High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice

Biochimica et Biophysica Acta 1733 (2005) 90 – 99 http://www.elsevier.com/locate/bba

Regular paper

High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice David Q.-H. Wanga,*, Lunan Zhangb, Helen H. Wanga a

Department of Medicine, Liver Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, 330 Brookline Avenue, Boston, Massachusetts, 02215, USA b Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, 02115, USA Received 14 September 2004; received in revised form 3 November 2004; accepted 8 December 2004 Available online 23 December 2004

Abstract The study of chylomicron pathway through which it exerts its metabolic effects on biliary cholesterol secretion is crucial for understanding how high dietary cholesterol influences cholelithogenesis. We explored a relationship between cholesterol absorption efficiency and gallstone prevalence in 15 strains of inbred male mice and the metabolic fate of chylomicron and chylomicron remnant cholesterol in gallstone-susceptible C57L and gallstone-resistant AKR mice. Our results show a positive and significant ( Pb0.0001, r=0.87) correlation between percent cholesterol absorption and gallstone prevalence rates. Compared with AKR mice, C57L mice displayed significantly greater absorption of cholesterol from the small intestine, more rapid plasma clearance of chylomicrons and chylomicron remnants, higher activities of lipoprotein lipase and hepatic lipase, greater hepatic uptake of chylomicron remnants, and faster secretion of chylomicron remnant cholesterol from plasma into bile. All of these increased susceptibility to cholesterol gallstone formation in C57L mice. We conclude that genetic variations in cholesterol absorption efficiency are associated with cholesterol gallstone formation in inbred mice and cholesterol absorbed from the intestine provides an important source for biliary hypersecretion. Differential metabolism of the chylomicron remnant cholesterol between C57L and AKR mice clearly plays a crucial role in the formation of lithogenic bile and gallstones. D 2004 Elsevier B.V. All rights reserved. Keywords: Bile; Bile flow; Biliary cholesterol secretion; Intestinal cholesterol absorption; Lymph; Nutrition; Gallstone

1. Introduction Epidemiological studies have shown that cholesterol gallstones are prevalent in cultures consuming a bWesternQ diet with high cholesterol, and the incidence of cholesterol gallstones in North America and European countries is

Abbreviations: Acat2, acyl-CoA:cholesterol acyltransferase gene 2; FPLC, fast performance liquid chromatography; HDL, high density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LDL, low density lipoprotein; QTL, quantitative trait locus; VLDL, very low density lipoprotein * Corresponding author. Tel.: +1 617 667 0561; fax: +1 617 975 5071. E-mail address: [email protected] (D.Q.-H. Wang). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.12.005

significantly higher than that in the developing nations [1– 4]. Several studies have found an association between the increased incidence of cholesterol gallstones in China and a bwesternizationQ of the traditional Chinese diet [5–7]; that is, excessive consumption of high cholesterol food. In Japan, cholesterol gallstones once were rare, but over the past 30 years with the adoption of Western-type dietary habits, the incidence has increased markedly [8–10]. However, studies on the effect of dietary cholesterol on biliary cholesterol metabolism in the human gave conflicting results, showing that high dietary cholesterol either increases [11–13] or does not affect cholesterol saturation of bile [14,15]. On basis of these observations, we hypothesized that the efficiency of intestinal cholesterol

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absorption may play a major regulatory role in the response of biliary cholesterol secretion to high dietary cholesterol and contribute to the formation of cholesterol gallstones. Furthermore, consideration of lipoprotein metabolism is often divided into endogenous and exogenous pathways. The endogenous pathway has received great attention because of the well-documented evidence demonstrating that plasma high density lipoprotein (HDL) rather than very low density lipoprotein (VLDL) and low density lipoprotein (LDL) preferentially provides unesterified cholesterol to the liver for secretion into bile [16–19]. In contrast, the metabolism of exogenous chylomicrons (i.e., lipoproteins of intestinal origin) is so rapid that it is difficult to measure alterations in this pathway [20,21]. Thus, the second purpose of this study was to test the hypothesis that the metabolic responses to high dietary cholesterol through an exogenous chylomicron pathway are different between gallstone-susceptible C57L and gallstone-resistant AKR mice. We found that there is a positive and significant correlation between the efficiency of cholesterol absorption and the prevalence of cholesterol gallstone formation in 15 strains of inbred male mice. This suggests that the high efficiency of intestinal cholesterol absorption and high dietary cholesterol are two independent risk factors for cholesterol gallstone formation. Furthermore, our results showed that compared with AKR mice, C57L mice displayed significantly higher efficiency of intestinal cholesterol absorption, more rapid plasma clearance rates of chylomicrons and chylomicron remnants, higher plasma activities of lipoprotein lipase and hepatic lipase, greater uptake of chylomicron remnants by the liver, and faster hepatic secretion of chylomicron remnant-derived cholesterol from plasma into bile. We conclude, therefore, that differential metabolism of cholesterol carried in chylomicron remnants between C57L and AKR mice has an important effect in inducing biliary cholesterol hypersecretion as well as enhancing susceptibility to cholesterol gallstone formation.

2. Materials and methods 2.1. Animals and diets Male strains of inbred A/J, AKR/J (AKR), BALB/cByJ (BALB), BDP/J (BDP), C3H/HeJ (C3H), C57BL/6J (C57BL), C57L/J (C57L), DBA/2J (DBA), FVB/NJ (FVB), SJL/J (SJL), SM/J (SM), SWR/J (SWR), 129S3/ SvImJ (129S3), and 129X1/SvJ (129X1) mice, 6–8 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, ME), and 129/SvEv (129/S) mice were obtained from Charles River Laboratories (Wilmington, MA). C57L strain is homozygous for susceptible Lith alleles, and AKR strain for resistant Lith alleles [22]. All animals were maintained in a temperature-controlled room (22F1 8C)

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with a 12-h day cycle (6 AM–6 PM), and were provided free access to water and normal mouse chow containing trace (b0.02%) cholesterol (Harlan Teklad Laboratory Animal Diets, Madison, WI). 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. 2.2. Measurement of cholesterol absorption by fecal dual– isotope ratio method The above-mentioned 15 strains of inbred male mice (n=5 per group) were given intragastrically by gavage 1 ACi of [14C]cholesterol (NEN Life Science Products, Boston, MA) and 2 ACi of [3H]sitostanol (American Radiolabeled Chemicals, St. Louis, MO) in 150 Al of medium-chain triglyceride (Mead Johnson, Evansville, IN) according to published methods [23,24]. The ratio of the two radiolabels in the fecal extracts and the dosing mixture was used for the calculation of percent cholesterol absorption. 2.3. Gallstone study Additional groups of the above-mentioned 15 strains of male mice (n=10 per group) were fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butter fat for 8 weeks. Non-fasted mice were anesthetized with an intraperitoneal injection of 35 mg/kg pentobarbital. After cholecystectomy was performed, fresh gallbladder bile was examined for mucin gel, solid and liquid crystals, and gallstones, which were defined according to previously established criteria [22]. The gallstone prevalence rate for each mouse strain at 8 weeks on the lithogenic diet was calculated using the following equation: gallstone prevalence rate (%)=(total number of mice forming gallstones/ total number of mice studied)100. 2.4. Preparation of radiolabeled chylomicrons and chylomicron remnants Our preliminary study showed that chylomicron sizes and their lipid and lipoprotein compositions, but not their concentrations, were similar between AKR and C57L mice, and hence, we chose C57L strain for the lymph donor. After the mesenteric lymph duct and the duodenum were cannulated via PE-10 polyethylene catheters, respectively, the animal was infused through the duodenal catheter with a lipid emulsion containing medium-chain triglyceride, 100 ACi of [3H]cholesterol (NEN Life Science Products, Boston, MA), 0.1% (w/w) cholesterol, 0.5% taurocholate and 0.25% egg yolk lecithin by an infusion pump at a rate of 300 Al/h. The lymph was collected into a heparinized tube at room temperature (22F1 8C). The fresh lymph was layered under 0.9% NaCl (d=1.006 g/ml) in cellulose acetate tubes, and ultracentrifuged in a SW-41 swinging bucket rotor (Beckman Instrument, Palo Alto,

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CA) at 100,000g for 45 min at 17 8C. The floating layer of chylomicrons was dispersed in 0.15 M NaCl at 4 8C, and used within 24 h of collection. To prepare radiolabeled chylomicron remnants, nonfasted mice were anesthetized with pentobarbital. For the functional evisceration, the portal vein, the hepatic artery, and the superior mesenteric artery were doubly ligated and cut off between two ligatures. The whole intestine, spleen and pancreas were removed, and the liver was freed from all its ligaments. The tissues between the liver and the right adrenal gland were ligated and cut off so that the liver attached only to the inferior vena cava. The abdominal wall was closed. 250 Al of radiolabeled chylomicrons was intravenously injected into the functionally hepatectomized and eviscerated mice, and the animal was maintained under anesthesia for 1 h, and then a blood sample was taken from the heart into a heparinized microtube. The fresh plasma was layered under 0.9% NaCl in cellulose acetate tubes and ultracentrifuged in a SW-41 swinging bucket rotor at 100,000g at 17 8C for 12 h, dialyzed against 0.15 M NaCl solution, and used within 24 h of collection. Radioactive chylomicron remnants prepared by this procedure were labeled in both the unesterified cholesterol (~30%) and the cholesterol ester (~70%) fractions as examined by thin layer chromatography and counted by a liquid scintillation spectrometer (Beckman Instruments, San Ramon, CA). 2.5. Plasma clearance of chylomicrons and chylomicron remnants, and hepatic uptake of chylomicron remnants The plasma clearance studies commenced by injecting identical amounts of radiolabeled chylomicrons in a volume of 100 Al as a bolus into the right jugular vein of AKR and C57L mice (n=5 per group). At 0.1, 5, 10, 15, and 30 min after injection, exactly 50 Al of blood samples was collected from the left jugular vein. The fractions of the injected doses of tritium remaining in the plasma at each time point were used for calculating the clearance rate of chylomicrons. In additional groups of AKR and C57L mice (n=5 per group), identical amounts (100 Al) of radiolabeled chylomicron remnants were injected as a bolus into the right jugular vein. Blood samples (50 Al) were taken from the left jugular vein at 0.1, 5, 10, 15, and 30 min after injection. The fractions of the injected doses of tritium remaining in the plasma at each time points were used for calculating the clearance rate of chylomicron remnants. To study hepatic uptake of chylomicron remnants, after the last blood sample (i.e., 30 min after the injection) was harvested from the mouse, the liver was perfused for 1 min with 0.15 M NaCl through the portal vein to remove residual blood from the organ. The liver was then weighed and homogenized in 30 ml of CHCl3–CH3OH (2:1, v/v) for the measurement of tissue radioactivity.

2.6. Cannulation of the common bile duct and collection of hepatic biles To determine the transport rate of radiolabeled cholesterol in chylomicron remnants from plasma into bile, the common bile duct was cannulated as previously described [25]. Following cholecystectomy as well as successful catheterization and flow of fistula bile, identical amounts of [3H]cholesterol-labeled chylomicron remnants in a volume of 100 Al were injected via jugular vein as a bolus to chow- or lithogenic diet-fed AKR and C57L mice (n=5 per group). Bile samples were collected by gravity at 1-h intervals for 12 h. Animals were kept anesthetized with an intraperitoneal injection of 17 mg/kg pentobarbital every 2 h, and were given a small amount (100 Al) of 0.9% NaCl via the abdominal cavity to maintain hydration. During surgery and hepatic bile collection, mouse body temperature was maintained at 37F0.5 8C with a heating lamp and monitored with a thermometer. Total radioactivity in bile samples was counted in the liquid scintillation spectrometer. Also, radiolabeled cholesterol and bile salts in bile were separated by thin layer chromatography [26], and then quantified by the liquid scintillation spectrometer. Bile cholesterol levels were determined by HPLC [22]. 2.7. Fast performance liquid chromatography (FPLC) analysis At 14 days on chow or the lithogenic diet, fresh plasma was obtained from AKR and C57L mice (n=5 per group) that were fasted overnight (i.e., in the fasting state), or 1 h after feeding (i.e., in the postprandial state). Pooled plasma (250 Al) was fractionated by FPLC gel filtration (Bio-Rad Laboratories, Hercules, CA) on a Superose 6 column [19]. The column was eluted at a flow rate of 0.5 ml/min with a buffer containing 154 mM NaCl, 1 mM EDTA, and 3 mM NaN3 at pH 7.2. Sixty 0.5-ml fractions were collected and 100-Al aliquots from each fraction were used for measurement of cholesterol. 2.8. Postheparin lipase assay Postheparin plasma was obtained from blood drawn 10 min after an intravenous injection of 100 U/kg heparin into fasting mice. Total plasma lipoprotein lipase activity was determined in triplicate using a radiolabeled triolein emulsion according to published methods [27]. Hepatic lipase was measured after the inhibition of lipoprotein lipase with 1 M NaCl [28,29]. One milliunit (mU) of activity was defined as the amount that generated 1 nmol free fatty acids per minute at 37 8C, and plasma activities are expressed as mU per milliliter of plasma.

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2.9. Statistical analysis All data are expressed as meansFS.D. Statistically significant differences among groups of mice were assessed by Student’s t-test or Mann–Whitney U tests. Analyses were performed with SuperANOVA software (Abacus Concepts, Berkeley, CA). Statistical significance was defined as a twotailed probability of less than 0.05.

3. Results 3.1. Relationship between intestinal cholesterol absorption efficiency and gallstone prevalence Fig. 1A shows the cholesterol absorption efficiency determined by the fecal dual–isotope ratio method in 15 strains of inbred male mice on chow. In agreement with previous study [24] as measured by the plasma dual–isotope ratio method, we observed that there were marked differences among mouse strains with respect to intestinal cholesterol absorption efficiency: C3H (24F2%), A/J (29F2%), AKR (31F4%), BDP (31F3%), and BALB (32F3%) strains showed the lowest cholesterol absorption; SJL (33F3%), DBA (34F4%), SM (35F2%), 129/S (36F3%), 129X1 (38F5%), 129S3 (40F3%), and SWR (42F2%) strains gave intermediate values; and FVB (47F4%), C57BL (49F3%), and C57L (50F4%) strains displayed the highest values for cholesterol absorption. Fig. 1B exhibits that at 8 weeks on the lithogenic diet, gallstone prevalence rates are the lowest in C3H (10%), A/J (10%), AKR (20%), BDP (30%), and 129/S mice (30%). In contrast, gallstone prevalence rates were highest in the C57L (80%), C57BL (70%), SWR (60%), FVB (60%), DBA (60%), and BALB mice (50%), with SJL (40%), SM (40%), 129X1 (40%), and 129S3 mice (40%) showing intermediate values for gallstone prevalence rates. As shown in Fig. 1C, the efficiency of intestinal cholesterol absorption correlates positively and significantly (r=0.87, Pb0.0001) with the prevalence rates of cholesterol gallstones among inbred mice. 3.2. Plasma and biliary responses to high cholesterol diets The FPLC analysis revealed that on chow, C57L mice in the fasting state (Fig. 2A) or in the postprandial state (Fig. 2B) exhibited essentially similar amounts of cholesterol in the chylomicron, VLDL, LDL, and HDL fractions compared with AKR mice. Moreover, at 14 days on the lithogenic diet and in the fasting state (Fig. 2C), VLDL and LDL cholesterol levels were remarkably increased and HDL cholesterol levels were decreased strikingly in both strains of mice. Furthermore, VLDL, LDL, and HDL cholesterol concentrations were slightly higher in C57L mice than in AKR mice. It should be emphasized that in the postprandial state and on the lithogenic diet (Fig. 2D),

Fig. 1. Relationship between percent cholesterol absorption and gallstone prevalence rate in 15 strains of inbred male mice. (A) The cholesterol absorption efficiency for each strain (n=5 per group) was determined by the fecal dual–isotope ratio method [23]. Consistent with previous studies [24] as measured by the plasma dual–isotope ratio methods, cholesterol absorption efficiency varies from low to high among these inbred mice. (B) At 8 weeks on the lithogenic diet, gallstone prevalence rates are the lowest in C3H, A/J, AKR, BDP, and 129/S strains. In contrast, gallstone prevalence rates are the highest in C57L, C57BL, SWR, FVB, DBA, and BALB strains, with SJL, SM, 129X1, and 129S3 strains showing intermediate values for gallstone prevalence rates. (C) These results display a significant and positive correlation between percent cholesterol absorption and gallstone prevalence rate, suggesting that high efficiency of intestinal cholesterol absorption per se is an independent risk factor for cholesterol gallstone formation.

there were marked increases in the fraction of chylomicron cholesterol in C57L mice compared with those in AKR mice. We further examined the distribution of [3H]cholesterol in plasma by the FPLC method at 30 min after intragastrical administration of the lipid emulsion, i.e., in the postprandial state (Fig. 3). Of special note is that whatever chow or the lithogenic diet was fed, C57L mice displayed strikingly higher radioactivity values for chylomicrons in the plasma compared with AKR mice, suggesting that the former mice

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Fig. 2. Fast performance liquid chromatography (FPLC) profiles of plasma lipoproteins from AKR and C57L mice fed 14 days with chow or the lithogenic diet in the fasting state (i.e., after a fast of 12 h) and in the postprandial state (i.e., 1 h after feeding). Plasma was pooled (250 Al from five mice for each mouse strain) and subjected to FPLC analyses. The cholesterol content of each fraction was determined by enzymatic assay and expressed as microgram per fraction. On chow and (A) in the fasting state or (B) in the postprandial state, C57L mice exhibit essentially similar amounts of cholesterol in the chylomicron, VLDL, LDL, and HDL fractions compared with AKR mice. Moreover, at 14 days on the lithogenic diet and (C) in the fasting state or (D) in the postprandial state, VLDL and LDL cholesterol levels were remarkably increased and HDL cholesterol levels are strikingly decreased in both strains of mice. Of special note is that (D) in the postprandial state and on the lithogenic diet, chylomicron cholesterol levels are markedly higher in C57L mice than in AKR mice.

absorb more cholesterol from the intestine than the latter does. Furthermore, no radioactivity was found in the fractions of plasma VLDL, LDL, or HDL. We also studied changes in biliary cholesterol outputs in response to high dietary cholesterol at 14 days of the feeding during the first

hour of biliary washout following the interruption of the enterohepatic circulation. On chow, biliary cholesterol outputs (9.7F0.7 Amol/h/kg) in C57L mice were significantly ( Pb0.01) higher than those in AKR mice (5.0F1.0 Amol/h/kg). In contrast, feeding the lithogenic diet significantly ( Pb0.05) increased biliary cholesterol outputs in C57L mice (17.0F1.1 Amol/h/kg) and AKR mice (8.1F1.0 Amol/h/kg) compared with the chow diet, favoring C57L mice than AKR mice ( Pb0.0001). These observations suggest that intestinal chylomicrons provide a major cholesterol source to liver for secretion into bile upon the lithogenic diet. 3.3. Plasma clearance and hepatic uptake of intravenously administered lymph chylomicrons and chylomicron remnants

Fig. 3. In the postprandial state, the FPLC analysis exhibits the distribution of [3H]cholesterol in plasma at 30 min after intragastrical administration of the lipid emulsion. Of note is that whatever chow or the lithogenic diet is fed, C57L mice show markedly higher values for radiolabeled chylomicrons in the plasma compared with AKR mice. No radioactivity is detected in the fractions of plasma VLDL, LDL, or HDL.

Fig. 4A shows plasma clearance rates of [3H]cholesterol-labeled chylomicrons in mice. The tritium activity reduced rapidly in the plasma of mice for the first 5 min after the intravenous injection, and 62F6% and 33F4% of the injected radiolabeled chylomicrons remained in the plasma of AKR and C57L mice, respectively. Subsequently, radioactivity in the plasma continued to reduce in C57L mice, but remained nearly constant in AKR mice. By 30 min, only 13F5% of the radioactivity was detected

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(Fig. 4B) were cleared from the circulation with 87F7% of the injected dose being recovered in the livers of C57L mice. However, AKR mice displayed that 59F6% of the injected chylomicron remnants recovered in the livers with 68F7% of the injected dose being cleared from the circulation (Fig. 4B). 3.4. Lipoprotein lipase and hepatic lipase activities As expected [20,21], the disappearance of chylomicrons from the plasma is explained by rapid exhepatic processing of chylomicrons to chylomicron remnants by lipoprotein lipase and hepatic lipase, with subsequent uptake by the liver. Therefore, we examined the plasma activities of lipoprotein lipase and hepatic lipase in chow-fed AKR and C57L mice. Our results showed that the plasma activities of lipoprotein lipase (614F102 mU/ml) and hepatic lipase (93F23 mU/ml) in C57L mice were significantly ( Pb0.05) higher than those (410F82 mU/ml and 59F13 mU/ml) in AKR mice. 3.5. Transport of radiolabeled cholesterol in chylomicron remnants from plasma into bile

Fig. 4. Plasma clearance rates of [3H]cholesterol-labeled chylomicrons and chylomicron remnants as functions of time after the intravenous injection in chow-fed AKR and C57L mice (n=5 per group). (A) Radiolabeled chylomicrons and (B) chylomicron remnants are removed from plasma at faster rates in C57L mice than in AKR mice. Thirty minutes after injection, 87F5% (chylomicrons) and 94F3% (chylomicron remnants) of the injected dose are cleared from the plasma of C57L mice. In contrast, AKR mice display significantly slower plasma clearance rates of chylomicrons (49F4%) and chylomicron remnants (68F7%).

in the plasma of C57L mice, being significantly ( Pb0.001) lower than that in AKR mice (51F4%). This suggests that radiolabeled chylomicrons are removed more rapidly from the plasma of C57L mice compared with those in AKR mice. Furthermore, C57L mice displayed rapid plasma clearance rates of [3H]cholesterol-labeled chylomicron remnants (Fig. 4B), and by 30 min after injection, only 6F3% of the radioactivity was recovered in the plasma of C57L mice. In contrast, radioactivity disappeared at a slower rate from the circulation in AKR mice, and by 30 min, 32F7% of the injected dose was still present in the circulation. Taken together, our results indicate that both [3H]cholesterol-labeled chylomicrons and chylomicron remnants are removed from plasma in a significantly shorter time in C57L mice than in AKR mice. We also examined the hepatic uptake of radiolabeled chylomicron remnants. At 30 min after the intravenous injection, 94F3% of radiolabeled chylomicron remnants

As shown in Fig. 5A, in mice on chow, the chylomicron remnant-derived radioactivity appears rapidly in bile and rises sharply in the first hour. The tritium activity reached their highest values with 5.2F0.2% of the injected dose in C57L mice and 2.4F0.1% in AKR mice at 2 h after the injection. The analysis of biliary secretion on an hourly basis showed that the secretion rates of radioactivity decreased gradually over 12 h, and the radioactivity in bile was much lower in AKR mice than in C57L mice. It should be emphasized that compared with the standard rodent chow diet, feeding the lithogenic diet greatly augmented hepatic secretion of the chylomicron remnantderived radiolabeled cholesterol, mostly due to the dietary cholic acid as discussed elsewhere [30]. Consequently, the radioactivity recovered in bile was markedly higher in lithogenic diet-fed mice than that in chow-fed mice. Fig. 5B compares the cumulative biliary secretion of radioactivity in AKR with that in C57L mice. The cumulative biliary outputs established that whatever chow or the lithogenic diet was fed, the total amounts of radioactivity recovered in bile at 12 h after injection were significantly ( Pb0.0001) higher in C57L mice (34.8F0.7% in chow and 70.5F2.0% in the lithogenic diet) compared with those in AKR mice (16.7F1.7% in chow and 39.1F3.6% in the lithogenic diet). Analysis of the bile compositions of C57L and AKR mice revealed that at all time points, the majority of the radioactivity was present in cholesterol and bile salts. About half of radioactivity was in the form of unesterified cholesterol, and half was present in the form of bile salts as determined by thin layer chromatography [26], suggesting that the chylomicron remnant-derived cholesterol is

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Fig. 5. Biliary secretion of radiolabeled cholesterol in chylomicron remnants in chow- or lithogenic diet-fed AKR and C57L mice (n=5 per group). Bile samples are collected at 1-h intervals for a total of 12 h. (A) Over 12 h, chylomicron remnant cholesterol is transported from plasma into bile more rapidly in C57L mice than in AKR mice. It should be emphasized that compared with the standard rodent chow diet, feeding the lithogenic diet greatly augments hepatic secretion of the chylomicron remnant-derived radiolabeled cholesterol, mostly due to the dietary cholic acid as discussed elsewhere [30]. As a result, the radioactivity recovered in bile is markedly higher in lithogenic diet-fed mice than that in chow-fed mice. (B) The cumulative biliary outputs establish that whatever chow or the lithogenic diet is fed, the total amounts of radioactivity recovered in bile at 12 h after injection are significantly higher in C57L mice (34.8F0.7% in chow and 70.5F2.0% in the lithogenic diet) than in AKR mice (16.7F1.7% in chow and 39.1F3.6% in the lithogenic diet).

preferentially used for biliary secretion as cholesterol and bile salts.

4. Discussion Cholesterol gallstone formation is a multifactorial disease influenced by a complex interaction of genetic and environmental factors and the primary pathophysiologic event is biliary cholesterol hypersecretion from the liver. Several chromosome regions containing Lith genes have been identified by quantitative trait locus (QTL) analysis in inbred mice [31]. Furthermore, the majority of the environmental factors are probably related to Western-type dietary habits [1–4], including excess cholesterol and saturated fat consumption as well as high caloric intake [2,32]. The goal of the present studies was to explore a relationship between cholesterol absorption efficiency and gallstone prevalence and the metabolic fate of chylomicron and chylomicron

remnant cholesterol in mice. The most important findings are that (i) genetic variations in cholesterol absorption efficiency are associated with cholesterol gallstone formation in inbred mice; and (ii) there are marked differences in the metabolic responses to high dietary cholesterol between AKR and C57L mice, and upon feeding the lithogenic diet, dietary cholesterol augments biliary secretion of cholesterol and lithogenicity of bile through the chylomicron pathway more effectively in C57L mice than in AKR mice. Based on epidemiological evidence, most, but not all, studies suggested that high cholesterol diet often is invoked as an important risk factor in the pathogenesis of cholesterol gallstones because high cholesterol diet could increase the saturation of bile with cholesterol. Furthermore, the incidence of cholelithiasis in North America and European countries with Western diets is significantly higher than that in the developing countries [1–4]. Therefore, the effect of high dietary cholesterol on the composition of bile and the formation of gallstones has been extensively studied in humans and several animal species such mice, prairie dogs, and squirrel monkeys. However, some conflicting results from human studies were reported, showing that high dietary cholesterol either increases [11–13] or does not affect cholesterol saturation of bile [14,15]. These contradictory studies may be due to the fact that there are interindividual variations in intestinal cholesterol absorption efficiency in humans [33–37]. So, we test the hypothesis that intestinal cholesterol absorption efficiency is associated with cholesterol gallstone formation. In the present study, we found a positive and significant correlation between the efficiency of cholesterol absorption and the prevalence of cholesterol gallstone formation in 15 strains of inbred male mice. Our results suggest that the high efficiency of intestinal cholesterol absorption and the high dietary cholesterol are two independent risk factors for cholesterol gallstone formation. It should be emphasized that other genetic and pathophysiological factors such as Lith genes, increased cholesterol biosynthesis and reduced bile salt synthesis in the liver, biliary cholesterol hypersecretion, rapid cholesterol crystallization in bile, mucin hypersecretion and accumulation in the gallbladder, and gallbladder stasis also play a critical role in gallstone formation in this unique gallstonesusceptible mouse model [22,25,30,38]. Because hepatic hypersecretion of biliary cholesterol is an important prerequisite for cholesterol gallstone formation, we further investigated chylomicron remnant metabolism and its role in biliary lipid secretion. We observed that chylomicron remnant-derived radiolabeled cholesterol was removed more rapidly from plasma, and by 12 h, twofold higher radioactivity appeared in the biles of C57L mice compared with AKR mice, whatever chow or the lithogenic diet was fed. Our results were consistent with the previous findings [22,25] that C57L mice display significantly higher biliary cholesterol secretion and more rapid supersaturation of bile with cholesterol than AKR mice. Dijk and co-

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workers [39] found that radiolabeled chylomicrons injected intravenously to chow-fed rats are removed rapidly from plasma and the radioactivity appears in bile within 15 min, and 25% of the radioactivity is present as unesterified cholesterol. Furthermore, they [39] showed that lactoferrin, a glycoprotein that specifically inhibits the hepatic uptake of chylomicron remnants via the apolipoprotein E receptor, reduces markedly total amounts of the radioactivity recovered in bile, suggesting that chylomicron remnants are responsible for delivering cholesterol of dietary origin to bile. Two rat studies [40,41] found that the secretion of biliary cholesterol after intravenous injection of radiolabeled chylomicron remnants was essentially similar to those observed using radiolabeled chylomicrons, with a rapid clearance of radioactivity from plasma and appearance in bile in the first hour. Taken together, our results support the concept that the liver is very efficient to secrete lipoprotein cholesterol of intestinal origin into bile. Therefore, high dietary cholesterol through the chylomicron pathway could provide an important source of excess cholesterol molecules for secretion into bile, thereby inducing cholesterol-supersaturated bile. On the other hand, it is reasonable to assume that inhibiting cholesterol absorption and/or hepatic uptake of chylomicron remnants could induce a significant decrease in biliary cholesterol secretion and saturation. The lack of cholesterol ester synthesis in the small intestine due to targeted deletion of the acyl-CoA:cholesterol acyltransferase gene 2 (Acat2) causes a marked reduction in intestinal cholesterol absorption and complete resistance to dietinduced cholesterol gallstones [42]. Moreover, reduced biliary cholesterol secretion and gallstone prevalence in lithogenic diet-fed apolipoprotein E knockout mice may be explained by decreased availability of chylomicron-derived cholesterol in the liver for biliary cholesterol secretion [43]. These studies suggest that these animals fail to deliver cholesterol of intestinal origin into bile and to form gallstones. These results [42,43] are consistent with our findings that cholesterol derived from the intestine via the chylomicron and chylomicron remnant pathway influences biliary cholesterol secretion, and high dietary cholesterol through this pathway enhances cholelithogenesis [44]. Interestingly, a recent study [45] found that the deficiency of hepatic lipase does not affect the availability of lipoprotein-derived cholesterol for biliary secretion and expression levels of hepatic lipase are not necessary for diet-induced gallstone formation in mice. This suggests that hepatic lipase could not be a rate-controlling enzyme for the regulation of biliary cholesterol secretion, and hence, targeted deletion of hepatic lipase could not influence gallstone formation. Furthermore, it should be emphasized that the lack of hepatic lipase does not influence the metabolism of chylomicron and chylomicron remnants in mice [45]. A possible explanation is that lipoprotein lipase has a major effect on the metabolism of chylomicron and chylomicron remnants in the absence of hepatic lipase in

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mice. Clearly, additional studies are required to pursue this tantalizing observation. Although our studies showed several cholesterol transport-related molecules mediating diet-derived lipoprotein cholesterol metabolism, their structural genes [31,46] do not map to mouse chromosomes 2 and 19 and all are excluded as candidate genes for Lith1 or Lith2 that are identified in C57L strain [47,48]. Hitherto, a limited number of inbred mice were studied by QTL analysis and 12 Lith genes are identified in the mice studied [31]. Although the genes encoding for the proteins involved in intestinal cholesterol absorption and chylomicron (remnant) metabolism have not been identified as Lith genes, some candidate genes that are related to these proteins have been proposed [1,31]. Hence, further studies in new strains of inbred mice as well as transgenic and knockout mice are required to confirm the functions of these proposed candidate genes on intestinal cholesterol absorption, chylomicron metabolism, biliary cholesterol secretion, and gallstone formation. In conclusion, the high efficiency of intestinal cholesterol absorption is an independent risk factor for cholesterol gallstone formation, which greatly enhances biliary cholesterol hypersecretion on the murine lithogenic diet. Furthermore, high cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol significantly increase biliary cholesterol secretion, cholesterol saturation of bile, and gallstone formation in gallstone-susceptible C57L mice. If similar mechanisms operate in humans, then the elucidation of the molecular events underlying intestinal cholesterol absorption efficiency and hepatic secretion of chylomicron remnant cholesterol into the bile may lead to new insights into the understanding of biliary responsiveness to dietary cholesterol and offer a new approach to the prevention of cholesterol gallstone formation.

Acknowledgments We are greatly indebted to Dr. Martin C. Carey (Brigham and Women’s Hospital, Boston, MA) and Dr. Beverly Paigen (The Jackson Laboratory, Bar Harbor, ME) for valuable suggestions at the initiation of this study. D.Q.-H. Wang is a recipient of a New Scholar Award from the Ellison Medical Foundation (1999–2003). This work was supported in part by a grant from the Ellison Medical Foundation (to D.Q.-H. W.) and a research grant DK54012 (to D.Q.-H. W.) from the National Institutes of Health (US Public Health Service).

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