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J. Biol. Chem., Vol. 276, Issue 51, 48058-48065, December 21, 2001

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Disruption of the Sterol Carrier Protein 2 Gene in Mice Impairs Biliary Lipid and Hepatic Cholesterol Metabolism^*

Michael Fuchs^§, Andrea Hafer, Christian Münch, Frank Kannenberg^¶, Sandra Teichmann, Jürgen Scheibner, Eduard F. Stange, and Udo Seedorf^¶

From the  Division of Gastroenterology, Department of Medicine I, Medical University of Lübeck, D-23538 Lübeck, Germany and the ^¶ Institute for Arteriosclerosis Research and Institute for Clinical Chemistry and Laboratory Medicine, University of Münster, D-48129 Münster, Germany

Received for publication, July 17, 2001, and in revised form, October 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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/6^Scp2(/) 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/6^Scp2(/) 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/6^Scp2(/) mice. When C57BL/6^Scp2(/) 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1, 2) and in humans (3). The plasma membrane contains up to 90% of total cell cholesterol (4), 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 (5) and carrier proteins such as sterol carrier protein 2 (Scp2)¹ (6) and the Niemann Pick type C1 (Npc1) protein (7).

Scp2 is a soluble lipid transfer protein, which is capable of cholesterol transport in vitro (6, 8, 9). Although localized predominantly to peroxisomes, substantial amounts of Scp2 are present in cytosol (10, 11), suggesting that Scp2 may be involved in intracellular cholesterol transport in vivo. Indeed, several lines of evidence support this notion: (i) Scp2 antisense treatment of rats reduced and delayed biliary cholesterol secretion (6); (ii) diosgenin-induced hypersecretion of cholesterol into bile in rats was associated with increased hepatic Scp2 expression (6); (iii) adenovirus-mediated overexpression of Scp2 in mice led to increased biliary cholesterol and bile salt secretion rates (12); (iv) cytosolic levels of Scp2 were elevated in livers of genetically cholesterol gallstone-susceptible mice, even before gallstones had formed (13). These findings, together with elevated hepatic SCP2 levels in human gallstone carriers (14), 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 the Scp2 gene (C57BL/6^Scp2(/) mice) (15) 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/6^Scp2(/) 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Standard molecular biological techniques were applied, and sequencing was performed by the dideoxy chain termination method (16). DNA modifying enzymes, molecular weight markers, and Taq DNA polymerase were purchased from Roche Molecular Biochemicals (Mannheim, Germany). [-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 against L-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).

Animals-- Male C57BL/6 mice were obtained from Charles River Laboratories Inc. (Wilmington, MA). Mice with targeted disruption of the Scp2 gene (C57BL/6^Scp2(/)) 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 (15) 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 -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.

Lipid and Lipoprotein Analysis-- Hepatic biles were stored at 20 °C until use for lipid analyses. Phospholipids were measured with a standard enzymatic method (13). Biliary cholesterol and bile salts were analyzed employing gas chromatography mass spectrometry as described in detail elsewhere (15). 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 (17) 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 (18), and fractions of 0.5 ml were collected. Total cholesterol concentrations of each fraction and of plasma were determined employing a commercial colorimetric assay.

RT-PCR-- 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 (19, 20). 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 (21).

Northern Blot Analysis-- cDNA encoding a fragment (394 bp) of mouse Fabpl (22) 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).

Western Blot Analysis-- Liver homogenate and cytosol were prepared (13), and protein concentrations were assayed using bovine serum albumin as standard (13). Equal quantities of mouse liver homogenate or cytosol (50-100 µg) were subjected to reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13). 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.

Measurement of Bile Acid Pool Size and Composition-- Separate groups of chow-fed C57BL/6 (n = 3) and C57BL/6^Scp2(/) (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 (23). Following filtration, the volume was adjusted to a final volume of 100 ml of which an aliquot of 20 ml was taken to dryness (24). Bile salt composition of the dried extract was analyzed employing gas chromatography mass spectrometry as described in detail elsewhere (15). Together with the recovery of the radiolabeled internal standard, the bile salt pool size was calculated.

Measurement of Intestinal Cholesterol Absorption-- A dual-isotope ratio method was employed for the measurement of intestinal cholesterol absorption (24). 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.

Statistics-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gallstone Formation, Body and Liver Weight, and Hepatic Cholesterol Content-- 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/6^Scp2(/) mice (36 ± 3g) was significantly (p < 0.05) higher compared with wild-type animals (26 ± 1g) (15). 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/6^Scp2(/) 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/6^Scp2(/) 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/6^Scp2(/) 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).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of the lithogenic diet on the hepatic cholesterol concentrations. Male C57BL/6 and C57BL/6Scp2(/) mice were fed either the chow or the lithogenic diet for 3 months. Livers were harvested and hepatic total, free (white) and esterified cholesterol (black) concentrations were measured enzymatically. Each stacked bar represents the mean ± 1 S.D. of four mice that received the chow or the lithogenic diet. *, p < 0.05, compared with C57BL/6 mice.

Biliary Lipid Secretion Rates-- 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/6^Scp2(/) mice (cholesterol: 5.3 ± 2.3; phospholipids: 30 ± 8). Compared with chow-fed C57BL/6^Scp2(/) 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/6^Scp2(/) 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/6^Scp2(/), 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.

View this table: [in this window] [in a new window]   Table I Effect of the lithogenic diet on biliary lipid secretion rates and bile flow 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.

Plasma Lipoprotein Profiles-- 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/6^Scp2(/) animals (60 mg/dl). In response to the lithogenic diet, total plasma cholesterol levels in C57BL/6 and C57BL/6^Scp2(/) mice increased to 129 and 110 mg/dl, respectively. Under these conditions, VLDL/LDL cholesterol levels increased substantially in C57BL/6 and C57BL/6^Scp2(/) mice, but this was less pronounced in C57BL/6^Scp2(/) mice. HDL cholesterol levels decreased upon feeding the lithogenic diet to 12 and 34 mg/dl in C57BL/6 and C57BL/6^Scp2(/) animals, respectively. This finding indicates that SCP-2 expression may regulate lipoprotein cholesterol metabolism.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of the lithogenic diet on the distribution of plasma lipoprotein cholesterol by particle size. Mice fed a chow () or lithogenic diet () were briefly anesthetized with Fluothane, and blood was collected from the retro-orbital sinus. Equal volumes of plasma from six animals (panel A, C57BL/6; panel B, C57BL/6Scp2(/)) performed in triplicate were pooled, and lipoproteins were size-fractionated by fast performance liquid chromatography. The approximate retention times for mouse VLDL, LDL, and HDL are indicated by the brackets.

Lipoprotein Receptor and Apolipoprotein Expression in Liver-- Modifications in the plasma lipoprotein cholesterol distribution may have been caused by changes in hepatic lipoprotein synthesis and/or receptor-mediated lipoprotein cholesterol clearance. We therefore evaluated the hepatic expression of key cell surface receptors and apolipoproteins in lipoprotein metabolism. Scp2 deficiency did not change steady-state mRNA expression of the HDL receptor Srb1 (Fig. 3). After feeding the lithogenic diet, Srb1 mRNA levels increased significantly by 251% (p < 0.001), whereas no change occurred in C57BL/6 ^Scp2(/) mice. As shown by Western blot analysis under basal conditions, Scp2 deficiency did not influence Srb1 protein expression. When challenged with the lithogenic diet, steady-state Srb1 levels (Fig. 3) decreased in both groups by 32-34% (p > 0.05). Hepatic Ldlr mRNA levels were similar in C57BL/6^Scp2(-1-) and C57BL/6^Scp2(/) mice fed the control diet. When put on the lithogenic diet, C57BL/6^Scp2(/) mice responded with a significant (p < 0.01) 31% suppression of Ldlr mRNA levels, but no change was observed for C57BL/6 mice (data not illustrated). The steady-state expression of ApoA1 and ApoE, major ligands for the HDL and LDL receptor in mice, was investigated next. ApoA1 protein expression was significantly reduced to 67% (p < 0.05) in C57BL/6^Scp2(/) mice fed the control diet. When challenged with the lithogenic diet, ApoA1 protein levels were not suppressed in mice lacking Scp2 protein expression, whereas a significant 35% (p < 0.05) decrease was noticed in C57BL/6 mice (Fig. 4). Similar to the situation for ApoA1, ApoE protein levels in chow-fed C57BL/6^Scp2(/) mice were significantly (p < 0.01) decreased by 50% compared with C57BL/6 mice. In response to the lithogenic diet, no significant difference in ApoE protein expression was detected for both groups of mice. These results show that Scp2 deficiency can influence hepatic apolipoprotein and lipoprotein receptor expression.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of the lithogenic diet on Srb1 gene expression. Liver homogenate and total liver RNA were prepared from mice fed a chow (black bar) and lithogenic diet (white bar). Steady-state Srb1 mRNA levels were determined by multiplex RT-PCR, and Srb1 protein levels were measured with Western blotting. Signals corresponding to Srb1 mRNA were normalized employing 18S rRNA. Each bar represents the mean ± 1 S.D. for four to six mice. *, p < 0.001, compared with chow diet.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of the lithogenic diet on steady-state Apoa1 and Apoe protein levels. Liver homogenate was prepared from mice fed a chow and lithogenic diet. Steady-state protein levels were measured with Western blotting. Each bar represents the mean ± 1 S.D. for four to six mice that received chow (black) or the lithogenic diet (white). *, p < 0.05, compared with chow diet.

Hepatic Lipid Regulatory Enzymes-- Alterations in the expression of lipid regulatory enzymes may regulate the availability of free cholesterol for excretion into bile. Therefore, we employed RT-PCR to study steady-state mRNA levels of Hmgcr, the rate-limiting enzyme in cholesterol biosynthesis and Acat2, which is required for cholesteryl ester formation. As shown in Fig. 5, basal steady-state Hmgcr mRNA levels were 4-fold (p < 0.01) elevated in C57BL/6^Scp2(/) mice. In response to the lithogenic diet, Hmgcr mRNA levels were significantly (p < 0.01) suppressed by 82% in C57BL/6 mice. In contrast, the decrease of Hmgcr mRNA levels in C57BL/6^Scp2(/) mice was only 21% (p > 0.05). On standard chow diet we did not find a significant difference in Acat2 mRNA expression (Fig. 5) among C57BL/6 and C57BL/6^Scp2(/) mice. When the mice were fed the lithogenic diet, Acat2 mRNA levels increased significantly (p < 0.01) by 57% and 78%, respectively. These findings are in line with increased hepatic cholesterol synthesis in C57BL/6^Scp2(/) mice. Consistent with a lower hepatic free cholesterol content, Scp2 deficiency is associated with an insufficient suppression of hepatic cholesterol synthesis in response to the lithogenic diet.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of the lithogenic diet on steady-state Hmgcr and Acat2 mRNA levels. Total liver RNA was prepared from mice fed a chow and lithogenic diet. Steady-state Hmgcr and Acat2 mRNA levels were determined by multiplex RT-PCR. Signals corresponding to Hmgcr and Acat2 mRNA were normalized employing 18 S rRNA. Each bar represents the mean ± 1 S.D. for four to six mice that received chow (black) or the lithogenic diet (white). *, p < 0.01, compared with chow diet; **, p < 0.01, compared with chow-fed C57BL/6Scp2(/).

Putative Cholesterol-transporting Proteins-- Increased hepatic Scp2 expression and enhanced hepatocellular cholesterol trafficking occurs in gallstone-susceptible C57L/J mice fed a lithogenic diet (13). Although we measured a comparable increase in cytosolic Scp2 levels in C57BL/6 mice fed the lithogenic diet (data not illustrated), biliary cholesterol secretion rates were not decreased in C57BL/6^Scp2(/) mice. To investigate whether other putative cholesterol-transporting proteins may maintain hepatocellular cholesterol transport, we studied steady-state Npc1 and Fabpl expression. As determined by multiplex RT-PCR, lack of Scp2 protein expression did not alter Npc1 mRNA levels and no change was observed when the mice received the lithogenic diet (data not illustrated). As depicted in Fig. 6, we observed a dramatic 16-fold (p < 0.001) induction of steady-state Fabpl mRNA levels in chow-fed Scp2-deficient mice. When challenged with the lithogenic diet, Fabpl mRNA expression levels in C57BL/6^Scp2(/) mice increased slightly by 27% (p > 0.05). Although C57BL/6 mice responded to the lithogenic diet with a 2-fold increase of Fabpl mRNA expression, this did not reach statistical significance. The corresponding steady-state L-Fabp protein levels in chow-fed C57BL/6 mice were 6-fold lower (p < 0.01) compared with C57BL/6^Scp2(/) animals. Whereas L-Fabp protein levels increased 3-fold (p < 0.01) in C57BL/6 mice fed the lithogenic diet, no further elevation was noticed in C57BL/6^Scp2(/) mice. This finding, together with similar biliary cholesterol secretion rates in chow-fed animals, supports the concept of a compensatory hepatic up-regulation of Fabpl gene expression in C57BL/6^Scp2(/) mice.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of the lithogenic diet on Fabpl gene expression. Liver cytosol and total liver RNA were prepared from mice fed a chow (black bar) and lithogenic diet (white bar). Steady-state Fabpl mRNA levels were determined by multiplex RT-PCR, and L-FABP protein levels were measured with Western blotting. Signals corresponding to Fabpl mRNA were normalized employing 18 S rRNA. Each bar represents the mean ± 1 S.D. for four to six mice. *, p < 0.01, compared with chow diet; **, p < 0.01, compared with chow-fed C57BL/6Scp2(/).

Bile Salt Pool Size and Composition-- To assess whether disruption of the Scp2 gene alters size and composition of the bile salt pool, we extracted bile salts from gallbladder, liver, intestine, and their contents from C57BL/6 and C57BL/6^Scp2(/) mice fed the standard chow diet. Although C57BL/6 mice had a mean bile salt pool size that was 3-fold higher than in C57BL/6^Scp2(/) animals, this did not reach statistical significance. However, the bile salt pool composition of mice with disruption of the Scp2 gene was 32-fold (p < 0.05) enriched with 23-norcholate whereas chenodeoxycholate was reduced by 90% (p < 0.001). The muricholate and cholate content of the bile salt pool did not differ significantly among the two groups of mice. This suggests that Scp2 gene expression may alter size and composition of the bile salt pool.

Intestinal Cholesterol Absorption-- Scp2 is expressed not only in liver but also in the intestine and, based on in vitro studies, has been proposed to participate in cholesterol absorption (25-27). To address this possibility, we measured intestinal cholesterol absorption in vivo under conditions where the mice received diets with a low and high cholesterol content. As shown in Fig. 7, Scp2 deficiency slightly reduced the percentage of cholesterol that is absorbed from the intestine (65% versus 73%; p > 0.05). When the lithogenic diet was given to C57BL/6 and C57BL/6^Scp2(/) mice, cholesterol absorption decreased significantly (p < 0.001) to 27% and 38%, respectively. This indicates that adaptive changes of intestinal cholesterol absorption in response to the lithogenic diet are not impaired in mice with disruption of the Scp2 gene.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Effect of the lithogenic diet on intestinal cholesterol absorption. The percentage of intestinal cholesterol absorption was measured by a fecal dual-isotope ratio method. Each bar represents the mean ± 1 S.D. of six mice that received the chow (black) or the lithogenic diet (white). *, p < 0.001, compared with chow diet.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Scp2 gene codes via alternative transcription initiation at two separated promoters (28) for two proteins, sterol carrier protein X (Scpx) and sterol carrier protein 2 (Scp2). Scpx has been shown to play a critical role in -oxidation of the cholesterol side chain in bile acid synthesis (29). Cytosolic Scp2 expression in liver correlated with biliary cholesterol secretion rates in C57L/J mice (13), which is in agreement with Scp2 mediated intracellular cholesterol trafficking (6, 12). We now show that C57BL/6^Scp2(/) mice are characterized by abnormalities of biliary lipid secretion and hepatic lipid metabolism, which appear to be attributed to deficiency of Scpx and Scp2.

Compared with chow-fed C57BL/6 mice, cholesterol and phospholipid output into bile was similar in C57BL/6^Scp2(/) mice. In contrast, bile salt secretion was 3-fold higher in wild-type animals. Most likely, this is a result of (i) an impaired bile salt synthesis caused by Scpx deficiency (29) and (ii) a more hydrophilic bile salt pool with substantially reduced chenodeoxycholate levels. Despite a lower bile salt secretion, bile flow was 1.5-fold higher in C57BL/6^Scp2(/) mice, which is most likely caused by an increase in the bile salt-independent bile flow.

In response to the lithogenic diet, bile flow increased significantly only in C57BL/6 mice related to a substantial increase in bile salt secretion. In both groups of mice, not only bile salt but also biliary phospholipid secretion increased to a similar extent in response to the lithogenic diet. Whereas C57BL/6 mice fed the lithogenic diet substantially increased biliary cholesterol secretion, this was much less pronounced in C57BL/6^Scp2(/) mice. The amount of cholesterol secreted into bile depends in part on a complex interrelationship with intestinal cholesterol absorption (30), bile salt secretion rate, the hydrophobicity of the cycling bile salt pool, and the availability of free cholesterol in a metabolic pool of the hepatocyte that is still ill defined (31). Although in vitro studies have suggested that Scp2 may participate in cholesterol absorption from the intestine (26), cholesterol absorption was not decreased in C57BL/6^Scp2(/) mice, irrespective of the diet used. Therefore, a decreased cholesterol flux from the intestine to the liver apparently does not account for the decreased biliary cholesterol secretion rate. Instead, the reduced bile salt secretion rate may have contributed to the lower cholesterol secretion rate. Because the cycling bile salt pool of both groups of mice fed the lithogenic diet consisted to ~96% of cholate,² the lower cholesterol output in C57BL/6^Scp2(/) mice apparently is not attributable to a bile salt pool with increased hydrophobicity. Scp2 expression in liver cytosol correlates with biliary cholesterol secretion in mice (13) and studies with cultured McA-RH7777 rat hepatoma cells transfected with Scp2 (32) and human fibroblasts (6) suggested a role of Scp2 in regulating intracellular cholesterol distribution. Therefore, our results are consistent with decreased cholesterol transport from liver into bile, as has been shown recently in rats treated with Scp2 antisense oligonucleotides (33). In addition, elevated cholesterol/phospholipid ratios of hepatic bile, which reflect the lipid composition of vesicles secreted at the canalicular membrane (34) together with altered cholesterol/bile salt and phospholipid/bile salt coupling, indicate that structural changes at the level of the canalicular membrane (12) may have contributed to the altered biliary cholesterol secretion rate. Support comes from the observation that Scp2 may regulate cholesterol distribution between different kinetic domains of the plasma membrane (35, 36). Clearly, this aspect warrants further investigation, especially because altered plasma membrane lipid composition appears to influence trans-bilayer transport of biliary constituents (37).

Impaired biliary cholesterol secretion in C57BL/6^Scp2(/) mice was unmasked only when the animals received a cholesterol enriched lithogenic diet. This indicates that hepatocellular cholesterol trafficking in C57BL/6^Scp2(/) mice cannot be compensated under conditions with an increased flux of dietary cholesterol to the liver. As inferred from studies with human fibroblasts (6), we cannot exclude compensatory up-regulation of vesicular transport of cholesterol. Npc1, which is involved in cytosolic processing of LDL-derived lysosomal cholesterol (38), is not induced and can be excluded to compensate for the Scp2 deficiency. Induction of Fabpl gene expression in livers of C57BL/6^Scp2(/) mice may represent a compensatory mechanism to transport cholesterol intracellularly. This assumption is supported by in vitro experiments demonstrating the capability of L-Fabp to bind cholesterol and fluorescent sterols (39-41). Although controversial (42, 43), this view is supported by observations in diosgenin-fed rats, where biliary cholesterol hypersecretion (33) was associated with induction of Fabpl gene expression (44). Further experiments beyond the scope of this investigation, e.g. employing mice with targeted disruption of the Fabpl gene or adenoviral mediated L-Fabp overexpression should help to resolve the putative role of L-Fabp in hepatocellular cholesterol trafficking in vivo.

In C57BL/6 mice, the lithogenic diet increased hepatic total cholesterol concentration. Because hepatic Hmgcr mRNA levels in mice closely reflect hepatic cholesterol synthesis (45), substantially decreased Hmgcr mRNA levels indicated feedback inhibition of cholesterol synthesis. In keeping with previous results (15), total hepatic cholesterol contents were not influenced in C57BL/6^Scp2(/) mice. Instead, the hepatic cholesteryl ester content was increased in both groups of mice, which related to elevated Acat2 mRNA levels. Unexpectedly, hepatic Hmgcr mRNA expression was 4-fold induced in C57BL/6^Scp2(/) mice, suggesting that hepatic cholesterol synthesis was induced. Whether this is related to the induction of Fabpl gene expression (46, 47) remains to be elucidated. Another interesting finding was the failure to down-regulate Hmgcr mRNA in C57BL/6^Scp2(/) mice that received the lithogenic diet. Increased Acat2 mRNA levels and a disproportionally high cholesteryl ester formation yielded a hepatic free cholesterol content apparently not high enough to mediated feedback suppression of cholesterol synthesis. This explanation is supported by studies with Scp2-overexpressing mice where an increased hepatic content of free cholesterol was associated with decreased cholesterol synthesis (12).

Studies with Scp2-transfected rat hepatoma cells (32) and mice (12) demonstrated inhibition of ApoA1 and ApoE gene expression. Reduced ApoA1 protein levels in these studies may have resulted in decreased HDL cholesterol secretion, especially because ApoA1 synthesis may determine formation of HDL particles (48). This is in line with our results of decreased plasma HDL cholesterol levels and reduced hepatic ApoA1 protein levels in C57BL/6 mice fed the lithogenic diet. Based on the Scp2 transfection studies (12, 32), one would predict elevated apoprotein levels in livers of C57BL/6^Scp2(/) mice. However, this was clearly not the case. We speculate that the altered intracellular cholesterol distribution and availability may have contributed because changes in apolipoprotein expression are known to depend on the cholesterol content in putative regulatory cholesterol pools (49, 50).

Preferential biliary secretion of HDL cholesterol compared with non-HDL cholesterol is supported by an inverse relationship between plasma HDL cholesterol levels and biliary cholesterol in mice with variations in the hepatic expression of the HDL receptor Srb1 (21, 51-53). Despite increased Srb1 mRNA levels and in line with previous studies in C57BL/6 mice (45), Srb1 protein levels were not induced in response to the lithogenic diet. This contrasts the situation in C57L mice, where biliary cholesterol hypersecretion is associated with hepatic up-regulation of Srb1 expression during gallstone formation (21).

Collectively, compelling evidence has now accumulated supporting the concept that, at least in mice, Scp2 plays a role in hepatic cholesterol and lipoprotein metabolism and also in biliary lipid secretion. Whether L-Fabp may act as a cytosolic cholesterol-binding protein in vivo deserves further investigation and may help to elucidate the molecular mechanisms of hepatocellular cholesterol trafficking leading to the identification of targets to prevent diseases related to abnormal hepatic cholesterol metabolism such as gallstone disease or atherosclerosis.

	ACKNOWLEDGEMENTS

We thank Nadine Katzberg and Janine Pertack for excellent technical assistance.

	FOOTNOTES

^* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Fu 288/2; by Medical Faculty Project N10, Medical University of Lübeck; and by the "Interdisziplinäres Klinisches Forschungszentrum" of the Medical Faculty, University of Münster (Project A4).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Medicine I, University of Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. Tel.: 49-731-500-33824/24327; Fax: 49-731-500-24302; E-mail: michael.fuchs@medizin.uni-ulm.de.

Published, JBC Papers in Press, October 22, 2001, DOI 10.1074/jbc.M106732200

² M. Fuchs, unpublished results.

	ABBREVIATIONS

The abbreviations used are: Scp2, sterol carrier protein 2; Acat2, acyl-CoA:cholesterol acyltransferase 2; ApoA1, apoprotein AI; ApoE, apoprotein E; Fabp, fatty acid-binding protein; HDL, high density lipoprotein; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; LDL, low density lipoprotein; L-Fabp, fatty acid-binding protein of liver; Npc1, Niemann Pick type C1; RT, reverse transcription; Scpx, sterol carrier protein X; Srb1, scavenger receptor B class I; VLDL, very low density lipoprotein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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