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Correspondence to:
Ephraim Sehayek, To whom correspondence should be addressed.
We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals.
These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis. — Sehayek, E., J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow. Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice. J. Lipid Res. 2001. 42: 1250;–1256.
Supplementary key words:
bile acids, cholesterol absorption, colon cancer, dietary cholesterol, LDL-receptor knockout
Dietary cholesterol plays an important role in determining the plasma levels of cholesterol and the risk of cardiovascular diseases. Numerous clinical, epidemiological, and experimental studies show that whereas increased cholesterol intake increases the plasma levels of cholesterol and the risk of cardiovascular diseases, low cholesterol consumption provokes the opposite effects (1). These studies form the scientific basis for recommendations for low cholesterol intake as part of the dietary prevention of cardiovascular diseases in Western and Westernizing countries (2).
To exert its metabolic effects dietary cholesterol must be absorbed from the intestinal lumen. The absorption of dietary cholesterol is a complex process that is initiated by emulsification of dietary cholesterol, transfer of emulsified cholesterol (within the intestinal lumen) from the oil phase into the micellar phase, and uptake by mucosal enterocytes (3). The transfer of dietary cholesterol from the oil phase into biliary-derived intestinal micelles is an obligatory step. Specifically, the bile acids in intestinal micelles have a critical role in this process. Studies of genetically manipulated mice clearly show that alterations in bile acid pool and composition dramatically affected the absorption of dietary cholesterol (4) (5). Yet, although manipulations of bile acid pool and composition are potentially beneficial in reducing the absorption of dietary cholesterol, ample data in the literature suggest that certain bile acids may promote intestinal tumor formation (6).
Studies that addressed the effects of different bile acids on dietary cholesterol absorption suggested that whereas hydrophobic bile acids may improve absorption, hydrophilic bile acids may have an opposite effect (7). For example, studies that addressed the effect of the hydrophilic bile acid hyodeoxycholic acid (HDCA), which abounds in the bile of pigs and is also detected in rats, show that this compound suppresses the absorption of dietary cholesterol (7) (8). Moreover, in some species HDCA was also reported to stimulate liver cholesterol biosynthesis (8) (9) (10), lower plasma cholesterol levels (8) (10) and promote biliary cholesterol secretion (9) (10). The present study was designed to examine i) the metabolism of HDCA in mice, ii) the effect of this bile acid on plasma lipoprotein response to dietary cholesterol and atherosclerosis, and iii) the effect of HDCA on intestinal tumor formation.
Animals and diets
Cholesterol absorption measurements
Urinary, plasma, and biliary bile acid composition
Plasma and lipoprotein cholesterol
Measurements of liver cholesterol content and activities of hydroxymethylglutaryl-CoA reductase (HMGR), cholesterol 7
VLDL turnover
Quantitative atherosclerosis measurements
Intestinal tumorigenesis
Statistical analysis
We examined the effect of HDCA on dietary cholesterol absorption in wild-type C57BL/6 males. In these experiments mice were fed for 3 weeks with a chow diet supplied with increasing amounts of HDCA. As shown in Fig 1, increasing amounts of HDCA progressively decreased dietary cholesterol absorption rates. A 30% decrease, in absolute terms, was observed in animals fed 0.5% HDCA. Moreover, as shown in Fig 1, 50% of this decrease was already achieved in animals fed 0.05%;–0.1% HDCA and more than 95% of the decrease was observed in animals consuming 0.25% HDCA. These results suggest that HDCA efficiently suppresses the absorption of dietary cholesterol.
Previous studies of humans showed that HDCA is subjected to enterohepatic recycling and that nonconjugated HDCA is partially glucuronated and excreted in the urine (18). We examined the metabolism of HDCA in mice. As shown in Fig 2A, increasing amounts of HDCA in the diet progressively increased the fraction of HDCA in total bile acids in gallbladder bile. It is interesting to note that approximately 60% of the increment in biliary HDCA content was achieved by 0.1% HDCA in the diet and that the increment approached a plateau at 0.25% HDCA. We also examined the plasma concentrations of HDCA in the peripheral blood. As shown in Fig 2B, feeding increasing amounts of HDCA progressively increased the plasma concentrations of HDCA. Finally, HDCA was not detected in the urine of chow-fed animals whereas the concentration in 1% HDCA-fed animals reached 105 ± 58 µg/ml (mean ± SD). These studies clearly show that, in mice, HDCA is subjected to enterohepatic recycling, reaches the systemic circulation, and is partly subjected to urinary secretion.
The suppression of dietary cholesterol absorption prompted us to examine the effect of HDCA on liver cholesterol metabolism. Compared with chow-fed mice the livers of 0.5% HDCA-fed mice had decreased total cholesterol (chow-fed mice, 2.03 ± 0.1 mg/g wet liver weight; 0.5% HDCA-fed mice, 1.77 ± 0.1 mg/g wet liver weight; P = 0.018) and cholesteryl ester (chow-fed mice, 0.28 ± 0.08 mg/g wet liver weight; 0.5% HDCA-fed mice, 0.15 ± 0.04 mg/gm wet liver weight; P = 0.045). Moreover, increasing amounts of HDCA in the diet progressively stimulated the activity of HMGR ( Fig 3A), which reached a 4-fold increase in 1% HDCA-fed animals. It is interesting to note that the HMGR activity was directly correlated with the plasma levels of HDCA (Fig 3B; R 2 = 0.85; P < 0.0001). In contrast, HDCA displayed no effect on the liver activities of cholesterol 7
The suppression of dietary cholesterol absorption, depletion of liver cholesterol content, and stimulation of hepatic HMGR activity led us to postulate that HDCA may alter the plasma lipoprotein response to dietary cholesterol and atherosclerosis. For these studies we selected the C57BL/6 LDL receptor knockout (LDLR-KO) mouse as a model. In these animals, as in wild-type mice, 0.5% HDCA decreased by 30% the absorption of dietary cholesterol (74.4 ± 7.7% in chow-fed LDLR-KO mice; 42.5 ± 8.5% in 0.5% HDCA-fed LDLR-KO mice). Next, we fed LDLR-KO males with four different diets: rodent chow, chow supplemented with 0.5% HDCA, chow supplemented with 0.5% cholesterol, or chow supplemented with 0.5% cholesterol and 0.5% HDCA. After 3 weeks of feeding, plasma samples were analyzed for cholesterol levels. Compared with chow-fed animals the animals fed chow plus 0.5% HDCA had a 21% decrease in plasma cholesterol levels (chow-fed LDLR-KO animals, 216 ± 39 mg/dl; 0.5% HDCA-fed LDLR-KO animals, 171 ± 21 mg/dl; P < 0.0006). Moreover, animals fed 0.5% cholesterol displayed 3-fold increased plasma cholesterol levels compared with chow-fed animals whereas 0.5% cholesterol plus 0.5% HDCA-fed animals had a 62% decrease in plasma cholesterol levels compared with the 0.5% cholesterol-fed group (0.5% cholesterol-fed group, 660 ± 130 mg/dl; 0.5% cholesterol plus 0.5% HDCA-fed group, 252 ± 35 mg/dl; P < 0.00001). As shown in Fig 4, Superose gel chromatography revealed that regardless of the amount of cholesterol in the diet the addition of HDCA decreased the plasma VLDL and LDL cholesterol content and increased the buoyancy of LDL. These results clearly indicate that HDCA decreases the plasma levels of atherogenic lipoproteins by mechanisms that are independent of the LDL receptor pathway. To determine whether the decrease in VLDL-LDL cholesterol levels can be attributed to increased VLDL catabolism we studied the turnover of VLDL in chow- and 0.5% HDCA-fed LDLR-KO mice. As shown in Fig 5, the plasma VLDL decay curves in the two groups were superimposable, suggesting that the decrease in VLDL cholesterol cannot be explained by changes in VLDL catabolism.
Next, we examined the effect of HDCA on atherosclerosis formation in LDLR-KO mice. After being fed the four diets for 12 weeks, animals were killed and the atherosclerosis lesion area in the aortic root was quantified. As shown in Fig 6, compared with chow-fed animals, the animals fed chow plus 0.5% HDCA had a 50% decreased atherosclerotic lesion area (chow-fed animals, 10,827 ± 5,354 µm2; 0.5% HDCA-fed animals, 4,295 ± 2,384 µm2; NS). Moreover, compared with chow-fed animals, animals fed 0.5% cholesterol displayed a 10-fold increase in atherosclerosis area whereas 0.5% cholesterol plus 0.5% HDCA-fed animals had an 80% decrease in lesion area (0.5% cholesterol-fed animals, 108,539 ± 22,343 µm2; 0.5% cholesterol plus 0.5% HDCA-fed animals, 18,131 ± 6,129 µm2; P < 0.001).
Bile acids have been implicated in contributing to the occurrence of colon cancer. We examined the effect of HDCA on tumor formation in C57BL/6 ApcMin mice, a mutant rodent model with spontaneous intestinal tumor development. In these experiments we fed ApcMin mice for 16 weeks with either chow or chow supplied with 0.5% HDCA. After 16 weeks of feeding animals were killed and intestinal tumor number as well as tumor volume were measured. As shown in Table 1, HDCA had no effect on either the overall or segmental number of tumors in the intestines. Moreover, HDCA tended to decrease the size of tumors and this was particularly evident and statistically significant in the small intestine (78. 6 ± 18.6 and 57.3 ± 26.1 mm3 in chow- and HDCA-fed groups, respectively; P < 0.05). These results suggest that, in this mouse model, HDCA does not promote intestinal tumor formation.
In the present study we examined the metabolism of HDCA and its effect on plasma cholesterol levels, atherosclerosis, and intestinal tumor formation in mice. We found that HDCA i) efficiently suppresses dietary cholesterol absorption; ii) depletes the liver content of total cholesterol and cholesteryl ester, iii) reaches the systemic circulation and undergoes urinary excretion, iv) stimulates liver cholesterol biosynthesis, v) decreases plasma cholesterol levels of atherogenic lipoproteins, vi) decreases atherosclerosis formation, and vii) does not promote intestinal tumorigenesis.
Previous studies have shown that HDCA, provided to animals in a single dose, decreases the absorption of cholesterol from the intestine (7) (8). In the present study we carefully examined the dose response of dietary cholesterol absorption to HDCA. Our results clearly show that HDCA efficiently suppresses the absorption of dietary cholesterol and that approximately 50% of this suppression is already observed in animals fed 0.05;–0.1% HDCA (Fig 1). Moreover, the observation that the percentage of HDCA in the gallbladder bile (Fig 2A) and absorption rates (Fig 1) plateau at a comparable dose of HDCA suggest that HDCA may change the physicochemical properties of intestinal micelles and leads to decreased solubilization and absorption of dietary cholesterol.
As shown by others (8) (9) and as shown in Fig 3A, HDCA efficiently stimulates cholesterol biosynthesis in the liver. It is appealing to assume that the decrease in cholesterol absorption rates decreases liver cholesterol content, thereby stimulating the activity of HMGR. It is noteworthy, however, that whereas the decrease in dietary cholesterol absorption plateaus at 0.25% HDCA in the diet (Fig 1) the activity of HMGR is further increased in animals treated with higher amounts of HDCA (Fig 3A). These findings suggest that cholesterol absorption is not the sole mechanism for stimulated HMGR. It is interesting that the activity of HMGR was strongly and directly correlated with plasma levels of HDCA (R 2 = 0.85, P < 0.0001; Fig 3B). It is possible that circulating plasma HDCA is subjected to uptake by liver parenchymal cells and that the recycling across these cells stimulates, by as yet to be determined mechanisms, the activity of HMGR.
Previous studies have shown that HDCA decreases plasma cholesterol levels in the hamster (8) (10). In the present study we tested the effect of HDCA on plasma cholesterol metabolism in LDLR-KO mice. It is interesting that, regardless of the amounts of cholesterol in the diet, treatment with HDCA decreased plasma levels of VLDL and LDL cholesterol and increased the buoyancy of LDL particles (Fig 4). These observations clearly indicate that the decrease in VLDL-LDL cholesterol is brought about by mechanisms that are independent of the LDL-receptor catabolic pathway. Moreover, the fact that HDCA has no effect on the removal of VLDL from the circulation (Fig 5) precludes the possibility that alternative pathways (i.e., the LDL receptor pathway or proteoglycans) may contribute to this decrease. It is likely, therefore, that the decrease in cholesterol absorption decreases the flux of intestinally derived cholesterol through the liver and secretion of cholesterol in VLDL. This, in turn, may explain the decrease in plasma levels of VLDL cholesterol and the changes in LDL cholesterol and buoyancy.
The diet-induced model of atherosclerosis in mice involves feeding the bile acid cholic acid along with high cholesterol and high fat in the diet. In this model cholic acid is essential for the induction of atherosclerotic lesions, although the exact mechanism is not certain. Here we show that feeding the hydrophilic bile acid HDCA to LDLR-KO mice dramatically decreases atherosclerosis formation (Fig 6). Obviously, the response of plasma VLDL-LDL cholesterol to HDCA has an important role in determining this effect. Yet, whereas in chow-fed animals HDCA decreased atherosclerosis by 50% the corresponding decrease in plasma cholesterol was only 21%. Moreover, whereas in 0.5% cholesterol-fed animals HDCA decreases atherosclerosis by 80% the corresponding decrease in plasma cholesterol levels was only 62%. Although, in mice, the relationship between plasma cholesterol levels and atherosclerosis is nonlinear we cannot exclude the possibility that HDCA may decrease atherosclerosis through mechanisms other then plasma cholesterol metabolism. It is possible that the capacity of HDCA to reach the systemic circulation may have a direct effect on the arterial wall and its cholesterol-independent effects (i.e., stimulation of HMGR) may affect atherogenesis through lipoprotein-independent mechanisms.
Clearly, the above-described HDCA-dependent beneficial effects could have been largely offset by potential increased risk of tumors formation in the gastrointestinal tract (6). Here we have used C57BL/6 ApcMin mice, which develop spontaneous tumors in their gastrointestinal tract, and show that HDCA has no effect on the number of tumors and even decreases the tumor size in this model (Table 1). These findings suggest that HDCA is probably safe in terms of gastrointestinal tumorigenesis.
In the present study we show that, in mice, HDCA effectively inhibits the absorption of dietary cholesterol, decreases the levels of plasma atherogenic lipoproteins, and suppresses atherosclerosis formation. If humans respond to HDCA in a similar fashion then this bile acid may serve as an efficient agent in suppressing plasma cholesterol levels and atherosclerosis formation.
Abbreviations: HDCA, hyodeoxycholic acid; HMGR, hydroxymethylglutaryl-CoA reductase; LDLR-KO, LDL receptor knockout.
The authors acknowledge the support of NIH grant DK26756 (S.S.).
Manuscript received January 30, 2001; and in revised form March 29, 2001
Copyright © 2001 by Lipid Research, Inc.
Original Article
Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice
Ephraim Sehayeka,
Jennie G. Onoa,
Elizabeth M. Duncana,
Ashok K. Battab,
Gerald Salenb,
Sarah Sheferc,
Lien B. Neguyenc,
Kan Yangd,
Martin Lipkind, and
Jan L. Breslowa
a Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, NY 10021
b Medical Services, Veterans Affairs Medical Center, East Orange, NJ 07018
c Department of Medicine and Liver Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103
d Strang Cancer Prevention Center and New York Hospital-Cornell Medical Center, New York, NY 10021
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Wild-type, LDL receptor knockout (LDLR-KO), and ApcMin males, on the C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor, ME) and studied at 10;–13 weeks of age. Animals were housed in a humidity- and temperature-controlled room with a 12-h dark-light cycle (6 AM;–6 PM light-dark cycle) at the Laboratory Animal Research Center at Rockefeller University (New York, NY) and fed for 3 weeks with Picolab Rodent Chow 20 (5053) pellets containing 0.02% (w/w) cholesterol or the same diet fortified with cholesterol to a final concentration of 0.5% (w/w) (TD 97275; Harlan Teklad, Madison, WI). HDCA (Sigma, St. Louis, MO) was dissolved in ethanol, poured on the pelleted chow and 0.5% cholesterol diet, and the ethanol was evaporated [final content of 0.05%;–1% (w/w) HDCA]. All experiments were approved by the Institutional Animal Care and Research Advisory Committee of Rockefeller University.
Cholesterol absorption was determined in wild-type and LDLR-KO animals, using a modified form of the "dual isotope single meal feeding" method as described previously (11). Briefly, after 3 weeks of feeding animals were placed in metabolic cages, fasted for 4;–6 h (toward the end of the light cycle), and administered an intragastric bolus of olive oil containing [14C]cholesterol (Du Pont, Wilmington, DE) and ß-[3H]sitostanol (American Radiolabeled Chemicals, St. Louis, MO). Feces were collected for 24 h, dried, and homogenized, and lipids were extracted and counted for 14C and 3H. The percentage absorption of dietary cholesterol was calculated according to the formula: % cholesterol absorption = {1 - [fecal (14C dpm/3H dpm)]/[administered (14C dpm/3H dpm)]} x 100.
For urine collection mice were placed in metabolic cages and urine was collected for 24;–48 h. For plasma and biliary bile acids animals were fasted for 4;–6 h (during the middle of the light cycle) and anesthetized, after which the abdominal cavity was exposed through a ventral incision and the gallbladder bile was aspirated. The thoracic cavity was then exposed and blood was aspirated through ventricular puncture into an EDTA-containing syringe. The gallbladder bile was stored at 4°C and cholesterol, phospholipids, and bile acid concentrations were analyzed within 24;–48 h as previously described (12). For bile acids analysis nor-deoxycholic acid (10 µg in 100 µl of methanol) was added to aliquots of bile, plasma, or urine together with 1 ml of 3 N sodium hydroxide and the contents were heated in an autoclave at 110°C for 3 h (13). After cooling, products were diluted with 1 ml of water and extracted first with n-hexane (4 x 2 ml) and then with ethyl acetate (2 x 2 ml). The aqueous phase was cooled on ice, acidified with 50% hydrochloric acid to pH 1, and extracted with ethyl acetate (4 x 3 ml). The organic layer was washed with water to neutrality and evaporated at 55°C under nitrogen. The residue was converted into the methyl ester-trimethylsilyl ether derivatives and analyzed as previously described (14). Derivatized bile acids were dissolved in 100 µl of hexane and 1;–5 µl was injected into a gas-liquid chromatography column.
Plasma cholesterol levels were determined enzymatically with a Sigma kit as described above for biliary cholesterol. Lipoprotein cholesterol profiles were determined by on-line postcolumn analysis of Superose 6 gel-filtered plasma as previously described (15). 
-hydroxylase, and sterol 27-hydroxylase
Animals were fasted for 4;–6 h (during the middle of the light cycle) and then anesthetized. The abdominal cavity of each was exposed, the liver was harvested, and the total and free cholesterol content was determined by gas-liquid chromatography as previously described (12). The content of cholesteryl ester was calculated by subtracting the value for free cholesterol from the total cholesterol content after KOH hydrolysis. On liver harvesting, tissue samples were snap frozen in liquid nitrogen and used for the measurements of HMGR, cholesterol 7-hydroxylase, and sterol 27-hydroxylase activities as previously described (11).
VLDL was isolated from 0.5% cholesterol-fed LDLR-KO mice. Animals were fasted for 4;–6 h and blood was drawn through a ventricular puncture into EDTA-containing syringes. VLDL (d = 1.006) was isolated by ultracentrifugation at 40,000 rpm for 12 h at 4°C [Beckman (Fullerton, CA) rotor 42.2Ti and centrifuge L8-55M], washed once again in the same density, and labeled with [cholesteryl-1,2-3H]cholesteryl oleyl ether (Amersham Pharmacia Biotech, Piscataway, NJ). Animals were lightly anesthetized and injected with labeled VLDL in 0.1 ml of phosphate-buffered saline via the retro-orbital plexus. Blood was collected periodically via the opposite retro-orbital plexus; plasma was immediately isolated and counted for 3H. The radioactivity at 0.5 min postinjection was defined as 100% of injected radioactivity and fractional clearance rates were calculated with a two-compartment model as described (16).
Animals were fed for 12 weeks with the indicated diet. At 16 weeks of age animals were fasted, anesthetized, and blood was collected via ventricular puncture into EDTA-containing syringes. The circulatory system was perfused with phosphate-buffered saline by intraventricular injection. The heart, containing the aortic root, was fixed in phosphate-buffered formalin and processed for aortic root quantitative atherosclerosis assay as previously described (17).
ApcMin males were fed with chow or 0.5% HDCA. After 16 weeks of feeding, animals were anesthetized, blood was collected by ventricular puncture, and necropsy was carried out. The entire gastrointestinal tract was removed and opened longitudinally, intestinal contents were washed with ice-cold phosphate-buffered saline (pH 7.2), and fixed overnight in 10% neutral buffered formalin. The specimens were examined under a dissecting microscope, tumor number was recorded, tumor volume was calculated after measuring the length, width, and thickness of the lesion, using an eye-piece dissecting microscope grid, and tumor incidence and number were calculated per animal.
The differences in percent cholesterol absorption were analyzed with a one-way ANOVA parametric test and Newman-Keuls multiple comparison posttest. Differences in liver cholesterol content and plasma cholesterol levels were analyzed with an unpaired Student t-test. A one-way ANOVA nonparametric test and Kruskal-Wallis posttest were applied in analyzing group differences in atherosclerosis. Correlations between plasma HDCA and HMGR were analyzed with the linear regression tool in the Analysis TulPak (Excel 2000®; Microsoft, Redmond, WA). Finally, the Fisher exact probability test was used for the analysis of tumor incidence whereas the Mann-Whitney test was used to analyze the effect of HDCA on intestinal tumor number and volume.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Figure 1.
Effect of HDCA on cholesterol absorption in C57BL/6 wild-type mice. Five different groups of C57BL/6 males were fed a chow diet supplied with 0%;–0.5% HDCA. After 3 weeks animals were placed in metabolic cages and administered a gastric bolus of 100 µl of olive oil containing [14C]cholesterol and ß-[3H]sitostanol, feces were collected, and percent cholesterol absorption was calculated as described in Materials and Methods (means ± SD, n = 5 per group). * P < 0.05 and ** P < 0.001 compared to 0% HDCA.
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Figure 2.
Effect of HDCA on gallbladder bile and plasma HDCA concentrations in wild-type mice. Animals were treated as described in the legend to Fig 1. After 3 weeks gallbladder bile was aspirated, plasma samples were drawn, and the amounts of HDCA in the gallbladder bile (A) and concentrations in plasma (B) were determined as described in Materials and Methods (means ± SD, n = 5 males per group). -hydroxylase or sterol 27-hydroxylase, or the concentrations of biliary cholesterol, phospholipids, and total bile acids (data not shown).
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Figure 3.
Effect of HDCA on liver HMGR activity (A) and relationship of HMGR activity to plasma HDCA (B) levels in wild-type mice. Animals were treated as described in the legend to Fig 1. After 3 weeks, animals were fasted, plasma samples were drawn, livers were harvested, and HMGR activity was determined as described in Materials and Methods (means ± SD, n = 5 males per group).
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Figure 4.
Effect of HDCA on plasma lipoprotein cholesterol profile in C57BL/6 LDLR-KO mice. Four different groups of males were fed with either chow (open circles), chow supplied with 0.5% HDCA (closed circles), 0.5% cholesterol (open squares), or 0.5% cholesterol and 0.5% HDCA (closed squares). After 3 weeks animals were fasted, plasma samples were isolated, the plasma of animals in each group were pooled, and plasma lipoprotein cholesterol profiles were analyzed as described in Material and Methods (n = 6 males in each group).
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Figure 5.
Effect of HDCA on VLDL turnover in LDLR-KO mice. Animals were fed for 3 weeks with either chow (open circles) or chow supplied with 0.5% HDCA (closed circles) and injected with [cholesteryl-1,2-3H]cholesteryl oleyl ether-labeled VLDL. Blood samples were drawn, and plasma was isolated and counted for 3H at the indicated time points and analyzed as described in Materials and Methods (means ± SD, n = 4 males in each group).
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Figure 6.
Effect of HDCA on aortic root atherosclerosis lesion area in LDLR-KO mice. Animals were fed with either chow (open circles), chow supplied with 0.5% HDCA (closed circles), 0.5% cholesterol (open squares), or 0.5% cholesterol and 0.5% HDCA (closed squares). After 12 weeks of feeding, at 16 weeks of age, animals were killed and aortic root atherosclerosis lesion area was quantified as described in Materials and Methods. The horizontal line is the median lesion area in each group (n = 9 or 10 males in each group).
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Table 1.
Effect of HDCA on intestinal tumorigenesis and tumor volume in C57BL/6 ApcMin mice
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES 
ACKNOWLEDGMENTS
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INTRODUCTION
MATERIALS AND METHODS
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REFERENCES
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