Reduced Susceptibility to Cholesterol Gallstone Formation in Mice That Do Not Produce Apolipoprotein B48 in the Intestine

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1 Reduced Susceptibility to Cholesterol Gallstone Formation in Mice That Do Not Produce Apolipoprotein B48 in the Intestine Helen H. Wang and David Q.-H. Wang It has been found that polymorphisms in the apolipoprotein (APO)-B gene are associated with cholesterol gallstones in humans. We hypothesized that APO-B plays a major regulatory role in the response of biliary cholesterol secretion to high dietary cholesterol and contributes to cholesterol gallstone formation. In the present study, we investigated whether lack of expression of intestinal Apob48 or Apob100 reduces susceptibility to cholesterol gallstones by decreasing intestinal absorption and biliary secretion of cholesterol in male mice homozygous for an APO- B48 only allele (Apob 48/48 ), an APO-B100 only allele (Apob 100/100 ), or a wild-type APO-B allele (Apob / ) before and during an 8-week lithogenic diet. We found that cholesterol absorption was significantly decreased as a result of the APO-B48 deficiency in Apob 100/100 mice compared with wild-type and Apob 48/48 mice, regardless of whether chow or the lithogenic diet was administered. Consequently, hepatic cholesterol synthesis was significantly increased in Apob 100/ 100 mice compared with wild-type and Apob 48/48 mice. On chow, the APO-B100 deficiency in Apob 48/48 mice with reduced plasma levels of LDL/VLDL but not HDL cholesterol induced relative hyposecretion of biliary bile salts and phospholipids accompanying normal biliary cholesterol secretion. Compared with Apob 48/48 and wild-type mice, lithogenic diet fed Apob 100/100 mice displayed significantly lower secretion rates of biliary cholesterol, but not phospholipid or bile salts, which results in significant decreases in prevalence rates, numbers, and sizes of gallstones. In conclusion, absence of expression of intestinal Apob48, but not Apob100, reduces biliary cholesterol secretion and cholelithogenesis, possibly by decreasing intestinal absorption and hepatic bioavailability. (HEPATOLOGY 2005;42: ) Studies in humans and inbred mice have demonstrated that a complex genetic basis determines the individual and strain predisposition to develop cholesterol gallstones in response to environmental factors. 1 The primary pathophysiological defect Abbreviations: APO, apolipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy- 3-methylglutaryl coenzyme A; mrna, messenger RNA. From the Department of Medicine, Liver Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, MA. Received April 5, 2005; accepted July 17, Supported in part by National Institutes of Health grant DK54012 (D.Q.- H.W.) (US Public Health Service). D.Q.-H.W. is a recipient of a New Scholar Award from the Ellison Medical Foundation ( ). Address reprint requests to: David Q.-H. Wang, M.D., Ph.D., Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., DA 601, Boston, MA dqwang@caregroup.harvard.edu; fax: Copyright 2005 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. involved in cholesterol gallstone formation is increased biliary secretion of cholesterol from the liver, which produces bile supersaturated with cholesterol. 2 Subsequently, biliary cholesterol precipitates as anhydrous and monohydrate crystals, which grow and agglomerate toward the formation of macroscopic stones in the gallbladder. Biliary cholesterol is mostly derived from circulating lipoproteins, which originate from the hepatic uptake of plasma high-density lipoprotein (HDL) and chylomicron remnants. 3-8 Thus, the potential interrelationship between lipoprotein metabolism related genes and cholesterol gallstones requires further investigation. In particular, it has been found that polymorphisms in the apolipoprotein (APO)-B gene are associated with cholesterol gallstones in humans Therefore, we hypothesized that APO-B plays a major regulatory role in the response of biliary cholesterol secretion to high dietary cholesterol and contributes to the formation of cholesterol gallstones. APO-B has two different isoforms, APO-B48 and APO-B100, and plays a particularly critical role in the 894

2 HEPATOLOGY, Vol. 42, No. 4, 2005 WANG AND WANG 895 assembly of triglyceride-rich lipoproteins. APO-B100 is mainly synthesized in the liver and has an obligatory structural role in the formation of triglyceride-rich very low-density lipoprotein (VLDL). 13 After triglyceride hydrolysis, most VLDL remnants are rapidly taken up by hepatocytes, but some are further metabolized to lowdensity lipoprotein (LDL) that remains in the plasma with a half-life of approximately 20 hours. 13 APO-B48 is principally produced by the intestine and is essential for the packaging of alimentary lipids into chylomicrons. 14,15 After intestinal chylomicrons enter the circulation via the thoracic duct, their triglyceride cores are hydrolyzed by lipoprotein lipase and hepatic lipase. The resulting chylomicron remnants, which are enriched with dietary cholesterol, are efficiently and rapidly cleared from plasma by hepatocytes with a half-life of less than 10 minutes Clearly, the two isoforms of APO-B display substantially different metabolic pathways. However, no information has been reported on the relative contributions of APO- B48 and APO-B100 containing lipoproteins to biliary cholesterol physiology and cholelithogenesis. In particular, the pathway for metabolism of dietary cholesterol involves the hepatic uptake of chylomicron remnants and the use of chylomicron cholesterol for biliary secretion. 5-8 Therefore, it is crucial to compare the effect of APO- B48 containing lipoproteins with that of APO-B100 containing lipoproteins on regulating both the hepatic availability of dietary cholesterol for bile secretion and the risk for diet-induced cholesterol gallstones. In the present study, we investigated intestinal cholesterol absorption, gene expression levels of intestinal and hepatic lipid transporters, hepatic cholesterol and bile salt biosyntheses, biliary lipid secretion, and cholesterol gallstone formation in lithogenic diet-fed APO-B48 only and APO-B100 only mice compared with identically treated control wild-type mice. Materials and Methods Chemicals Medium-chain triglyceride was purchased from Mead Johnson (Evansville, IN). Intralipid (20% wt/vol) was obtained from Pharmacia (Clayton, NC). Radioisotopes [1,2-3 H]cholesterol, [4-14 C]cholesterol, DL-[5-3 H]mevalonolactone, and DL-[3-14 C]hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) were purchased from NEN Life Science Products (Boston, MA). [5,6-3 H]sitostanol was obtained from American Radiolabeled Chemicals (St. Louis, MO). Genetically Modified Mice and Diets The breeding pairs of mice that synthesize exclusively APO-B48 (APO-B48 only mice or Apob 48/48 mice) or exclusively APO-B100 (APO-B100 only mice or Apob 100/100 mice) were purchased from the Jackson Laboratory (Bar Harbor, ME). These mouse models were originally generated by Farese and colleagues. 19 Mice that were homozygous for the wild-type APO-B allele (Apob / mice) were used as controls. Three groups of mice had an identical genetic background ( 50% C57BL/6J and 50% 129/SvJ), and both C57BL/6J and 129/SvJ strains are gallstone-susceptible strains. 8 All mice were bred in our colony at Beth Israel Deaconess Medical Center (Boston, MA). Male mice studied at 8 to 10 weeks of age were fed normal rodent chow (Harlan Teklad F6 Rodent Diet 8664, Madison, WI) containing trace ( 0.02%) amounts of cholesterol, or a semisynthetic lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butter fat for 8 weeks. 20 All animals were maintained in a temperature-controlled room (22 C 1 C) with a 12-hour-day cycle (6 A.M. to 6 P.M.). All procedures were in accordance with current National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Harvard University. Measurement of Intestinal Cholesterol Absorption Fecal Dual-Isotope Ratio Method. Nonfasted and nonanesthetized mice (n 10 per group) were administered via gavage an intragastric bolus of 150 L of medium-chain triglyceride containing 1 Ci of [ 14 C]cholesterol and 2 Ci of [ 3 H]sitostanol. The ratios of the two radiolabels in the fecal extract from the 4-day pooled feces and the dosing mixture were used for calculating the percent cholesterol absorption. 21 Plasma Dual-Isotope Ratio Method. Additional groups of chow-fed mice (n 10 per group) were injected intravenously with 2.5 Ci of [ 3 H]cholesterol in 100 L of Intralipid. Immediately, each animal was administered via gavage an intragastric bolus of 1 Ci of [ 14 C]cholesterol in 150 L of medium-chain triglyceride. To calculate the percent cholesterol absorption, the ratio of the two radiolabels in the plasma sample taken on the third day was assayed. 21 Cholesterol Balance Analysis Mice housed in individual metabolic cages with wire mesh bottoms were allowed to adapt to the environment for 2 weeks. When body weight, food ingestion, and fecal excretion were constant (i.e., in an apparent metabolic steady state), food intake was measured and feces were collected daily for the balance study. The mice (n 5 per group) were fed chow or the lithogenic diet for 7 days. A cholecystectomy was then performed; the common bile duct was cannulated, and hepatic bile was collected for the

3 896 WANG AND WANG HEPATOLOGY, October 2005 first hour of biliary secretion. Percent cholesterol absorption was calculated as described previously. 21 Microscopic Studies of Gallbladder Biles and Gallstones Before (day 0, on chow) and at 8 weeks on the lithogenic diet, fasted mice (n 20 per group) were anesthetized with an intraperitoneal injection of 35 mg/kg pentobarbital. After cholecystectomy, fresh gallbladder bile were examined for mucin gel, solid and liquid crystals, and gallstones, which were defined according to previously established criteria. 20 Determination of Biliary Lipid Outputs and Bile Salt Pool Sizes The first-hour collection of hepatic biles in additional groups of mice (n 5 per group) on chow (day 0) and fed the lithogenic diet for 8 weeks were used for biliary lipid secretion studies. 22 To determine the circulating bile salt pool size, an 8-hour biliary washout study was performed. 22 During hepatic bile collection, body temperature was maintained at 37 C 0.5 C with a heating lamp and monitored with a thermometer. Lipid Analyses Biliary phospholipids were determined as inorganic phosphorus using the method of Bartlett. 23 Total and individual bile salt concentrations, bile cholesterol, and cholesterol content in chow and gallstones were measured via high-performance liquid chromatography. 20,24 Cholesterol saturation indices in gallbladder and hepatic biles were calculated from the critical tables. 25 Cholesterol content in the small intestine and liver was measured by enzymatic methods. 20 Hydrophobicity indices of bile salts in hepatic biles were calculated according to Heuman s method. 26 Fecal neutral steroids were saponified and extracted, as well as measured via high-performance liquid chromatography. 21 Blood for lipid assays was obtained from mice fasted overnight, and plasma total cholesterol and HDL cholesterol levels were measured as described previously. 27 Non-HDL cholesterol levels were assessed according to arithmetic difference. Measurement of Small Intestinal Transit Time Small intestinal transit times in mice (n 5 per group) fed either chow or the lithogenic diet for 10 days were studied. Nonabsorbable [ 3 H]sitostanol was used as a reference marker. In brief, 2 Ci of [ 3 H]sitostanol in 100 L of medium-chain triglyceride were instilled into the small intestine via a previously fitted in situ externalized duodenal catheter. Exactly 30 minutes after instilling, the mice were anesthetized with pentobarbital. The abdomen was opened, and the stomach, small and large intestines, and cecum were removed. The small intestine was cut into 20 segments equally. The radioactivity in the individual segments was determined via liquid scintillation spectrometry. Samples of the stomach, cecum, and large intestine were also analyzed, but none ever showed appreciable radioactivity. Small intestinal transit time was evaluated by geometric center methods. 28 Determination of Activities of Hepatic HMG-CoA Reductase and Cholesterol 7 -Hydroxylase Liver samples were collected from nonfasted mice (n 5 per group) on chow or fed the lithogenic diet for 8 weeks. To minimize diurnal variations of hepatic enzyme activities, all procedures were performed between 9:00 and 10:00 A.M. Microsomal activities of HMG-CoA reductase were determined by measuring the conversion rate of [ 14 C]HMG-CoA to [ 14 C]mevalonic acid and with [ 3 H]mevalonolactone as an internal standard. 29 Hepatic activities of cholesterol 7 -hydroxylase were determined via high-performance liquid chromatography as described elsewhere. 29,30 Quantitative Real-Time Polymerase Chain Reaction Assay Total RNA was extracted from fresh liver and jejunum tissues of mice (n 4 per group) using RNeasy Midi (Qiagen, Valencia, CA). Reverse transcription was performed using the SuperScript II First-strand Synthesis System (Invitrogen, Carlsbad, CA) with 5 g of total RNA and random hexamers to generate complementary DNA. Primer Express Software (Applied Biosystems, Foster City, CA) was used to design the primers (Table 1). Real-time polymerase chain reaction assays for all samples were performed in triplicate. To obtain a normalized target value, the target amount was divided by the endogenous reference amount of rodent GAPDH as the invariant control. Fast-Performance Liquid Chromatography At 14 days on chow or the lithogenic diet, fresh plasma was obtained from fasted mice (n 5 per group). Pooled plasma (250 L) was fractionated by fast-performance liquid chromatography gel filtration (Bio-Rad Laboratories, Hercules, CA) on a Superose 6 column. 8 The column was eluted at a flow rate of 0.5 ml/minute with a buffer containing 154 mmol/l NaCl, 1 mmol/l EDTA, and 3 mmol/l NaN 3 at ph 7.2. Sixty 0.5-mL fractions were collected, and 100- L aliquots from each fraction were used for measurement of cholesterol.

4 HEPATOLOGY, Vol. 42, No. 4, 2005 WANG AND WANG 897 Table 1. Primer and Probe Sequences Used in mrna Quantification via Real-time Polymerase Chain Reaction Gene Accession No. Forward Reverse Probe Hmgcr M ATTCTGGCAGT 5 -CCTCGTCCTT 5 -CACCGACAAGA CAGTGGGAACT-3 CGATCCAATTT-3 AGCCTGCTGCCA-3 Cyp7a1 NM_ TTTGATGACATG 5 -AGGTTGCAGG 5 -CACCTTGTGATCC GAGAAGGCTAAG-3 AATGGTGTTTG-3 TCTGGGCATCTCA-3 Srebp-2 AF TGAAGCTGGCC 5 -CCACATCACTG 5 -CAAGCTCCTGAA AATCAGAAAA-3 TCCACCAGACT-3 GGGCATCGACCTG-3 Abcg5 AF CCTGCAGAGC 5 -GCATCGCTG 5 -AGCAGCCTCAC GACGTTTTTC-3 TGTATCGCAAC-3 TGTGCGCGAGA-3 Abcg8 AF TGGATAGTGC 5 -AATTGAATCTG 5 -CAAGCTGTCGTT CTGCATGGATC-3 CATCAGCCCC-3 CCTCCGGTGGTG-3 Abcb4 NM_ GGAAATCATTGG 5 -CACGGCCATA 5 -AGCCCGTGCTG TGTGGTAAGTCA-3 GCGGATATTTT-3 TTCTCTACTACGATCGC-3 Abcb11 NM_ TCTGGTACGGC 5 -GCTGCTATTAT 5 -AAGGCGAGTACA TCCAGACTTG-3 GACACAGAGGAAAAT-3 CACCAGGGACACTGATC-3 Npc1l1 AY CCACAGACCC 5 -GCTCGTCATGG 5 -CCCCTAAAAGCC TGTGGAACTGT-3 AAAGCCTTT-3 AAGCCCGGAAAG-3 Statistical Methods All data are expressed as the mean SD. Statistically significant differences among groups of mice were assessed via Student t test, Mann-Whitney U tests, or chisquare tests. If the F value was significant, comparison among groups of mice was further analyzed by a multiple comparison test. Analyses were performed with Super- ANOVA software (Abacus Concepts, Berkeley, CA). Statistical significance was defined as a two-tailed probability of less than.05. Results Plasma, Liver, and Small Intestine Lipids. In the chow-fed state (Table 2), plasma total and HDL cholesterol levels were similar in three groups of mice. However, plasma LDL/VLDL cholesterol concentrations were significantly reduced in Apob 48/48 mice compared with wildtype and Apob 100/100 mice. Furthermore, the lithogenic diet induced significant increases in plasma total and LDL/VLDL cholesterol concentrations and slight increases in HDL cholesterol concentrations in wild-type and Apob 48/48 mice. The results of fast-performance liquid chromatography profiles confirmed cholesterol distributions in plasma VLDL, LDL, and HDL in mice fed chow (Fig. 1A) or the lithogenic diet (Fig. 1B) as analyzed by chemical methods (Table 2). Moreover, no significant differences in liver cholesterol concentrations were detected among the three groups of mice. As shown in Table 2, small intestine total cholesterol levels were significantly higher in Apob 100/100 mice Table 2. Plasma, Liver, and Small Intestine Cholesterol Concentrations Parameter Wild-type Apob 48/48 Apob 100/100 Chow diet Plasma cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL and VLDL cholesterol (mg/dl) * Liver total cholesterol (mg/g) Small intestine total cholesterol (mg/g) Lithogenic diet Plasma cholesterol (mg/dl) ,,# HDL cholesterol (mg/dl) LDL and VLDL cholesterol (mg/dl) **,, Liver total cholesterol (mg/g) Small intestine total cholesterol (mg/g) ,**, NOTE. Values represent the mean SD of 5 animals per group. All mice were fed a normal rodent chow diet containing trace ( 0.02%) cholesterol or a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat for 8 weeks. Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein. *P.05; P.01; P.001; P.0001 compared with wild-type mice on chow. P.0001 compared with Apob 48/48 mice on chow. P.001; **P.0001 compared with Apob 100/100 mice on chow. P.01; P.05 compared with wild-type mice fed a lithogenic diet. #P.01; P.01 compared with Apob 48/48 mice fed a lithogenic diet.

5 898 WANG AND WANG HEPATOLOGY, October 2005 Fig. 1. Fast-performance liquid chromatography profiles of plasma lipoproteins from wild-type, Apob 48/48, and Apob 100/100 mice on chow or a lithogenic diet for 14 days. Plasma was pooled (250 L from 5 mice for each group) and subjected to fast-performance liquid chromatography analysis. The cholesterol content of each fraction was determined via enzymatic assay and expressed as micrograms per fraction. VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; Apob, apolipoprotein B. than in wild-type and Apob 48/48 mice, regardless of whether chow or the lithogenic diet was administered. Influence of APO-B48 and APO-B100 on Intestinal Cholesterol Absorption. On chow, cholesterol absorption efficiency was identical between wild-type and Apob 48/48 mice, both being significantly higher than Apob 100/100 mice as measured using the plasma dual-isotope ratio method (Fig. 2A). These results were further confirmed by two independent analyses: the fecal dualisotope ratio method (Fig. 2B) and the mass balance technique (Table 3). Of special note is that compared with the chow diet, cholesterol absorption measured by the mass balance method increased significantly in wild-type and Apob 48/48 mice fed the lithogenic diet, mostly because of dietary cholic acid, as discussed elsewhere. 20,21 Small Intestinal Transit Time. At 30 minutes after intraduodenal instillation of nonabsorbable [ 3 H]sitostanol, the distribution of radioactivity in the small intestine was similar in chow-fed mice, with peaks between segments 7 and 16. Mean values for the geometric centers of the distribution profiles were identical in Apob 48/48 ( ) and Apob 100/100 ( ) compared with wild-type mice ( ). Furthermore, feeding the lithogenic diet did not significantly change small intestinal transit times (geometric center ) in these mice. It suggests that the intestinal transit time is not responsible for the difference in cholesterol absorption efficiency in these mice. Expression Levels of Intestinal Sterol Transporters. In the chow-fed state (Fig. 3A), the relative messenger RNA (mrna) levels for the intestinal sterol transporters Abcg5/g8 were significantly higher in Apob 100/100 mice than in wild-type and Apob 48/48 mice. Feeding the lithogenic diet significantly increased expression levels of Abcg5/g8, and the three groups of mice displayed similar expression levels of these two transporters. Although expression levels of the intestinal Npc1l1 were reduced by the lithogenic diet (Fig. 3B) compared with the chow diet, no significant differences were found in these mice. Influence of APO-B48 and APO-B100 on Gallstone Prevalence and Lipid Composition of Gallbladder Biles. After 8 weeks on the lithogenic diet, gallstone prevalence rates were similar between wild-type (80%) and Apob 48/48 mice (80%), both being significantly (P.0001) greater than Apob 100/100 mice (20%). The sterols extracted from the stones of each mouse group contained only cholesterol, which constituted more than 99% of stone weight. The size of gallstones was mm in wild-type mice and mm in Apob 48/48 mice, being significantly (P.05) bigger than those in Apob 100/100 mice ( mm). In addition, the number of gallstones in wild-type and Apob 48/48 mice was mainly between 7 and 9, whereas in Apob 100/100 mice the corresponding values were 1 and 3. Of special note is that 80% of Apob 100/100 mice were gallstone-free. Table 4 shows that on chow, biliary cholesterol and phospholipid compositions were similar among the three groups of mice. However, there is a tendency for Apob 48/48 mice to display a markedly lower bile salt concentration. Consequently, the cholesterol saturation indices for Fig. 2. Influence of APO-B48 and APO-B100 on intestinal cholesterol absorption. Cholesterol absorption efficiency was determined via (A) plasma and (B) fecal dual-isotope ratio methods in chow-fed wild-type, Apob 48/48, and Apob 100/100 mice (n 10 per group). As measured using the plasma dual-isotope ratio method, intestinal cholesterol absorption was significantly lower in Apob 100/100 mice (5% 1%) than in wild-type (38% 3%) and Apob 48/48 mice (39% 5%). No differences in percent cholesterol absorption are found between two independent methods in mice. Apob, apolipoprotein B.

6 HEPATOLOGY, Vol. 42, No. 4, 2005 WANG AND WANG 899 Table 3. Cholesterol Balance Data in Wild-type, Apob 48/48, and Apob 100/100 Mice During Ingestion of Chow or Lithogenic Diet Under Metabolic Steady State Conditions Genotype Cholesterol Intake (mg/d) Biliary Cholesterol (mg/d) Steroid Excretion (mg/d) Absorbed Cholesterol (mg/d)* Cholesterol Absorption (%) Chow diet Wild-type Apob 48/ Apob 100/ , , Lithogenic diet Wild-type # Apob 48/ ** ** Apob 100/ ,,## ,***, ***,, , NOTE. Values represent the mean SD of 5 animals per group. All groups of mice were 8 weeks old and fed normal rodent chow containing trace amounts ( 0.02%) of cholesterol or a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat. *Absorbed cholesterol was determined by subtracting the daily fecal neutral steroid output from the daily cholesterol intake and the daily biliary cholesterol output as measured via HPLC. 21 The percent cholesterol absorption was calculated according to published methods. 21 P.0001; P.001; P.00001; P.01; #P.05; P.001 compared with wild-type mice on chow. **P.0001; P.05; P.001 compared with Apob 48/48 mice on chow. P.00001; P.05 compared with Apob 100/100 mice on chow. P.01; ***P.001; P.0001 compared with wild-type mice fed a lithogenic diet. ##P.05; P.01; P.001 compared with Apob 48/48 mice fed a lithogenic diet. Apob 48/48 mice were slightly higher than those in wildtype and Apob 100/100 mice. At 8 weeks on the lithogenic diet, pooled gallbladder biles of wild-type and Apob 48/48 mice became supersaturated with cholesterol. However, the cholesterol saturation indices for Apob 100/100 mice were markedly lower compared with those in wild-type and Apob 48/48 mice. Bile Flow and Biliary Lipid Secretion. We observed that bile flow rates during the first hour were identical in chow-fed wild-type ( L/min/kg), Apob 48/48 ( L/min/kg), and Apob 100/100 ( L/ min/kg) mice. Moreover, the lithogenic diet did not significantly alter bile flow rates in these mice. On chow (Fig. 4A), outputs of biliary bile salts and phospholipids were significantly lower in Apob 48/48 mice compared with those in wild-type mice. However, biliary cholesterol outputs were identical in the three groups of mice. Furthermore, the lithogenic diet significantly increased biliary cholesterol outputs in wild-type mice ( mol/hr/kg) and Apob 48/48 mice ( mol/hr/kg), both being significantly higher than Apob 100/100 mice ( mol/hr/kg). Although biliary phospholipid outputs were significantly increased by the lithogenic diet, there are not significantly statistical differences among three groups of mice. Additionally, biliary bile salt outputs were increased slightly in these mice fed the lithogenic diet compared with the chow diet, but these changes do not reach significantly statistical differences. The circulating bile salt pool sizes ( mol) were similar in the three groups of mice on chow. However, the total bile salt pool sizes ( mol) calculated from Fig. 3. Relative mrna levels for the intestinal sterol transporters Abcg5, Abcg8, and Npc1l1 as analyzed via real-time polymerase chain reaction techniques. These measurements were made using the jejunum RNA of mice fed chow or a lithogenic diet for 8 weeks. For each mrna expression level, the fold change for the Apob 48/48 and Apob 100/100 mice is expressed relative to chow-fed wild-type mice, which in each case is arbitrarily set at 1.0. The values represent the mean SD of the fold change found in these mice. mrna, messenger RNA; Apob, apolipoprotein B.

7 900 WANG AND WANG HEPATOLOGY, October 2005 Table 4. Lipid Compositions of Pooled Gallbladder Biles Genotype Cholesterol (mmol/l) Phospholipid (mmol/l) Bile Salt (mmol/l) Cholesterol (mol%) Phospholipid (mol%) Bile Salt (mol%) PL/(PL BS)* Total Lipid Concentration (g/dl) Cholesterol Saturation Index Chow diet Wild-type Apob 48/ Apob 100/ Lithogenic diet Wild-type Apob 48/ Apob 100/ NOTE. Values were determined from pooled gallbladder biles (n 20 per group). *PL/(PL BS), phospholipid divided by (phospholipid bile salt). These values represent the mean cholesterol saturation indices of the pooled gallbladder biles calculated from critical tables. 25 the circulating bile salt pool size plus the bile salt pool in the gallbladder were significantly (P.01) increased in the lithogenic state; this is mostly like due to enlarged gallbladders. However, the total bile salt pool sizes were similar in the three groups of mice. High-performance liquid chromatography analysis revealed that chow-fed mice exhibited similar bile salt compositions in hepatic biles, and the predominant bile salt Fig. 4. Total outputs ( mol/hr/kg body weight) of cholesterol, phospholipids, and bile salts during the first hour of biliary secretion in mice (n 5 per roup). (A) On a chow diet, secretion rates of biliary phospholipids (P.05) and bile salts (P.01) are significantly lower in Apob 48/48 mice than those in wild-type mice. However, total outputs of biliary cholesterol are basically similar among wild-type, Apob 48/48, and Apob 100/100 mice. (B) A lithogenic diet significantly (P.01) increased outputs of biliary cholesterol in wild-type and Apob 48/48 mice, but did not influence biliary cholesterol secretion rates in Apob 100/100 mice. Although outputs of biliary phospholipids were significantly (P.01) increased by a lithogenic diet compared with chow, the values for biliary phospholipid outputs were similar in the three groups of mice. Furthermore, no differences in biliary bile salt outputs were found in the three groups of mice between chow and a lithogenic diet. B.W., body weight; Apob, apolipoprotein B. species were taurocholate (46.8%-50.9%) and tauro- muricholate (42.3%-46.1%), with identical hydrophilicity indices ( 0.33 to 0.37). On the lithogenic diet, taurocholate (61.8%-70.2%) became the major bile salt, with a significant (P.01) increase in taurodeoxycholate (14.4%-19.2%) and taurochenodeoxycholate (7.5%- 10.9%) and a significant (P.001) decrease in tauro- -muricholate (2.3%-6.3%). Moreover, the hydrophobicity indices ( 0.07 to 0.09) were significantly (P.01) increased after 8 weeks on the lithogenic diet. Expression Levels of Hepatic Lipid Transporters. Under basal physiological conditions (Fig. 5A), the relative mrna levels for the hepatic lipid transporters Abcg5/g8, Abcb4, and Abcb11 were similar in the three groups of mice. However, on the lithogenic state (Fig. 5B), gene expression levels of all these transporters were increased. Furthermore, wild-type and Apob 48/48 mice displayed significantly higher expression levels of hepatic Abcg5/g8 compared with Apob 100/100 mice. However, no differences in gene expression levels of Abcb4 and Abcb11 were found in these mice. Hepatic Cholesterol and Bile Salt Synthesis. Figure 6 exhibits that on chow, expression levels of the hepatic sterol regulatory element-binding protein-2 (Srebp-2) are twofold higher in Apob 100/100 mice than wild-type and Apob 48/48 mice. Although the lithogenic diet significantly reduced mrna levels of Srebp-2 by 50% in mice, expression levels of Srebp-2 were still significantly higher in Apob 100/100 mice than in wild-type and Apob 48/48 mice. Figure 7 shows that Apob 100/100 mice display significantly higher enzymatic activities and mrna levels of hepatic HMG-CoA reductase compared with those in wild-type and Apob 48/48 mice, regardless of whether chow or the lithogenic diet was administered. Of note is that the lithogenic diet significantly reduced the activities and expression levels of hepatic HMG-CoA reductase in wild-type and Apob 48/48 mice, but to a lesser extent in Apob 100/100

8 HEPATOLOGY, Vol. 42, No. 4, 2005 WANG AND WANG 901 It has been observed that polymorphisms in the APO-B gene are associated with cholesterol gallstones in humans However, the metabolic abnormalities underlying the supersaturation of bile and the formation of cholesterol gallstones induced by APO-B expression and/or polymorphisms are not yet fully understood. Thus the use of Apob 48/48 and Apob 100/100 mice should help to elucidate whether their expression levels are critical for cholesterol gallstone formation and to dissect the potential pathophysiological roles of each APO-B subtype, APO-B48 and APO-B100, in the formation of gallstones. In the present study, we observed that mice with deficiency in Apob48 expression (i.e., Apob 100/100 mice) display a significantly reduced cholesterol absorption, and when switched from chow to a lithogenic diet, they still show a diminished cholesterol absorption as determined by the cholesterol balance analysis. Under high dietary cholesterol loads, a great amount of cholesterol is accumulated in the small intestine of Apob 100/100 mice, which results in increased expression levels of intestinal sterol efflux transporters Abcg5/g8. This suggests that the function of Abcg5/g8 may be regulated by the content of cholesterol within the enterocyte. Consequently, Apob 100/100 mice excrete a significantly greater amount of fecal neutral steroids than wild-type and Apob 48/48 mice, and increased excretion of these steroids may reduce cholesterol accumulation in the body. 31 Moreover, in an experiment with a small number of mice, Young et al 14 found that disruption of the APO-B gene significantly inhibits cholesterol absorption in mice that express a human APO-B transgene in the liver but do not synthesize any APO-B in the intestine. Therefore, our findings support the concept that APO-B48 is essential for the pack- Fig. 5. Gene expression levels of hepatic lipid transporters Abcg5, Abcg8, Abcb4, and Abcb11 as analyzed by real-time polymerase chain reaction techniques. These measurements were made using the liver RNA of mice on chow or fed the lithogenic diet for 8 weeks. For each mrna expression level, the fold-change for the Apob 48/48 and Apob 100/100 mice is expressed relative to chow-fed wild-type mice, which in each case is arbitrarily set at 1.0. The values represent the mean SD of the fold change found in these mice. mrna, messenger RNA; Apob, apolipoprotein B. mice. Although the activities and expression levels of hepatic cholesterol 7 -hydroxylase were significantly decreased by administering the lithogenic diet compared with chow, no significant differences were found among the three groups of mice. Discussion Fig. 6. Relative mrna levels of the hepatic sterol regulatory elementbinding protein-2 (Srebp-2) in mice (n 5 per group) fed chow or a lithogenic diet for 8 weeks. (A) On a chow diet, expression levels of Srebp-2 were twofold higher (P.01) in Apob 100/100 mice compared with those in wild-type and Apob 48/48 mice. (B) Although the lithogenic diet significantly reduced expression levels of Srebp-2 by 50% in all three groups of mice, the relative mrna levels for Srebp-2 were still significantly (P.01) higher in Apob 100/100 mice compared with wild-type and Apob 48/48 mice.

9 902 WANG AND WANG HEPATOLOGY, October 2005 Fig. 7. Enzymatic activities and mrna levels of HMG-CoA reductase and cholesterol 7 -hydroxylase in the livers of wild-type, Apob 48/48, and Apob 100/100 mice (n 5 per group) fed chow or a lithogenic diet for 8 weeks. The enzymatic activities and mrna levels of HMG-CoA reductase were significantly (P.05) higher in the livers of Apob 100/100 mice compared with those in wild-type and Apob 48/48 mice, regardless of whether (A) chow or (B) a lithogenic diet was administered. Furthermore, under a high cholesterol plus cholic acid load, enzymatic activities and mrna levels of HMG-CoA reductase were significantly (P.05) reduced in wild-type and Apob 48/48 mice, but to a lesser extent in Apob 100/100 mice. Compared with (C) chow, (D) a lithogenic diet significantly (P.05) reduced enzymatic activities and mrna levels of cholesterol 7 -hydroxylase in all mice. It is noteworthy that no significant differences were found in enzymatic activities and mrna levels among the three groups of mice. aging of alimentary lipids into chylomicrons and that expression levels of intestinal APO-B48 are crucial for cholesterol absorption. In the chow-fed state, mice with deficiency in Apob48 expression display an increased hepatic cholesterol biosynthesis by upregulating expression levels of hepatic HMG-CoA reductase and its activity, mostly as a result of the reduced delivery of dietary cholesterol from the intestine to the liver. Jung et al 32 also found an increased whole-body cholesterogenesis in APO-B knockout mice rescued with a human APO-B transgene in the liver, as measured by stable isotope incorporation techniques. These observations suggest an adaptive response of hepatic cholesterol synthesis to impaired hepatic influx of chylomicron remnant-derived cholesterol. This alteration could produce a corresponding reduction of biliary cholesterol secretion, although the increase of hepatic cholesterol biosynthesis may result in a partial normalization of secretion rates of biliary cholesterol in Apob 100/100 mice. However, the absence of APO-B48 expression does not induce an alteration in the hepatic synthesis and biliary secretion of bile salts in Apob 100/100 mice. Furthermore, we observed that plasma levels of LDL/VLDL cholesterol, but not HDL cholesterol, are reduced in chow-fed Apob 48/48 mice. These changes are associated with relative hyposecretion of biliary bile salt and phospholipid accompanying with normal secretion rates of biliary cholesterol, suggesting that biliary cholesterol output may not be related to the hepatic uptake of plasma LDL/VLDL. Furthermore, decreased biliary bile salt secretion in Apob 48/48 mice suggests that LDL/VLDL cholesterol may be primarily used for hepatic bile salt biosynthesis. Hillebrant et al. 33 showed that plasma level and hepatic uptake of LDL/ VLDL is of importance for biliary bile salt outputs, consistent with our results. Because the biliary secretion of phospholipids depends on bile acid secretion, biliary phospholipid output is reduced. Consistent with this, bile salt and phospholipid concentrations in gallbladder biles are decreased markedly, which induces relatively higher cholesterol saturation index values for Apob 48/48 mice. It has been found that dietary cholic acid decreases plasma HDL via downregulation of APO-AI levels and greatly enhances intestinal cholesterol absorption, mainly by increasing intraluminal micellar cholesterol solubilization. 37 Our current results show that under the high cholesterol plus cholic acid feeding conditions, biliary cholesterol secretion is significantly increased in wildtype and Apob 48/48 mice, which is consistent with previous

10 HEPATOLOGY, Vol. 42, No. 4, 2005 WANG AND WANG 903 studies. 38 Moreover, expression levels of Abcg5/g8 are significantly increased in these mice; thus increased function of Abcg5/g8 could induce hypersecretion of biliary cholesterol. 39 However, these effects on biliary cholesterol hypersecretion are not observed in Apob 100/100 mice. Therefore, these results suggest that the hepatic uptake of cholesterol from chylomicron remnants may be a major but not rate-limiting contributor to biliary cholesterol hypersecretion, particularly during diet-induced cholelithogenesis. 38 Furthermore, we found that cholesterol monohydrate crystals and liquid crystals are appreciably less frequent in Apob 100/100 mice compared with those in wild-type and Apob 48/48 mice. Also, prevalence rate, as well as size and number of gallstones in Apob 100/100 mice, are significantly lower. As inferred from our cumulative results, it is highly probable that lack of APO-B48 expression induces the inhibition of intestinal absorption and hepatic bioavailability, which results in contributing considerably less cholesterol to biliary secretion. Studies that underline the importance of chylomicron remnant cholesterol in murine cholelithogenesis are that disturbed hepatic uptake of chylomicron remnants in APO-E deficient mice 40 and impaired intestinal cholesterol esterification in acyl-coa: cholesterol acyltransferase 2 knockout mice 41 significantly reduce biliary cholesterol secretion and gallstone formation. More recently, we found that biliary secretion of chylomicron remnant cholesterol is more rapid in gallstone-susceptible C57L mice than in gallstone-resistant AKR mice. 8 Our results suggest that reduced biliary cholesterol secretion in Apob 100/100 mice, but not in Apob 48/48 mice, is most likely due to the impaired delivery of dietary cholesterol into the liver for secretion into bile. Therefore, our findings support the notion that during diet-induced gallstones, intestinal chylomicrons principally contribute to biliary cholesterol hypersecretion, and APO-B48 expression levels have a pivotal effect on determining the response of secretion rates of biliary cholesterol in mice. The current findings in mice suggest that genetic polymorphisms of APO-B48, but not APO-B100, may be associated with cholesterol gallstones in humans. Furthermore, our studies in mice should provide a framework for further investigation of the molecular mechanisms that determine how the reported common APO-B gene variants affect its functional activity in chylomicron remnant metabolism and stone formation in humans. In conclusion, although high cholesterol absorption efficiency is associated with increased plasma cholesterol concentrations in the Finnish population, 42 it has not been established whether intestinal cholesterol uptake and absorption efficiency influence cholesterol gallstone formation in humans. Epidemiological investigations have shown that cholesterol gallstones are prevalent in cultures consuming a Western diet with high cholesterol, and the incidence of cholesterol gallstones in North America and European countries is significantly higher than that seen in developing countries. In China and Japan, cholesterol gallstones once were rare, but over the past 30 years the incidence has increased markedly because of the adoption of Western-type dietary habits (i.e., excessive consumption of high cholesterol food) Recent animal studies 8 showed that the efficiency of intestinal cholesterol absorption may play a major regulatory role in the response of biliary cholesterol secretion to high dietary cholesterol and contribute to cholesterol gallstone formation. Therefore, our findings may provide some rationale for the development of intestinal APO-B48 specific inhibitors, which might provide an efficacious novel strategy for the prevention of cholesterol gallstones by inhibiting the intestinal absorption of cholesterol. References 1. Wang DQ-H, Afdhal NH. Genetic analysis of cholesterol gallstone formation: searching for Lith (gallstone) genes. Curr Gastroenterol Rep 2004;6: Paigen B, Carey MC. Gallstones. In: King RA, Rotter JF, Motulsky AG, eds. The Genetic Basis of Common Diseases. 1st ed. New York: Oxford University Press, 2002: Schwartz CC, Halloran LG, Vlahcevic ZR. Preferential utilization of free cholesterol from high-density lipoproteins for biliary cholesterol secretion in man. Science 1978;200: Robins SJ, Fasulo JM. High density lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile. J Clin Invest 1997;99: Dijk MCM, Pieters M, van Berkel TJC. Kinetics of biliary secretion of chylomicron remnant cholesterol (esters) in the rat. Eur J Biochem 1993; 211: Smit MJ, Kuipers F, Vonk RJ, Temmerman AM, Jäckle S, Windler EET. Effects of dietary cholesterol on bile formation and hepatic processing of chylomicron remnant cholesterol in the rat. HEPATOLOGY 1993;17: Bravo E, Guldur T, Botham KM, Mayes PA, Cantafora A. Hepatic uptake and processing of cholesterol and cholesteryl ester from chylomicron remnants: an in vivo study in the rat. Biochim Biophys Acta 1992;1123: Wang DQ-H, Zhang L, Wang HH. High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice. Biochim Biophys Acta 2005;1733: Juvonen T, Savolainen MJ, Kairaluoma MI, Lajunen LH, Humphries SE, Kesaniemi YA. Polymorphisms at the apob, apoa-i, and cholesteryl ester transfer protein gene loci in patients with gallbladder disease. J Lipid Res 1995;36: Han T, Jiang Z, Suo G, Zhang S. Apolipoprotein B-100 gene Xba I polymorphism and cholesterol gallstone disease. Clin Genet 2000;57: Fu X, Gong K, Shao X. The relationship between serum lipids, apolipoproteins level and bile lipids level, chemical type of stone. Zhonghua Yi Xue Za Zhi 1995;75: [in Chinese]. 12. Tan YF, Yang S, Yu RB, Shen C, Ding WL, Zhou WM, et al. Relationship among the XhaI and EcoRI locus polymorphisms of apolipoprotein B gene, serum lipid metabolism and gallstone disease. Zhonghua Yi Xue Za Zhi 2003;83: [in Chinese].

11 904 WANG AND WANG HEPATOLOGY, October Havel RJ, Kane JP. Introduction: structure and metabolism of plasma lipoproteins. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. Volume 2. 2nd ed. New York: McGraw-Hill, Inc., 1995: Young SG, Cham CM, Pitas RE, Burri BJ, Connolly A, Flynn L, et al. A genetic model for absent chylomicron formation: mice producing apolipoprotein B in the liver, but not in the intestine. J Clin Invest 1995;96: Hamilton RL, Wong JS, Cham CM, Nielsen LB, Young SG. Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency. J Lipid Res 1998;39: Jäckle S, Rinninger F, Greeve J, Greten H, Windler E. Regulation of the hepatic removal of chylomicron remnants and -very low density lipoproteins in the rat. J Lipid Res 1992;33: Cooper AD. Hepatic uptake of chylomicron remnants. J Lipid Res 1997; 38: Mahley RW, Hussain MM. Chylomicron and chylomicron remnant catabolism. Curr Opin Lipidol 1991;2: Farese RV Jr, Veniant MM, Cham CM, Flynn LM, Pierotti V, Loring JF, et al. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100. Proc Natl Acad Sci U S A 1996;93: Wang DQ-H, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile. J Lipid Res 1997;38: Wang DQ-H, Carey MC. Measurement of intestinal cholesterol absorption by plasma and fecal dual-isotope ratio, mass balance, and lymph fistula methods in the mouse: an analysis of direct versus indirect methodologies. J Lipid Res 2003;44: Wang DQ-H, Lammert F, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: pathophysiology of biliary lipid secretion. J Lipid Res 1999;40: Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959;234: Rossi SS, Converse JL, Hofmann AF. High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids. J Lipid Res 1987;28: Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19: Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res 1989;30: Nishina PM, Wang J, Toyofuku W, Kuypers FA, Ishida BY, Paigen B. Atherosclerosis and plasma and liver lipids in nine inbred strains of mice. Lipids 1993;28: Wang DQ-H, Paigen B, Carey MC. Genetic factors at the enterocyte level account for variations in intestinal cholesterol absorption efficiency among inbred strains of mice. J Lipid Res 2001;42: Lammert F, Wang DQ-H, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes. J Lipid Res 1999;40: Hylemon PB, Studer EJ, Pandak WM, Heuman DM, Vlahcevic ZR, Chiang JY. Simultaneous measurement of cholesterol 7 -hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14 C]cholesterol as substrate. Anal Biochem 1989;182: Kruit JK, Plosch T, Havinga R, Boverhof R, Groot PH, Groen AK, et al. Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice. Gastroenterology 2005;128: Jung HR, Turner SM, Neese RA, Young SG, Hellerstein MK. Metabolic adaptations to dietary fat malabsorption in chylomicron-deficient mice. Biochem J 1999;343: Hillebrant CG, Nyberg B, Einarsson K, Eriksson M. The effect of plasma low density lipoprotein apheresis on the hepatic secretion of biliary lipids in humans. Gut 1997;41: Srivastava RA, Srivastava N, Averna M. Dietary cholic acid lowers plasma levels of mouse and human apolipoprotein A-I primarily via a transcriptional mechanism. Eur J Biochem 2000;267: Hedrick CC, Hassan K, Hough GP, Yoo JH, Simzar S, Quinto CR, et al. Short-term feeding of atherogenic diet to mice results in reduction of HDL and paraoxonase that may be mediated by an immune mechanism. Arterioscler Thromb Vasc Biol 2000;20: Claudel T, Sturm E, Duez H, Torra IP, Sirvent A, Kosykh V, et al. Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element. J Clin Invest 2002;109: Wang DQ-H, Tazuma S, Cohen DE, Carey MC. Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse. Am J Physiol 2003;285:G494-G Wang DQ-H, Carey MC. Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI. Biochim Biophys Acta 2002;1583: Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest 2002;110: Amigo L, Quinones V, Mardones P, Zanlungo S, Miquel JF, Nervi F, et al. Impaired biliary cholesterol secretion and decreased gallstone formation in apolipoprotein E-deficient mice fed a high-cholesterol diet. Gastroenterology 2000;118: Buhman KK, Accad M, Novak S, Choi RS, Wong JS, Hamilton RL, et al. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nat Med 2000;6: Kesäniemi YA, Miettinen TA. Cholesterol absorption efficiency regulates plasma cholesterol level in the Finnish population. Eur J Clin Invest 1987; 17: Huang YC, Zhang XW, Yang RX. Changes in cholelithiasis in Tianjin in the past 30 years. Chin Med J 1984;97: Nakayama F, Miyake H. Changing state of gallstone disease in Japan. Am J Surg 1970;120: Nagase M, Tanimura H, Setoyama M, Hikasa Y. Present features of gallstones in Japan: a collective review of 2,144 cases. Am J Surg 1978;135: