days for C57BL/6J mice. Neither the rate of appearance of liquid and solid crystals nor the evolution of macroscopic cholesterol gallstones was influenced by the absence of PC-TP in knockout mice of either genetic background. To further characterize the influence of PC-TP on bile formation, we measured bile flow as well as biliary lipid concentrations and compositions in mice fed chow and the lithogenic diet. Because genetic background also influences biliary lipid secretion (17Wang 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.J. Lipid Res. 1999; 40: 2066-2079Abstract Full Text Full Text PDF PubMed Google Scholar), we chose a common phenotypic response as a basis for choosing the duration of the dietary cholesterol challenge in C57BL/6J versus FVB/NJ mice. When fed the lithogenic diet, the formation of liquid and solid cholesterol crystals in bile is associated with mucin accumulation (24Wang D. Q-H. Paigen B. Carey M.C. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile.J. Lipid Res. 1997; 38: 1395-1411Abstract Full Text PDF PubMed Google Scholar). In our experiments, occlusion of the gallbladder with mucin eventually prevented surgical cannulation. This occurred reproducibly at 7 days for C57BL/6J mice and at 18 h for FVB/NJ mice. Therefore, these time points were chosen to characterize biliary responses to the lithogenic diet in the two strains of mice in the presence or absence of PC-TP. In preliminary experiments, we also demonstrated by Western blot analysis that PC-TP expression in wild-type mice was not influenced by feeding the lithogenic diet for the specified periods of time (data not shown). Figure 2displays flow rates of hepatic biles. For Pctp−/− compared with wild-type C57BL/6J mice (Fig. 2A), bile flow rates were the same in chow-fed mice and in mice fed the lithogenic diet. For the same genotype, bile flow rates were higher in mice fed the lithogenic diet compared with the chow diet, but this difference only achieved statistical significance at each time point for Pctp−/− mice. In chow-fed mice with the FVB/NJ genetic background (Fig. 2B), bile flow rates were the same for the first 30 min, after which flow rates in Pctp−/− mice were reduced by ∼35% compared with littermate controls. The lithogenic diet did not influence bile flow in FVB/NJ mice of either genotype, nor were differences in bile flow observed between Pctp−/− and wild-type mice fed the lithogenic diet. Figure 3shows biliary lipid concentrations in hepatic biles. Phospholipids were readily detected in biles of chow-fed Pctp−/− C57BL/6J mice (Fig. 3A) at concentrations that were 1.3-fold higher compared with wild-type mice. The inset to Fig. 3A shows steady-state mRNA levels of Abcb4, which is rate limiting for biliary phospholipid secretion under physiological conditions (25Smith A.J. de Vree J.M. Ottenhoff R. Oude Elferink R.P. Schinkel A.H. Borst P. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (−/−) mice.Hepatology. 1998; 28: 530-536Crossref PubMed Scopus (122) Google Scholar). Expression in livers of C57BL/6J mice fed the chow diet was essentially unchanged in Pctp−/− mice. Challenge with the lithogenic diet increased biliary phospholipid concentrations in both genotypes. However, phospholipid concentrations in bile were 1.3-fold lower in the Pctp−/− mice after 7 days on the lithogenic diet. After challenge with the lithogenic diet, Abcb4 mRNA expression levels were similar in both wild-type and Pctp−/− mice (Fig. 3A, inset). In chow-fed FVB/NJ mice (Fig. 3B), phospholipid concentrations were 4-fold higher in Pctp−/− mice, whereas expression levels of Abcb4 mRNA did not differ (Fig. 3B, inset). After 18 h on the lithogenic diet, biliary phospholipid concentrations in wild-type mice increased to concentrations similar to those present in Pctp−/− mice fed chow. In Pctp−/− mice, biliary phospholipid concentrations did not change in response to the lithogenic diet. As shown in the inset to Fig. 3B, expression of Abcb4 mRNA was similar in Pctp−/− and littermate controls. In C57BL/6J wild-type and Pctp−/− mice fed the chow diet, biliary cholesterol concentrations did not differ (Fig. 3C), nor did expression levels of mRNA encoding Abcg5, one of two canalicular hemitransporters that largely control biliary cholesterol secretion (26Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (612) Google Scholar) (Fig. 3C, inset). After challenge with the lithogenic diet, cholesterol concentrations in biles of Pctp−/− mice were 1.3-fold lower than in wild-type littermate controls. However, Abcg5 mRNA expression was the same in the livers of both genotypes. For FVB/NJ mice fed chow, biliary cholesterol concentrations were 5.9-fold higher in the absence of PC-TP (Fig. 3D). After feeding the lithogenic diet, cholesterol concentrations in biles of Pctp−/− mice decreased, whereas concentrations in wild-type mice increased. As a result, final concentrations of cholesterol were similar in both genotypes. The inset to Fig. 3D shows that livers of chow-fed FVB/NJ Pctp−/− mice expressed Abcg5 mRNA at the same level as wild-type mice and that levels were similar in both genotypes after the dietary cholesterol challenge. Figure 3E demonstrates that biliary bile salt concentrations in chow-fed C57BL/6J mice were not affected by the absence of PC-TP. The same was true for the mRNA expression levels of Abcb11 (Fig. 3E, inset), the canalicular bile salt export pump that rate limits biliary bile salt secretion (27Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. The sister-P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar, 28Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 98: 2011-2016Crossref PubMed Scopus (285) Google Scholar). Bile salt concentrations increased in both Pctp−/− and wild-type mice after challenge with the lithogenic diet. Although bile salt concentrations were 1.3-fold lower in knockout mice, hepatic expression levels of Abcb11 mRNA were the same in Pctp−/− and wild-type mice (Fig. 3E, inset). In FVB/NJ mice fed chow, bile salt concentrations were markedly increased" @default.
• W1987127854 created "2016-06-24" @default.
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• W1987127854 creator A5016110387 @default.
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• W1987127854 date "2005-03-01" @default.
• W1987127854 modified "2023-10-14" @default.
• W1987127854 title "Impaired response of biliary lipid secretion to a lithogenic diet in phosphatidylcholine transfer protein-deficient mice" @default.
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