Potential conflict of interest: Nothing to report.
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 (Apob48/48), an “APO-B100 only” allele (Apob100/100), 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 Apob100/100 mice compared with wild-type and Apob48/48 mice, regardless of whether chow or the lithogenic diet was administered. Consequently, hepatic cholesterol synthesis was significantly increased in Apob100/100 mice compared with wild-type and Apob48/48 mice. On chow, the APO-B100 deficiency in Apob48/48 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 Apob48/48 and wild-type mice, lithogenic diet–fed Apob100/100 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;42:894–904.)
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 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.9–12 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 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 low-density 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.16–18 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.
Medium-chain triglyceride was purchased from Mead Johnson (Evansville, IN). Intralipid (20% wt/vol) was obtained from Pharmacia (Clayton, NC). Radioisotopes [1,2-3H]cholesterol, [4-14C]cholesterol, DL-[5-3H]mevalonolactone, and DL-[3-14C]hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) were purchased from NEN Life Science Products (Boston, MA). [5,6-3H]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 Apob48/48 mice) or exclusively APO-B100 (APO-B100 only mice or Apob100/100 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 μL of medium-chain triglyceride containing 1 μCi of [14C]cholesterol and 2 μCi of [3H]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 μCi of [3H]cholesterol in 100 μL of Intralipid. Immediately, each animal was administered via gavage an intragastric bolus of 1 μCi of [14C]cholesterol in 150 μL 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 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.
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 [3H]sitostanol was used as a reference marker. In brief, 2 μCi of [3H]sitostanol in 100 μL 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 7α-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 [14C]HMG-CoA to [14C]mevalonic acid and with [3H]mevalonolactone as an internal standard.29 Hepatic activities of cholesterol 7α-hydroxylase were determined via high-performance liquid chromatography as described elsewhere.29, 30
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 μg 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.
Table 1. Primer and Probe Sequences Used in mRNA Quantification via Real-time Polymerase Chain Reaction
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 μL) 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 NaN3 at pH 7.2. Sixty 0.5-mL fractions were collected, and 100-μL aliquots from each fraction were used for measurement of cholesterol.
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 chi-square tests. If the F value was significant, comparison among groups of mice was further analyzed by a multiple comparison test. Analyses were performed with SuperANOVA software (Abacus Concepts, Berkeley, CA). Statistical significance was defined as a two-tailed probability of less than .05.
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 Apob48/48 mice compared with wild-type and Apob100/100 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 Apob48/48 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).
Table 2. Plasma, Liver, and Small Intestine Cholesterol Concentrations
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 Apob48/48 mice on chow.
P < .001
P < .0001 compared with Apob100/100 mice on chow.
P < .01
P < .05 compared with wild-type mice fed a lithogenic diet.
P < .01
P < .01 compared with Apob48/48 mice fed a lithogenic diet.
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 Apob100/100 mice than in wild-type and Apob48/48 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 Apob48/48 mice, both being significantly higher than Apob100/100 mice as measured using the plasma dual-isotope ratio method (Fig. 2A). These results were further confirmed by two independent analyses: the fecal dual-isotope 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 Apob48/48 mice fed the lithogenic diet, mostly because of dietary cholic acid, as discussed elsewhere.20, 21
Table 3. Cholesterol Balance Data in Wild-type, Apob48/48, and Apob100/100 Mice During Ingestion of Chow or Lithogenic Diet Under Metabolic Steady State Conditions
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 Apob48/48 mice on chow.
P < .00001
P < .05 compared with Apob100/100 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 Apob48/48 mice fed a lithogenic diet.
At 30 minutes after intraduodenal instillation of nonabsorbable [3H]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 Apob48/48 (10.8 ± 0.5) and Apob100/100 (10.7 ± 0.7) compared with wild-type mice (11.7 ± 0.9). Furthermore, feeding the lithogenic diet did not significantly change small intestinal transit times (geometric center = 11.9-13.2) 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 Apob100/100 mice than in wild-type and Apob48/48 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 Apob48/48 mice (80%), both being significantly (P < .0001) greater than Apob100/100 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 0.28 ± 0.21 mm in wild-type mice and 0.23 ± 0.16 mm in Apob48/48 mice, being significantly (P < .05) bigger than those in Apob100/100 mice (0.18 ± 0.10 mm). In addition, the number of gallstones in wild-type and Apob48/48 mice was mainly between 7 and 9, whereas in Apob100/100 mice the corresponding values were 1 and 3. Of special note is that 80% of Apob100/100 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 Apob48/48 mice to display a markedly lower bile salt concentration. Consequently, the cholesterol saturation indices for Apob48/48 mice were slightly higher than those in wild-type and Apob100/100 mice. At 8 weeks on the lithogenic diet, pooled gallbladder biles of wild-type and Apob48/48 mice became supersaturated with cholesterol. However, the cholesterol saturation indices for Apob100/100 mice were markedly lower compared with those in wild-type and Apob48/48 mice.
Table 4. Lipid Compositions of Pooled Gallbladder Biles
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
Bile Flow and Biliary Lipid Secretion.
We observed that bile flow rates during the first hour were identical in chow-fed wild-type (106 ± 12 μL/min/kg), Apob48/48 (105 ± 19 μL/min/kg), and Apob100/100 (103 ± 14 μL/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 Apob48/48 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 (24.6 ± 4.6 μmol/hr/kg) and Apob48/48 mice (24.0 ± 5.1 μmol/hr/kg), both being significantly higher than Apob100/100 mice (12.3 ± 1.4 μmol/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 (3.1-3.5 μmol) were similar in the three groups of mice on chow. However, the total bile salt pool sizes (4.5-4.7 μmol) calculated from 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 species were taurocholate (46.8%-50.9%) and tauro-β-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- β-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 Apob48/48 mice displayed significantly higher expression levels of hepatic Abcg5/g8 compared with Apob100/100 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 Apob100/100 mice than wild-type and Apob48/48 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 Apob100/100 mice than in wild-type and Apob48/48 mice. Figure 7 shows that Apob100/100 mice display significantly higher enzymatic activities and mRNA levels of hepatic HMG-CoA reductase compared with those in wild-type and Apob48/48 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 Apob48/48 mice, but to a lesser extent in Apob100/100 mice. Although the activities and expression levels of hepatic cholesterol 7α-hydroxylase were significantly decreased by administering the lithogenic diet compared with chow, no significant differences were found among the three groups of mice.
It has been observed that polymorphisms in the APO-B gene are associated with cholesterol gallstones in humans.9–12 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 Apob48/48 and Apob100/100 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., Apob100/100 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 Apob100/100 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, Apob100/100 mice excrete a significantly greater amount of fecal neutral steroids than wild-type and Apob48/48 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 al14 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 packaging 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 al32 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 Apob100/100 mice. However, the absence of APO-B48 expression does not induce an alteration in the hepatic synthesis and biliary secretion of bile salts in Apob100/100 mice. Furthermore, we observed that plasma levels of LDL/VLDL cholesterol, but not HDL cholesterol, are reduced in chow-fed Apob48/48 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 Apob48/48 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 Apob48/48 mice.
It has been found that dietary cholic acid decreases plasma HDL via downregulation of APO-AI levels34–36 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 wild-type and Apob48/48 mice, which is consistent with previous 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 Apob100/100 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 Apob100/100 mice compared with those in wild-type and Apob48/48 mice. Also, prevalence rate, as well as size and number of gallstones in Apob100/100 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 mice40 and impaired intestinal cholesterol esterification in acyl-CoA:cholesterol acyltransferase 2 knockout mice41 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 Apob100/100 mice, but not in Apob48/48 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).43–45 Recent animal studies8 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.