Disruption of the Sterol Carrier Protein 2 Gene in Mice Impairs Biliary Lipid and Hepatic Cholesterol Metabolism
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m106732200
ISSN1083-351X
AutoresMichael Fuchs, Andrea Hafer, Christian Münch, Frank Kannenberg, Sandra Teichmann, Jürgen Scheibner, Eduard F. Stange, Udo Seedorf,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoHepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6Scp2 (−/−) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6Scp2 (−/−) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6Scp2 (−/−) mice. When C57BL/6Scp2 (−/−) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution. Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6Scp2 (−/−) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6Scp2 (−/−) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6Scp2 (−/−) mice. When C57BL/6Scp2 (−/−) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution. sterol carrier protein 2 acyl-CoA:cholesterol acyltransferase 2 apoprotein AI apoprotein E fatty acid-binding protein high density lipoprotein 3-hydroxy-3-methylglutaryl-CoA reductase low density lipoprotein fatty acid-binding protein of liver Niemann Pick type C1 reverse transcription sterol carrier protein X scavenger receptor B class I very low density lipoprotein Cholesterol gallstone disease is characterized by a perturbation of the physical-chemical balance of cholesterol solubility in bile with an unphysiological cholesterol saturation. Hypersecretion of unesterified cholesterol into bile appears to represent the key molecular mechanism in the gallstone-susceptible C57L/J mouse (1Wang D.Q.H. Lammert F. Paigen B. Carey M.C. J. Lipid Res. 1999; 40: 2066-2079Abstract Full Text Full Text PDF PubMed Google Scholar, 2Wang D.Q.-H. Lammert F. Cohen D.E. Paigen B. Carey M.C. Am. J. Physiol. 1999; 276: G751-G760Crossref PubMed Google Scholar) and in humans (3Apstein M.D. Carey M.C. Eur J. Clin. Invest. 1996; 26: 343-352Crossref PubMed Scopus (105) Google Scholar). The plasma membrane contains up to 90% of total cell cholesterol (4Lange Y. Swaisgood M.H. Ramos B.V. Steck T.L. J. Biol. Chem. 1989; 264: 3786-3793Abstract Full Text PDF PubMed Google Scholar), which implies that sterol trafficking in the cell is tightly controlled. Efforts to elucidate the complex molecular mechanisms of intracellular cholesterol transport suggested the contribution of vesicles (5Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (248) Google Scholar) and carrier proteins such as sterol carrier protein 2 (Scp2)1 (6Puglielli L. Rigotti A. Greco A.V. Santos M.J. Nervi F. J. Biol. 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Biol. Chem. 1995; 270: 18723-18726Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar); (ii) diosgenin-induced hypersecretion of cholesterol into bile in rats was associated with increased hepatic Scp2 expression (6Puglielli L. Rigotti A. Greco A.V. Santos M.J. Nervi F. J. Biol. Chem. 1995; 270: 18723-18726Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar); (iii) adenovirus-mediated overexpression of Scp2 in mice led to increased biliary cholesterol and bile salt secretion rates (12Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodriguez A. Kozarsky K. Quinones V. Rigotti A. Nervi F. Gastroenterology. 2000; 119: 1708-1719Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar); (iv) cytosolic levels of Scp2 were elevated in livers of genetically cholesterol gallstone-susceptible mice, even before gallstones had formed (13Fuchs M. Lammert F. Wang D.Q.H. Paigen B. Carey M.C. Cohen D.E. Biochem. J. 1998; 336: 33-37Crossref PubMed Scopus (58) Google Scholar). These findings, together with elevated hepatic SCP2 levels in human gallstone carriers (14Ito T. Kawata S. Imai Y. Kakimoto H. Trzaskos J.M. Matsuzawa Y. Gastroenterology. 1996; 110: 1619-1627Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), support the concept that Scp2 is involved in hepatocellular trafficking of cholesterol to the canalicular membrane for biliary secretion. The availability of mice with homozygous disruption of theScp2 gene (C57BL/6Scp2 (−/−) mice) (15Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (244) Google Scholar) allowed us to test the hypothesis that Scp2 deficiency impairs biliary lipid secretion and hepatic lipid metabolism. In the present study, we show impaired biliary lipid secretion, hepatic cholesterol synthesis and content, lipoprotein metabolism, intracellular cholesterol distribution, and bile salt metabolism in C57BL/6Scp2 (−/−) mice. This mouse model unmasked a putative but yet not appreciated role of liver fatty acid-binding protein (l-Fabp) in hepatic cholesterol metabolism and biliary lipid secretion. Standard molecular biological techniques were applied, and sequencing was performed by the dideoxy chain termination method (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). DNA modifying enzymes, molecular weight markers, and Taq DNA polymerase were purchased from Roche Molecular Biochemicals (Mannheim, Germany). [α-32P]dCTP (3000 mCi/mmol), [4-14C]cholesterol (60 mCi/mmol), and [24-14C]taurocholic acid (40 mCi/mmol) were from PerkinElmer Life Sciences (Frankfurt, Germany). [5,6-3H]Sitostanol (30Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Primers were obtained from MWG Biotech (Ebersberg, Germany). Rabbit anti-human APOAI and goat anti-human APOE antibodies were purchased from Calbiochem (Bad Soden, Germany). A polyclonal antibody raised against a peptide of the carboxyl terminus of mouse Srb1 was kindly provided by Dr. S. Azhar (Palo Alto, CA). An affinity-purified antibody againstl-Fabp was a generous gift from Drs. Wolfrum and Spener (University of Münster, Münster, Germany). Horseradish peroxidase-labeled secondary antibodies were from DAKO Chemicals (Hamburg, Germany). Unless otherwise indicated, materials were obtained from Sigma (Deisenhofen, Germany) or Biometra (Göttingen, Germany). Male C57BL/6 mice were obtained from Charles River Laboratories Inc. (Wilmington, MA). Mice with targeted disruption of the Scp2 gene (C57BL/6Scp2 (−/−)) by homologous recombination were kindly provided by Dr. Udo Seedorf (Institute for Arteriosclerosis Research, University of Münster, Münster, Germany) and bred to generate our own mouse colony. The animals were maintained under constant light-dark cycles (light from 6:00 a.m. to 6:00 p.m.) with free access to water and a standard chow diet (∼3% fat, <0.02% cholesterol; Altromin 1340, Altromin GmbH, Lage, Germany). Genotype analysis using DNA isolated from mouse tail tips (15Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (244) Google Scholar) was performed with all knockout mice of our colony. Twelve-week-old mice were put on a lithogenic diet (TD 90221, Teklad Research Diets, Madison, WI) consisting of 15% cocoa fat, 1.25% cholesterol, and 0.5% sodium cholate or on standard chow diet for 3 months. Despite significant amounts of cholic acid present in the lithogenic diet, none of the animals developed diarrhea. Liver histology and measurements of bilirubin, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transferase excluded hemolysis and significant liver disease under our experimental conditions. After 3 months on the diet, overnight fasted mice were anesthetized with pentobarbital (35 mg/kg body weight). The abdominal cavity was then opened by a midline incision to expose the gallbladder and biliary tract for visual inspection. To minimize the influence of circadian variations on our measurements, surgery was done between 9:00 and 10.00 a.m. If present, gallstones were visible to the unaided eye through the gallbladder wall. We next performed a cholecystectomy, followed by cannulation of the common bile duct with a PE-10 polyethylene catheter (Clay Adams, Parsippany, NJ). Hepatic biles were collected by gravity for 1 h, during which the body temperatures of the mice were kept constant with a heating lamp. Thereafter, livers were harvested, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C until use for mRNA and protein determinations. Throughout the experimental period, all animals received human care according to the criteria for the care and use of laboratory animals. Protocols were approved by the Institutional Animal Care and Use Committee. Euthanasia was consistent with recommendations of the American Veterinary Medical Association. Hepatic biles were stored at −20 °C until use for lipid analyses. Phospholipids were measured with a standard enzymatic method (13Fuchs M. Lammert F. Wang D.Q.H. Paigen B. Carey M.C. Cohen D.E. Biochem. J. 1998; 336: 33-37Crossref PubMed Scopus (58) Google Scholar). Biliary cholesterol and bile salts were analyzed employing gas chromatography mass spectrometry as described in detail elsewhere (15Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (244) Google Scholar). Bile flow rates were determined gravimetrically assuming a density of 1 g/ml and used to calculate biliary lipid secretion rates. To determine hepatic cholesterol contents, lipids were extracted from liver tissue (17Bligh E.G. Dyer W.J. Can. J. Biol. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42389) Google Scholar) and dissolved in isopropanol, and aliquots were used for colorimetric determination of total and free cholesterol. Prior to harvesting, livers were briefly flushed with saline to minimize the contribution from plasma cholesterol in determinations of hepatic cholesterol contents. For determinations of plasma lipoproteins following fasting for 12–16 h, blood was collected from the retro-orbital venous plexus into tubes containing 1 μl of 1 mm EDTA. Pooled plasma (400 μl total from up to 6 animals) was subjected to fast performance liquid chromatography using two Superose 6 columns (Amersham Biosciences, Freiburg, Germany) connected in series. Proteins were eluted using a buffer containing 154 mm NaCl, 1 mm EDTA, 0.02% NaN3 at 0,5 ml/min (18Jiao S. Cole T.G. Kitchens R.T. Pfleger B. Schonfeld G. Metabolism. 1990; 39: 155-160Abstract Full Text PDF PubMed Scopus (97) Google Scholar), and fractions of 0.5 ml were collected. Total cholesterol concentrations of each fraction and of plasma were determined employing a commercial colorimetric assay. Relative quantitation of transcript abundance employed reverse transcription-polymerase reaction. Because of its invariant expression across treatment conditions, 18 S ribosomal RNA was used as internal standard (19Suzuki T. Higgins P.J. Crawford D.R. BioTechniques. 2000; 29: 332-337Crossref PubMed Scopus (709) Google Scholar, 20Thellin O. Zorzi W. Lakaye B. de Borman B. Coumans B. Hennen G. Grisar T. Igout A. Heinen E. J. Biotechnol. 1999; 8: 291-295Crossref Scopus (1255) Google Scholar). Total mouse liver RNA was isolated using TRIzol reagent (Life Technologies, Inc., Munich, Germany) according to the manufacturer's instructions and reverse transcription was performed with the SuperScript II preamplification system (Life Technologies, Inc.). An aliquot of the reaction was subjected to PCR amplification using gene specific primers together with a "competimer"/primer mix specific for 18 S ribosomal RNA (QuantumRNA 18 S Internal Standards, Ambion, Austin, TX). Gene-specific cDNAs were amplified with the following primer pairs (the size of the resulting amplicon is given in parentheses):Hmgcr (882 bp): 5′-ATC ATC TTG GAG AGA TAA AAC TGC CA-3′ (sense), 5′-GGG ACG GTG ACA CTT ACC ATC TGT ATG ATG-3′ (antisense);Acat2 (423 bp): 5′-CAT CTC GCC GAA GGC GTT GAG-3′ (sense), 5′-CGC TGC GTG CTG GTC TTT GAG-3′ (antisense); Ldlr (210 bp): 5′-CTC CTC ATT CCC TCT GCC AGC CAT-3′ (sense), 5′-GAA GTC GAC ACT GTA CTG ACC ACC-3′ (antisense); Srb1 (590 bp): 5′-AGT GGG GGT GGG AGA GAA AC-3′ (sense), 5′-CAA GCC TGT GAG CCT GAA GC-3′ (antisense); Npc1 (457 bp): 5′-GCA TCT TCT GTT GCA GCA GC-3′ (sense), 5′-GGT TCT CAT TCC TTG CGC CA-3′ (antisense). Competimers are primers that can not be extended. The ratio of 18 S competimers to primers was adjusted such that the amount of 18 S rRNA product was within the same range as that of the mRNAs of interest. Amplified samples were subjected to densitometry following agarose gel electrophoresis with ethidium bromide staining. Initial validation of the RT-PCR protocol demonstrated similar results when compared with Northern blot analysis (21Fuchs M. Ivandic B. Müller O. Schalla C. Scheibner J. Bartsch P. Stange E.F. Hepatology. 2001; 33: 1451-1459Crossref PubMed Scopus (48) Google Scholar). cDNA encoding a fragment (394 bp) of mouse Fabpl (22Wolfrum C. Ellinghaus P. Fobker M. Seedorf U. Assmann G. Börchers T. Spencer F. J. Lipid Res. 1999; 40: 708-714Abstract Full Text Full Text PDF PubMed Google Scholar) was synthesized by reverse transcription-polymerase chain reactions employing total mouse liver RNA and the following primer set: 5′-AAA TTC TCT TGC TGA CTC-3′ (sense); 5′-AAC TTC TCC GGC AAG TAC-3′ (antisense). Employing a TA cloning kit (Invitrogen, The Netherlands), the resulting PCR product of 394 bp was subcloned into pCRII and sequenced. Identity was confirmed by data base comparison using the Basic Local Alignment Search Tool (BLAST). Mouse liver RNA (15 μg) was denatured with formaldehyde and formamide, electrophoresed on 1% (w/v) agarose gels, and transferred onto GeneScreenPlus membranes (PerkinElmer Biosciences). A radiolabeled probe of gel-purified Fabpl cDNAs was obtained employing a random primer labeling kit (Life Technologies, Inc.). The filters were hybridized overnight with the 32P-labeled probe at 42 °C. Following washing, filters were exposed to X-Omat LS films (Kodak, Stuttgart, Germany) at −80 °C with intensifying screens. Densitometry with a GS-700 imaging densitometer (Bio-Rad, München, Germany) using Molecular Analyst 1.5 software was employed to quantitate steady state mRNA levels. To normalize for equivalent loading of RNA, the filter was stripped and rehybridized with a Gapdh probe (CLONTECH, Heidelberg, Germany). Liver homogenate and cytosol were prepared (13Fuchs M. Lammert F. Wang D.Q.H. Paigen B. Carey M.C. Cohen D.E. Biochem. J. 1998; 336: 33-37Crossref PubMed Scopus (58) Google Scholar), and protein concentrations were assayed using bovine serum albumin as standard (13Fuchs M. Lammert F. Wang D.Q.H. Paigen B. Carey M.C. Cohen D.E. Biochem. J. 1998; 336: 33-37Crossref PubMed Scopus (58) Google Scholar). Equal quantities of mouse liver homogenate or cytosol (50–100 μg) were subjected to reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13Fuchs M. Lammert F. Wang D.Q.H. Paigen B. Carey M.C. Cohen D.E. Biochem. J. 1998; 336: 33-37Crossref PubMed Scopus (58) Google Scholar). Proteins were electrophoretically transferred onto 0.45-μm nitrocellulose membranes. Antigen detection was carried out using a 1:5000 dilution of a polyclonal antibody against mouse Srb1. Anti-human APOA1 and APOE antibodies were employed as 1:4000 and 1:1000 dilutions, respectively. The concentration of the antibody against l-Fabp was 2.5 μg/ml. Following incubation with horseradish peroxidase-labeled secondary antibodies (DAKO Chemicals), detection of immunoreactive proteins was accomplished with enhanced chemiluminescence (PerkinElmer Life Sciences) and quantified by scanning densitometry. Separate groups of chow-fed C57BL/6 (n = 3) and C57BL/6Scp2 (−/−) (n = 4) mice were used to measure the bile salt pool size and composition. After an overnight fast, mice were anesthetized with pentobarbital (35 mg/kg body weight). The abdominal cavity was opened by a midline incision to remove gallbladder, liver, and intestine. Organs and their contents were minced in 100 ml of 75% ethanol with 10 μl [24-14C]taurocholic acid added as internal standard. Bile salts were extracted by heating at 50 °C for 4 h (23Yu C. Wang F. Kan M. Jin C. Jones R.B. Weinstein M. Deng C.-X. McKeehan W.L. J. Biol. Chem. 2000; 275: 15482-15489Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Following filtration, the volume was adjusted to a final volume of 100 ml of which an aliquot of 20 ml was taken to dryness (24Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Bile salt composition of the dried extract was analyzed employing gas chromatography mass spectrometry as described in detail elsewhere (15Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (244) Google Scholar). Together with the recovery of the radiolabeled internal standard, the bile salt pool size was calculated. A dual-isotope ratio method was employed for the measurement of intestinal cholesterol absorption (24Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, six mice of each group were housed individually in wire cages and adjusted to this environment for 3 days. After an overnight fast, mice were anesthetized with diethylether just enough to numb the animal for a few seconds to prevent the mouse from biting the polyethylene tubing (PE 86; Clay Adams) used for gavage. Anesthetized animals were given an intragastric bolus of MCT oil containing a mixture of 1 μCi of [4-14C]cholesterol and 2 μCi of [5,6-3H]sitostanol. The total volume of the mixture was adjusted to the body weight of each animal (100 μl/25 g body weight). Following gavage, the mice were placed individually in clean cages to collect the feces for a period of 72 h. Aliquots of dried feces and the dosing mixture were extracted with chloroform-methanol (2:1 v/v), and the [14C]/[3H] ratio of each was determined for calculation of the percent cholesterol absorption. Food intake and weight of dried feces did not differ significantly among the groups studied. Data were analyzed by unpaired, two-tailed Student's t test. Results are expressed as the arithmetic mean ± 1 S.D., and p values < 0.05 were considered to be statistically significant. Cholesterol gallstones were present in gallbladders of all mice fed the lithogenic diet. Common bile duct stones with extrahepatic dilatation of the bile duct occurred in one C57BL/6 mouse, which was not used for further analysis. In contrast, gallstones did not form in animals that received the standard chow diet. This indicates that targeting Scp2 expression does not allow prevention of cholesterol gallstone formation in gallstone-susceptible C57BL/6 mice fed a lithogenic diet. The mean initial body weight of C57BL/6Scp2 (−/−) mice (36 ± 3g) was significantly (p < 0.05) higher compared with wild-type animals (26 ± 1g) (15Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (244) Google Scholar). When challenged with the lithogenic diet, body weights of C57BL/6 mice and Scp2-deficient mice increased to 30 ± 2g (p > 0.05) or remained constant, respectively. The mean initial liver weight (g/100 g body weight) in C57BL/6 and C57BL/6Scp2 (−/−) mice was 4.2 ± 0.4 and 4.2 ± 0.3, respectively. In response to the lithogenic diet, the mean liver weights of both groups increased (p < 0.001) to a similar extent to 8.3 ± 2.0 and 7.0 ± 1.1, respectively. The mean hepatic free cholesterol concentration (mg/g liver) of chow-fed C57BL/6Scp2 (−/−) mice was 2.09 ± 0.33 and significantly (p < 0.005) lower than in C57BL/6 mice (2.98 ± 0.09), reflected by a reduced total hepatic cholesterol concentration (2.73 ± 0.32 versus 3.96 ± 0.38; p < 0.05) (Fig.1). Hepatic total cholesterol contents increased 7–9-fold in response to the lithogenic diet in C57BL/6Scp2 (−/−) and C57BL/6 mice, respectively. This was attributable to a significant increase of the cholesteryl ester concentration, which was more pronounced in C57BL/6 mice (37.4 ± 6.6 versus 18.4 ± 8.7;p < 0.05). To measure biliary lipid secretion rates, hepatic bile was collected during the first hour of an acute bile fistula before and after feeding a lithogenic diet. As shown in Table I, biliary cholesterol and phospholipid secretion rates (nmol/min/100 g body wt) in chow-fed C57BL/6 mice were 2.6 ± 0.6 and 42 ± 17, respectively. Similar secretion rates were obtained for C57BL/6Scp2 (−/−) mice (cholesterol: 5.3 ± 2.3; phospholipids: 30 ± 8). Compared with chow-fed C57BL/6Scp2 (−/−) animals, C57BL/6 mice had 2.7-fold higher (p < 0.01) total bile salt secretion rates and an ∼2-fold lower (5.8 ± 0.8 versus 8.5 ± 1.6 μl/min/100 g body wt; p < 0.05) bile flow. Under control conditions, the cholesterol/phospholipid ratio of hepatic bile that reflects the lipid composition of canalicular vesicles was 3-fold lower in C57BL/6 mice. Additionally, the cholesterol/bile acid and phospholipid/bile acid coupling rate that mirrors the linkage of cholesterol and phospholipid to bile acid secretion was substantially lower in C57BL/6 mice. When C57BL/6 animals were fed the lithogenic diet, cholesterol and phospholipid secretion rates increased significantly to 56 ± 11 (p < 0.001) and 238 ± 62 (p < 0.01), respectively. We measured a comparable and significant (p < 0.001) increase of biliary phospholipid secretion, which amounted to 152 ± 45 in C57BL/6Scp2 (−/−) mice, but biliary cholesterol output was elevated only 3-fold (p < 0.001). As shown for biliary cholesterol and phospholipids, the lithogenic diet increased bile salt secretion significantly (p < 0.001) in both groups. Whereas bile flow remained almost constant in C57BL/6Scp2 (−/−), a significant (p < 0.01) increase to 15.0 ± 2.7 was noticed for C57BL/6 animals. In response to the lithogenic diet, the cholesterol/phospholipid ratio of hepatic bile increased 4-fold in C57BL/6 mice but did not increase in gene ablated mice. The cholesterol/bile acid ratio was identical among the two groups after feeding the lithogenic diet, whereas the phospholipid/bile acid coupling rate declined to a similar degree in both groups. These data indicate that impaired biliary cholesterol but not phospholipid and bile salt secretion in response to the lithogenic diet may reflect alterations at the level of the canalicular plasma membrane.Table IEffect of the lithogenic diet on biliary lipid secretion rates and bile flowStrainDietBiliary lipid secretion ratesBile flowCholesterolPhospholipidsBile saltsnmol/min/100 g body weightμl/min/100 g body weightC57BL/6Chow2.6 ± 0.642 ± 17258 ± 1105.8 ± 0.8Lithogenic56 ± 11ap < 0.001, compared to chow-fed mice.238 ± 62bp < 0.01, compared to chow-fed mice.2957 ± 708ap < 0.001, compared to chow-fed mice.15.0 ± 2.7bp < 0.01, compared to chow-fed mice.C57BL/6Scp2(−/−)Chow5.3 ± 2.330 ± 883 ± 40cp < 0.01, compared to chow-fed C57BL/6 mice.8.5 ± 1.6dp < 0.05, compared to chow-fed C57BL/6 mice.Lithogenic16.3 ± 2.4ap < 0.001, compared to chow-fed mice.152 ± 45ap < 0.001, compared to chow-fed mice.745 ± 298ap < 0.001, compared to chow-fed mice.9.9 ± 2.4C57BL/6 and C57BL/6Scp2(−/−) mice (n = 6–9) were fed a low cholesterol standard chow or a lithogenic diet for 12 weeks. Fasted animals were anesthetized with pentobarbital between 9:00 a.m. and 10:00 a.m. Following laparotomy and cannulation of the common bile duct, hepatic bile was collected for 1 h and biliary lipids were analyzed. Data are expressed as mean ± 1 S.D.a p < 0.001, compared to chow-fed mice.b p < 0.01, compared to chow-fed mice.c p < 0.01, compared to chow-fed C57BL/6 mice.d p < 0.05, compared to chow-fed C57BL/6 mice. Open table in a new tab C57BL/6 and C57BL/6Scp2(−/−) mice (n = 6–9) were fed a low cholesterol standard chow or a lithogenic diet for 12 weeks. Fasted animals were anesthetized with pentobarbital between 9:00 a.m. and 10:00 a.m. Following laparotomy and cannulation of the common bile duct, hepatic bile was collected for 1 h and biliary lipids were analyzed. Data are expressed as mean ± 1 S.D. Lipoprotein profiles of pooled plasma (Fig. 2) obtained from animals fed the control diet demonstrated that most cholesterol was carried in the HDL fraction. However, C57BL/6 mice exhibited lower HDL cholesterol (37 mg/dl) levels than C57BL/6Scp2 (−/−) animals (60 mg/dl). In response to the lithogenic diet, total plasma cholesterol levels in C57BL/6 and C57BL/6Scp2 (−/−) mice increased to 129 and 110 mg/dl, respectively. Under these conditions, VLDL/LDL cholesterol levels increased substantially in C57BL/6 and C57BL/6Scp2 (−/−) mice, but this was less pronounced in C57BL/6Scp2 (−/−) mice. HDL cholesterol levels decreased upon feeding the lithogenic diet to 12 and 34 mg/dl in C57BL/6 and C57BL/6Scp2 (−/−)animals, respectively. This finding indicates that SCP-2 expression may regulate lipoprotein cholesterol metabolism. Modifications in the plasma lipoprotein chol
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