1 Department of Medicine, Harvard Medical School, Gastroenterology Division, Brigham and Women's Hospital and Harvard Digestive Diseases Center, Boston, Massachusetts 02115; and 2 Jackson Laboratory, Bar Harbor, Maine 04609
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ABSTRACT |
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Cholic acid is a critical component of the
lithogenic diet in mice. To determine its pathogenetic roles, we fed
chow or 1% cholesterol with or without 0.5% cholic acid to C57L/J
male mice, which because of lith genes
have 100% gallstone prevalence rates. After 1 yr on the diets, we
measured bile flow, biliary lipid secretion rates, hepatic cholesterol
and bile salt synthesis, and intestinal cholesterol absorption. After
hepatic conjugation with taurine, cholate replaced most
tauro--muricholate in bile. Dietary cholic acid plus cholesterol
increased bile flow and biliary lipid secretion rates and reduced
cholesterol 7
-hydroxylase activity significantly mostly via
deoxycholic acid, cholate's bacterial 7
-dehydroxylation product but
did not downregulate cholesterol biosynthesis. Intestinal cholesterol
absorption doubled, and biliary cholesterol crystallized as phase
boundaries shifted. Feeding mice 1% cholesterol alone produced no
lithogenic or homeostatic effects. We conclude that in mice cholic acid
promotes biliary cholesterol hypersecretion and cholelithogenesis by
enhancing intestinal absorption, hepatic bioavailability, and phase
separation of cholesterol in bile.
genetics; phase diagrams; bile salt species; bile flow; microscopy; mucin; nutrition; 3-hydroxy-3-methylglutaryl-coenzyme A reductase; cholesterol 7- hydroxylase
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INTRODUCTION |
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CHOLESTEROL GALLSTONES develop in a majority of mice carrying lith genes on feeding a lithogenic diet containing cholesterol and cholic acid (2, 21, 34, 39), whereby the bile salt (BS) pool is enriched with taurocholate (TC). In an experiment with a small number of mice, Tepperman and co-workers (34) as well as Fujino and colleagues (14) found that feeding 1% cholesterol alone for 4-8 mo did not induce cholesterol gallstone formation. This indicates that the role of cholic acid is essential in the mouse gallstone model, but its mechanism is not well understood. In rats, Uchida and co-workers (36) observed that cholic acid is necessary for intestinal cholesterol absorption via marked increases in TC and taurodeoxycholate (TDC) in bile. Akiyoshi and colleagues (1) found that in diabetic mice enhanced cholesterol absorption was induced by increased synthesis and secretion of endogenous TC and that this plays an important role in formation of cholesterol gallstones even on a chow diet. In contrast, others (11, 30) have suggested that addition of cholic acid or TC to chow or 2% cholesterol diets does not influence intestinal cholesterol absorption. Furthermore, there are contradictory studies in the literature concerning percent cholesterol absorption in healthy inbred mice fed chow, which has been reported to vary from 20 to 80% with small interstrain differences (1, 7, 22, 33, 43). These findings suggest that the effect of cholic acid on cholesterol absorption and gallstone formation in the mouse should be reevaluated. Therefore, using gallstone-susceptible C57L/J mice (21), we explored the role of cholic acid on intestinal cholesterol absorption, hepatic cholesterol, and BS synthesis as well as cholesterol gallstone formation. We investigated whether long-term feeding of 1% cholesterol without cholic acid could induce the same biliary and hepatic enzymatic phenotypes as observed when mice were fed the lithogenic diet. Our findings suggest that, through forming TC, cholic acid has multiple effects at pathophysiological, biochemical, and physicochemical levels, all of which appear important in murine cholesterol gallstone formation.
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MATERIALS AND METHODS |
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Chemicals.
Intralipid (20%, wt/vol) was obtained from Pharmacia (Clayton, NC),
and medium-chain triglyceride oil was from Mead Johnson (Evansville,
IN). All radiochemicals were purchased from DuPont NEN (Boston, MA).
The radiochemical purities of
[1,2-3H]cholesterol
and [4-14C]cholesterol
were >97% as determined by HPLC analyses. The radiochemical purity
of
DL-[3-14C]hydroxy-3-methylglutaryl
(HMG)-CoA was verified to be >97% by paper chromatography in
butanol-glacial acetic acid-water (7:2:3, vol/vol/vol).
The purity of
DL-[5-3H]mevalonolactone,
used as an internal standard, was >99% by TLC in
toluene-acetone-acetic acid (20:10:1, vol/vol/vol). For HPLC analyses
of BS species and cholesterol, all reagents were HPLC grade and
obtained from Fisher Scientific (Fair Lawn, NJ). BS standards were
obtained from Sigma Chemical (St. Louis, MO) and Calbiochem-Behring
(San Diego, CA), with the exception of the taurine conjugates of -
and
-muricholates: 3
,6
,7
-trihydroxy-5
-cholanoate (tauro-
-muricholate or T-
-MC) and
3
,6
,7
-trihydroxy-5
-cholanoate (tauro-
-muricholate or
T-
-MC), respectively, which were provided generously by Tokyo Tanabe
(Tokyo, Japan; courtesy of H. Sugata). Purity of
individual BS by HPLC was >98% (8, 37). All other chemicals and
solvents were American Chemical Society or reagent grade quality
(Fisher Scientific, Medford, MA).
Animals and diets. Male C57L/J mice, 4-6 wk old, were bred at The Jackson Laboratory (Bar Harbor, ME) and were homozygous for susceptible lith alleles (21). All animals were maintained in a temperature-controlled room (22 ± 1°C) with regular 12:12-h day-night cycles (6 AM-6 PM). Mice were allowed to adapt to the environment for at least 2 wk before lithogenic diet feeding and were provided free access to water and normal mouse chow. Throughout the experimental periods, mice were fed Purina Laboratory Chow, which contains trace cholesterol (<0.02%) (Mouse Diet 1401, S. Hanky Road, St. Louis, MO) or a semisynthetic lithogenic diet (27) each 100 g, which contains 1 g cholesterol, 15 g butter fat, 2 g corn oil, 50 g sucrose, 20 g casein, and essential vitamins and minerals with or without 0.5 g cholic acid. Once mice reached 8 wk of age, they were divided into 3 groups (n = 20 each) fed 1) chow containing <0.02% cholesterol, 2) the semisynthetic diet containing 1% cholesterol, or 3) the semisynthetic diet containing 1% cholesterol and 0.5% cholic acid. All experiments were executed according to accepted criteria for the care and experimental use of laboratory animals, and euthanasia was consistent with recommendations of the American Veterinary Medical Association. All protocols were approved by the Institutional Animal Care and Use Committees of Harvard University and The Jackson Laboratory.
Collection of gallbladder biles and gallstones and microscopic
studies.
After 1 yr of feeding, surgery was performed on mice that were fasted
overnight but had free access to water. Animals were weighed and
anesthetized with an intraperitoneal injection of 35 mg/kg
pentobarbital (Abbott Laboratories, North Chicago, IL). Surgery
commenced at 9 AM and was performed under sterile conditions through an
upper midline incision. After cholecystectomy, gallbladder volume was
measured by weighing the whole gallbladder and equating gallbladder
weight (including stones) with gallbladder volume. Gallbladders were
then opened, and 5 µl of fresh gallbladder bile were examined for
mucin gel, cholesterol crystals, liquid crystals, and gallstones
(37-39). Liquid crystals and solid crystals as well as gallstones
were defined according to previously established criteria (37-39).
After pooled gallbladder biles were ultracentrifuged at 100,000 g for 30 min at 37°C and filtered
through a preheated (37°C) Swinnex-GS filter assembly containing a
0.22 µm filter (Millipore Products Division, Bedford, MA), samples
were frozen and stored at 20°C for further lipid analyses.
Cannulation of common bile duct and collection of hepatic biles.
Additional groups of mice (n = 5 each)
fed chow (<0.02%), 1% cholesterol, or 1% cholesterol plus 0.5%
cholic acid were used for biliary lipid secretion studies. In brief,
the lower end of the common bile duct was ligated, and the common bile
duct was cannulated below the entrance of the cystic duct via a PE-10
polyethylene catheter with an inside diameter of 0.28 mm and an outside
diameter of 0.61 mm (Becton Dickinson Primary Care Diagnostics, Becton Dickinson, Sparks, MD). After successful catheterization and flow of
fistula bile, the cystic duct was doubly ligated and cholecystectomy was performed. Hepatic bile was collected by gravity for the first hour. After fresh hepatic biles were examined by polarizing light microscopy and their volumes determined by weighing (39), all samples
were frozen and stored at 20°C for further lipid analyses. During surgery and hepatic bile collection, mouse body temperature was
maintained at 37 ± 0.5°C with a heating lamp and monitored with
a thermometer.
Determination of cholesterol absorption. Four groups of mice (n = 5 each) were fed either chow containing <0.02% cholesterol, 1% cholesterol, 1% cholesterol plus 0.5% cholic acid, or 0.5% cholic acid for 2 days, which was sufficient to reach a TC steady state in bile (39). Cholesterol absorption was determined by the dual-isotope plasma ratio method described by Zilversmit and Hughes (46) and Turley et al. (35) with major modifications in methods for intravenous injection, intragastric administration, as well as lipid volumes and radiolabeled cholesterol given to each mouse. In brief, nonfasted mice were anesthetized lightly by intraperitoneal injection of 35 mg/kg pentobarbital. An 0.4-cm incision was made on the right or left side of the neck, and the jugular vein was exposed. Exactly 2.5 µCi of [3H]cholesterol dissolved completely in 100 µl of Intralipid was injected intravenously directly into the jugular vein using a 100-µl Hamilton syringe fitted with a 30-gauge needle and carried out over 1 min to prevent cardiac arrest. The incision was closed tightly with 3-0 silk sutures. After this procedure, a feeding needle with round tip (18 gauge, 50 mm in length) was inserted completely into the stomach of the mouse, and then by gavage the animal was given an intragastric dose of 1 µCi of [14C]cholesterol fully dissolved in 150 µl of medium-chain triglyceride oil. With this protocol, no problems were encountered with mice regurgitating gastric contents during and after the recovery period. After dosing, mice were returned to the animal room where they were free to eat chow or the appropriate semisynthetic diets for an additional 3 days. Mice were then anesthetized lightly as described and were bled from the heart into a microtube containing heparin (Elkins-Sinn, Cherry Hill, NJ) as anticoagulant. Aliquots of plasma were obtaining by centrifugation at 10,000 g for 30 min at room temperature. To determine the proportions of [14C]- and [3H]cholesterol doses remaining in plasma after 72 h, 100-µl plasma aliquots and the original dosing mixture were added directly to 10 ml EcoLite (ICN Biomedicals, Costa Mesa, CA). The vials were shaken vigorously for 10 min and counted in a liquid scintillation spectrometer (Beckman Instruments, San Ramon, CA). The ratio of the two radiolabels in plasma was used for calculating the percent cholesterol absorption using the following expression
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Measurement of activities of hepatic HMG-CoA reductase and
cholesterol 7-hydroxylase.
Liver samples were obtained from nonfasted mice after 1 yr of feeding
chow, 1% cholesterol, or 1% cholesterol plus 0.5% cholic acid. To
minimize diurnal variations of hepatic enzyme activities, all
procedures were performed between 8 and 9 AM. Microsomal activities of
HMG-CoA reductase were determined by measuring the conversion rate of
[14C]HMG-CoA to
[14C]mevalonic acid
using a radiochemical assay (10, 44). Products were quantified by
liquid scintillation counting with
[3H]mevalonolactone as
internal standard. Protein concentration was determined by the assay of
Bradford (4). Hepatic activities of cholesterol 7
-hydroxylase were
determined by the HPLC-based assay system of Hylemon et al. (19).
Lipid analyses. Biliary phospholipids were determined as inorganic phosphorus by the method of Bartlett (3). Total BS and individual BS concentrations were measured by HPLC according to the methods of Rossi et al. (31). Bile cholesterol, as well as cholesterol content in chow and gallstones, were determined by HPLC (15, 39). Cholesterol saturation indexes (CSI) in gallbladder and hepatic biles were calculated from the critical tables (5). Relative lipid compositions of mouse gallbladder and hepatic biles were plotted on condensed phase diagrams appropriate to their mean total lipid concentrations. The phase boundaries and crystallization pathways were extrapolated from model bile systems according to relative and total lipid concentrations developed for TC at 37°C (6, 37).
Statistical methods. Data are expressed as means ± SD. Differences among groups of mice fed chow, 1% cholesterol, 1% cholesterol plus 0.5% cholic acid, or 0.5% cholic acid were assessed for statistical significance by Student's t-test. Statistical significance is defined as a two-tailed probability of <0.05.
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RESULTS |
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Prevalence and characteristics of gallstones and mucin gel. After 1 yr of being fed chow or 1% cholesterol, both groups of mice had clear gallbladder biles and no mucus gel was detected. Moreover, they did not form either liquid crystals or solid cholesterol crystals or gallstones. In contrast, 100% of mice fed 1% cholesterol plus 0.5% cholic acid formed gallstones. The extracted sterols from these stones contained only cholesterol, which constituted >99% of stone weight. Most mice formed seven to nine gallstones. On average, the size of gallstones was 0.86 ± 0.26 mm in diameter. Gallbladders from all mice fed 1% cholesterol plus 0.5% cholic acid had layers of thick mucus gel (100%) containing cholesterol monohydrate crystals (100%) and liquid crystals (75%) (37-39).
Gallbladder volumes. At 1 yr, gallbladder volumes were similar between mice fed chow (26 ± 8 µl) and 1% cholesterol (23 ± 9 µl), and both were significantly (P < 0.01) smaller than those of mice (35 ± 12 µl) fed 1% cholesterol plus 0.5% cholic acid.
Lipid compositions of gallbladder and hepatic biles.
Table 1 shows biliary lipid compositions of
pooled gallbladder biles (n = 20) and
individual hepatic biles (n = 5 each
per group) at 1 yr. In general, biliary lipid compositions of
gallbladder and hepatic biles were identical between mice fed chow or
1% cholesterol alone. There were no significant differences in total
lipid concentration (gallbladder biles 6.6-8.7 g/dl and hepatic
biles 1.3-1.7 g/dl) among the three groups of mice. Compared with
mice fed chow or 1% cholesterol, mice fed 1% cholesterol plus 0.5%
cholic acid had marked increases in mole percent cholesterol and
lecithin but decreases in percent BS (Table 1). Moreover, in pooled
gallbladder biles apparent CSI (Table 1) were similar between mice fed
chow (CSI 0.52) and 1% cholesterol (CSI 0.64), and both were
significantly lower (P < 0.05) than
mice fed 1% cholesterol plus 0.5% cholic acid (CSI 1.74).
Nevertheless, if an urso-correction (5) is carried out for the
diminution in cholesterol solubility by T--MC in the gallbladder
biles, CSI values are 0.89 in chow and 1.14 in 1% cholesterol fed
groups, respectively; whereas in mice fed 1% cholesterol plus 0.5%
cholic acid, CSI increased to 1.85. Mean apparent CSI values of hepatic
biles (Table 1) were not significantly different between chow (1.13 ± 0.26) and 1% cholesterol feeding (1.41 ± 0.17), but both
were significantly (P < 0.05) lower than 1% cholesterol
plus 0.5% cholic acid feeding (1.91 ± 0.45; Table 1). An
urso-correction factor is unavailable for such dilute biles (39).
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Bile flow and biliary lipid secretion rates.
Figure 3 summarizes mean bile flow rates on
the different diets at 1 yr for the first hour following interruption
of the enterohepatic circulation. In mice fed 1% cholesterol alone,
bile flow (285 ± 23 µl · h1 · 100 g body wt
1) was similar
to that in mice fed chow (310 ± 86 µl · h
1 · 100 g body wt
1) and both were
significantly smaller (P < 0.05)
than in mice fed 1% cholesterol plus 0.5% cholic acid (599 ± 105 µl · h
1 · 100 g body wt
1).
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Influence of cholic acid feeding on intestinal cholesterol
absorption.
Percent cholesterol absorption, calculated from the plasma ratio of
tracer [14C]- and
[3C]cholesterol after
3 days of dosing, is shown in Fig. 5.
Normal mouse chow contains only <0.02% cholesterol, so that the
addition of 1% cholesterol by weight to the diet markedly increased
oral intake. However, there was no significant difference in the
cholesterol absorption between mice on chow (34 ± 7%) and those
fed 1% cholesterol (27 ± 9%). In contrast, dietary cholic acid
significantly increased (P < 0.001)
cholesterol absorption to 55 ± 5% in mice fed 1% cholesterol plus
0.5% cholic acid and to 63 ± 7% in mice fed 0.5% cholic acid without cholesterol compared with mice fed chow or 1% cholesterol alone.
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Effects of feeding 1% cholesterol with or without 0.5% cholic acid
on hepatic cholesterol and BS synthesis.
Figure 6,
top, shows the activities of hepatic
HMG-CoA reductase, the rate-limiting enzyme in cholesterol
biosynthesis. The values, although somewhat higher on the chow diet (33 ± 9 pmol · min1 · mg
1)
compared with those fed 1% cholesterol (26 ± 8 pmol · min
1 · mg
1)
and 1% cholesterol plus 0.5% cholic acid (30 ± 6 pmol · min
1 · mg
1),
were not significantly different between the groups. In contrast, Fig.
6, bottom, shows that after 1 yr of
feeding 1% cholesterol plus 0.5% cholic acid, cholesterol
7
-hydroxylase activities decreased significantly
(P < 0.05) to 1.8 ± 1.3 pmol · min
1 · mg
1
compared with 5.2 ± 2.4 pmol · min
1 · mg
1
with chow and 5.0 ± 2.9 pmol · min
1 · mg
1
with 1% cholesterol.
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DISCUSSION |
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Genetically determined cholesterol gallstone formation in mice can
provide important insights into pathogenesis and genetic modifiers of
cholesterol gallstone disease in humans. Because there is exceptionally
close homology between mouse and human genomes (9), the inbred mouse is
a most valuable research tool for investigating human gallstone genes.
Cholesterol cholelithiasis does not occur spontaneously in healthy
inbred mice, and the addition of 1% cholesterol and 0.5% cholic acid
to an otherwise well-balanced rodent mouse diet is essential for
inducing cholesterol gallstone formation (2, 21, 34, 39). Other small
experimental animals such as prairie dogs form cholesterol gallstones
after feeding the same amount of cholesterol without exogenous BS (24)
because their principal BS is TC. However, mice have a more hydrophilic BS pool composition with 46-54% TC and 34-42% T--MC
(Ref. 39 and Fig. 2), compared with the 91-94% TC of prairie dogs
(24). Humans also exhibit high cholate levels in bile with 33-43%
cholate (17, 38) conjugated with glycine and taurine in a 3/1 ratio. Feeding cholic acid is expected therefore to make the mouse BS pool
more like humans or prairie dogs because of an increase in TC and
decrease in T-
-MC concentrations (39). Nonetheless, the
pathobiological and physiological effects of these changes in BS pool
composition of mice and the other roles of cholic acid, if any, on
murine cholesterol cholelithogenesis have never been defined.
Therefore, we investigated the effects of the lithogenic diet
containing cholic acid on cholesterol absorption, hepatic cholesterol
homeostasis, biliary secretion, and gallstone formation in mice fed
special diets for 1 yr to observe maximum possible effects. The most
important findings induced by cholic acid in our study were
1) increased intestinal cholesterol
absorption and biliary cholesterol outputs, as well as a lithogenic
phase change in bile, all of which apparently contribute to cholesterol supersaturation and rapid cholesterol crystallization,
2) reduced activity of the
rate-limiting enzyme in BS biosynthesis (cholesterol 7
-hydroxylase)
and thereby disruption of the catabolic conversion of cholesterol to BS
and presumably contributing to the high cholesterol secretion rates,
thereby further favoring cholesterol supersaturation, and
3) promotion of mucin gel formation,
cholesterol crystals, and gallstones as well as the enlarged
gallbladders only when both cholesterol and cholic acid were added to
the diet, whereas addition of cholesterol alone did not reproduce these
biliary phenotypes.
Cholic acid replaces T--MC with TC, which enhances
intestinal cholesterol absorption.
In the natural state, 40% or more of the BS pool of mice fed chow is
composed of T-
-MC (Refs. 1, 39 and Fig. 2), and under these
conditions cholesterol is only modestly absorbed from the intestine
(Fig. 5). The reason apparently is that T-
-MC is a very poor
cholesterol solubilizer with similar properties to the taurine
conjugate of ursodeoxycholic acid (UDCA) (5, 25, 37). In fact compared
with TC, T-
-MC may be considered an "inhibitor" of intestinal
cholesterol absorption. It is known that in cholesterol gallstone
patients (16, 28) administration of UDCA reduces intestinal cholesterol
absorption significantly, and this has been confirmed by us in
gallstone-susceptible C57L/J mice (40). After feeding 1% cholesterol
plus 0.5% cholic acid, we have observed (39) that BS compositions in
bile were altered dramatically after 1 day of feeding, with TC
increasing to 70-80% and T-
-MC decreasing to 3-10%.
These profound changes in BS hydrophobicity induced by cholic acid in
bile and the intestine should markedly increase micellar cholesterol
solubility (6, 37). Because cholic acid increased cholesterol
absorption from the intestine significantly via TC, this should elevate
the cholesterol content of the liver (36), which in turn would increase
bioavailability of cholesterol to enhance biliary cholesterol
hypersecretion. Because insulin is apparently necessary for T-
-MC
synthesis in mice (1), diabetes mellitus increases synthesis and
secretion of endogenous TC in bile because of a spontaneous decrease in T-
-MC content. Therefore, alloxan-diabetic mice (1) fed 1% cholesterol without cholic acid form cholesterol gallstones
reproducibly and spontaneously. We also observed that, in each dietary
group (Fig. 5), the percent cholesterol absorption is slightly less than that in mice without dietary cholesterol, but the differences were
not statistically significant. Most likely this difference is
secondary to the effect of the radioisotope dilution on specific activity of cholesterol in the upper small intestine.
Cholic acid elicits a lithogenic phase change in bile with enlarged
gallbladder and accumulation of mucin gel.
Because the more hydrophobic BS (TC) replaced the hydrophilic BS
(T--MC), all crystallization pathways are shifted to the right on
the bile phase diagram, i.e., to higher lecithin contents (39). This
phase change facilitates solid cholesterol crystallization (37, 38)
during lithogenesis of cholesterol-supersaturated biles in mice fed
cholesterol plus cholic acid. In contrast, when a hydrophilic BS is
fed, all crystallization pathways would be shifted to lower lecithin
contents and cholesterol crystallization is delayed markedly (37, 38).
In fact, the addition of 0.1-0.3% hyocholic acid, also a very
hydrophilic BS (8), to a murine lithogenic diet prevents cholesterol
monohydrate crystals from forming in mice (12) and similarly
hyodeoxycholic acid, which is of approximate hydrophilicity to UDCA
(8), has been shown to prevent cholesterol gallstone formation in
hamsters (41). Of interest is that, compared with hepatic biles (Fig.
1), the lipid compositions of gallbladder biles from mice fed chow or cholesterol alone became appreciably less saturated with cholesterol and also contained lower moles percent lecithin. In contrast, the
relative lipid compositions of gallbladder and hepatic biles from mice
fed cholesterol plus cholic acid remained indistinguishable (Fig. 1).
This is consistent with the concept that the gallbladder absorbs
cholesterol and indeed some lecithin from bile (20, 26), but this
function is likely to be damaged in mice fed cholesterol plus cholic
acid, as evidenced by both mucin production and absence of changes in
relative lipid compositions (Fig. 1). Furthermore, we found that
gallbladder size was significantly enlarged by ~50% (see
RESULTS) in mice fed the lithogenic
diet for 1 yr compared with those fed for 8 wk (39), probably from
cholesterol toxicity to smooth muscle cells (45). The enlarged
gallbladder is most likely hypomotile and may facilitate cholesterol
crystallization and gallstone formation (13). Also, feeding cholesterol
plus cholic acid to mice is known to cause an acute inflammatory
reaction in the gallbladder wall with mucosal hyperplasia and mucin
hypersecretion (42). It is likely also that cholesterol-supersaturated
bile, or TC, or both may stimulate mucin production and secretion (23). Thereafter, by forming a cholesterol-enriched gel layer (24, 39), mucin
may provide an environment for continuous cholesterol crystal growth
and trapping of crystals as well as precluding further mucosal
absorption of cholesterol molecules (Fig. 1).
Response of hepatic cholesterol and BS synthesis to long-term
feeding of 1% cholesterol plus 0.5% cholic acid.
Dietary cholesterol suppresses hepatic cholesterol synthesis in the rat
by >95%, whereas cholic acid does so to a lesser extent (18).
Consistent with our previous studies (21), this effect was less
pronounced in C57L/J mice fed cholesterol or cholesterol plus cholic
acid and did not reach significance. Impaired downregulation of hepatic
cholesterol synthesis in C57L/J mice fed 1% cholesterol plus 0.5%
cholic acid (21) may contribute to biliary cholesterol hypersecretion
and the formation of cholesterol-supersaturated biles. Although the
lithogenic diet reduced cholesterol 7-hydroxylase activities in mice
(29), feeding cholic acid alone also suppressed hepatic BS synthesis in
rats (18). The current results showed that feeding cholesterol alone
did not influence activities of cholesterol 7
-hydroxylase. This
suggests that a diminished conversion of cholesterol to BS induced by
cholic acid and its deoxycholic acid metabolite (Fig. 2) may contribute
to increased cholesterol bioavailability and augmented hepatic
cholesterol secretion.
Addition of cholesterol or cholic acid alone to chow diet does not induce cholesterol gallstone formation. We observed that feeding cholesterol alone did not influences gallbladder size, hepatic secretion of biliary lipids, biliary CSI values, or mucin accumulation in the gallbladder, nor did it induce cholesterol gallstone formation. Also reported by Fujino and colleagues (14) is that feeding 0.5% cholic acid alone did not induce cholesterol gallstone formation in mice. Furthermore, they (14) observed that feeding 1.0% cholic acid induced high contents of free fatty acids (mainly palmitic acid) in hepatic biles possible due to hepatocyte toxicity, as might have been anticipated when feeding 1.5% cholic acid induced mouse mortality (14).
In summary, we have continued our pathophysiological investigation of the inbred mouse, which is now emerging as a model par excellence for the study of cholesterol gallstone disease because the genetic resources are so rich and quantitative trait loci for several lith genes have been discovered (21). In this study, we have focused critically on the essential role of 0.5% cholic acid for cholelithogenesis by a lithogenic diet, whereas feeding 1% cholesterol alone even for 1 yr is nonlithogenic. We show here that by suppressing T- ![]() |
ACKNOWLEDGEMENTS |
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This work was supported in part by research and center National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34584 to D. Q.-H. Wang, DK-48873 to D. E. Cohen, DK-51553 to B. Paigen, and DK-36588, DK-34854, and DK-52911 to M. C. Carey.
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FOOTNOTES |
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D. Q.-H. Wang is a recipient of an Industry Research Scholar Award from American Digestive Health Foundation/American Gastroenterological Association (1996-1999). F. Lammert was supported by a Postdoctoral Fellowship (La 997/2-1) from Deutsche Forschungsgemeinschaft.
This paper was presented in part at the annual meeting of the American Association of the Study for Liver Diseases, Chicago, IL, 1997, and published as an abstract (Hepatology 26: 404A, 1997).
Present addresses: F. Lammert, Dept. of Internal Medicine III, Univ. of Technology at Aachen, Aachen, D-52074, Germany; D. E. Cohen, Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Q.-H. Wang, Dept. of Medicine, Gastroenterology Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: dwang{at}rics.bwh.harvard.edu).
Received 4 August 1998; accepted in final form 24 November 1998.
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