Introduction

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The enterohepatic circulation of bile acids, which is an important physiological component of the digestive system, requires efficient bile acid absorption by the ileum. Bile acids are synthesized from cholesterol in the liver and secreted with bile into the small intestine, where they facilitate absorption of fat, fat-soluble vitamins, and cholesterol (Hofmann, 1994a). After functioning as detergent agents in the lumen of the intestine, bile acids are reabsorbed and returned back to the liver via portal circulation. In the liver, bile acids are resecreted into bile (Hofmann, 1994b). This efficient circulation of bile acids is dependent upon transporters located in both the liver and intestine. Less than 5% of the circulating bile acid pool typically escapes reabsorption and is eliminated in the feces on a daily basis (Oelkers et al., 1997). Although some bile acids are absorbed by diffusion across the entire length of the small intestine, the majority of bile acids is absorbed via active transport in the terminal ileum (Duane et al., 2000). Ileal bile acid transport is mediated by the apical sodium-codependent bile acid transporter (ASBT). ASBT is a 348-amino acid protein localized on the apical surface of epithelial cells lining the ileum (Wong et al., 1995). The human ASBT gene (SLC10A2) is located on chromosome 13q33 (Wong et al., 1996) and has been sequenced by Dawson et al. (Oelkers et al., 1997; Craddock et al., 1998). Furthermore, ASBT was recently cloned from the human, hamster, and rat (Wong et al., 1994, 1995; Shneider et al., 1995).

ASBT plays a critical role in the intestinal reclamation of bile salts secreted by the liver (Chen et al., 2001). Although complete disruption of this process leads to congenital primary bile acid malabsorption (Oelkers et al., 1997), partial inhibition via ileal bypass or by resin therapy causes an increase in bile acid synthesis and an ~20% reduction in serum cholesterol levels (Miettinen and Lempinen, 1977). In contrast to their beneficial cholesterol-lowering effects, ASBT inhibitors have the potential to raise plasma triglycerides (TGs), similar to what has been reported for the bile acid sequestrant cholestyramine (Angelin et al., 1986). A study conducted by Duane et al. (2000) reported that reduced absorption of bile acid, due to a diminished expression of the ASBT in the human ileum, contributes to hypertriglyceridemia. Overall, further understanding of an ASBT inhibitor's effect on intestinal bile acid absorption, on plasma and hepatic cholesterol levels, and on plasma lipoprotein composition is needed to design an effective therapeutic agent for treatment of hypercholesterolemia.

1-[4-[4[(4R,5R)-3,3-Dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy] butyl]-4-aza-1-azoniabicyclo[2.2.2]octane methanesulfonate, salt) (SC-435) was recently identified from the H-14 assay as a potent ASBT inhibitor (Rapp et al., 2001). This compound, which has been shown to block ASBT expressed in baby hamster kidney cells, has an IC₅₀ value of 1.2 ± 0.2 nM for taurocholate uptake and more than 66,000-fold selectivity for ASBT compared with alanine uptake, which also uses a sodium-dependent transporter, under identical conditions. Overall, SC-435 has been shown to be a specific, nonabsorbable, inhibitor of bile acid reabsorption in the distal ileum (Huff et al., 2001; Rapp et al., 2001).

The current study was conducted to investigate 1) the effects of the ASBT inhibitor SC-435 on cholesterol and lipoprotein metabolism in guinea pigs, 2) whether a dose-dependent response exists with regard to the ASBT inhibitor, and 3) some of the mechanisms by which the ASBT inhibitor lowers plasma LDL-cholesterol. Furthermore, the activity of enzymes associated with cholesterol absorption was measured to better understand hepatic cholesterol homeostasis and lipoprotein secretion. Based on current knowledge of reducing intestinal bile acid absorption, we hypothesized that the ASBT inhibitor would lower plasma cholesterol concentrations in guinea pigs. The guinea pig was used as the animal model for this study because of its similarities to humans in terms of hepatic cholesterol and lipoprotein metabolism (Fernandez, 2001). Important similarities include a high LDL-to-HDL ratio; higher concentrations of free than esterified cholesterol in the liver; similar activities of main enzymes regulating cholesterol metabolism; comparable rates of hepatic cholesterol synthesis and catabolism; and, most importantly, similar responses to dietary and drug treatments by lowering plasma LDL-cholesterol (Fernandez, 2001).

	Materials and Methods

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Materials. Cholesterol oxidase, cholesterol esterase, peroxidase, and cholesterol and TG kits were purchased from Roche Applied Science (Mannheim, Germany). Free cholesterol and phospholipid kits were obtained from Wako Pure Chemicals (Tokyo, Japan). Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, NADP, phosphatidylcholine, and the bile acid kit were obtained from Sigma-Aldrich (St. Louis, MO). Oleoyl-[1-¹⁴C]coenzyme A was obtained from Amersham Biosciences (Piscataway, NJ). 7-Hydroxycholesterol and 7-hydroxycholesterol were purchased from Steraloids (Wilton, NH). [¹⁴C]Cholesterol was obtained from PerkinElmer Life Sciences (Boston, MA). The ASBT inhibitor was provided by Pharmacia Corporation (St. Louis, MO).

Diets. Diets were prepared and pelleted by Research Diets, Inc. Isocaloric diets were designed to meet the nutritional requirements of the guinea pigs. All diets were equal in composition except for the amount of ASBT inhibitor. The ASBT inhibitor concentrations in the four diets were as follows: 0.0, 0.0018, 0.009, and 0.036% (Table 1). The amount of cholesterol in the diets was maintained at 0.17% to raise plasma cholesterol concentrations to more readily detect ASBT inhibitor effects. Dietary cholesterol of 0.17% in this model corresponds to an absorbed amount equal to the daily cholesterol synthesis rates (Lin et al., 1992) in guinea pigs and is equivalent to 1200 mg/day for a human diet. The fat mix was rich in lauric and myristic acids, known to cause endogenous hypercholesterolemia in guinea pigs (Roy et al., 2000).

Animals. Male Hartley guinea pigs weighing between 250 and 300 g were purchased from Harlan (Indianapolis, IN). Animals were randomly allocated to one of the four treatments, 10 guinea pigs per group, for a total of 4 weeks, an amount of time known to result in steady-state plasma cholesterol levels. Two guinea pigs were housed per cage in a light cycle room (light from 7:00 AM to 7:00 PM) at 72°C. Diet and water were provided ad libitum. At the end of the feeding period, animals fasted for approximately 12 h before blood collection. To measure fecal bile acids and food consumption, six guinea pigs from each group were housed individually for 48 h. During this time, feces were collected and diets were weighed daily to determine the amount of food consumed. All animal experiments were conducted in accordance with U.S. Public Health Service/U.S. Department of Agriculture guidelines. Experimental protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.

Lipoprotein Isolation. After the fasting period, guinea pigs were anesthetized under halothane vapors and blood was obtained via heart puncture. Plasma samples were collected and preservation cocktail was added to the samples (0.5 ml/100 ml aprotinin, 0.1 ml/100 ml phenylmethylsulfonyl fluoride, and 0.1 ml/100 ml sodium azide). One milliliter of plasma from each animal was stored at 4°C for analyzing plasma lipids, whereas the rest was used for lipoprotein isolation.

Plasma and Hepatic Lipids. Plasma total cholesterol, TG, and HDL cholesterol were determined using enzymatic analysis (Allain et al., 1974). Plasma TG was determined by blanking free glycerol (Carr et al., 1993). HDL cholesterol was analyzed after precipitation of apoB-containing lipoproteins with dextran sulfate (Warnick et al., 1982) with a modification (Fernandez et al., 1999).

Lipoprotein Characterization. VLDL and LDL compositions were calculated by measuring phospholipids, TG (Carr et al., 1993), protein (Markwell et al., 1978), and free and total cholesterol (Allain et al., 1974). Esterified cholesterol was calculated as the difference between total cholesterol and free cholesterol. The number of LDL molecules was calculated based on one apolipoprotein-B per LDL (apoB molecular mass 412,000 kDa). The molecular weights for TG, free and esterified cholesterol, and phospholipids were 885.4, 386.6, 646, and 734, respectively (Fernandez et al., 1995). LDL diameters were calculated according to Van Heck and Zilversmit (1991).

Hepatic Microsome Isolation. Microsomes were isolated as described previously (Fernandez et al., 1995). Briefly, livers obtained from guinea pigs on different diets were pressed through a tissue grinder, placed in a cold buffer (50 mM KH₂PO₄, 0.1 mol/l sucrose, 50 mM KCl, 50 mM NaCl, 30 mM EDTA, and 2 µM dithiothreitol, pH 7.2), and homogenized with a Potter-Elvehjem homogenizer. The microsomal fraction was obtained after two centrifugations at 10,000g for 15 min (JA-20 rotor in a J2-21 centrifuge; Beckman Coulter, Inc.), and 1-h centrifugation at 100,000g at 4°C. Samples were further homogenized and centrifuged for one additional hour at 100,000g at 4°C. Microsomal pellets were resuspended in buffer, homogenized, and stored at 70°C. Protein content in microsomes was measured according to Markwell et al. (1978).

ACAT Activity. Hepatic ACAT (EC 2.3.1.26) activity was measured by the incorporation of [¹⁴C]oleoyl-CoA in cholesteryl ester in hepatic microsomes isolated from the four groups of animals according to Smith et al. (1986). No exogenous cholesterol was added. Hepatic microsomes (0.8-1.0 mg of protein/assay) were preincubated with albumin (84 mg/ml) and buffer (50 mM KH₂PO₄, 0.1 mol/l sucrose, 50 mM KCl, 30 mM EDTA, and 50 mM NaF) to a final volume of 0.18 ml for 5 min at 37°C. Oleoyl-[1-¹⁴C]coenzyme A (500 µM) (0.15 Gbq/pmol) was added, and the samples were incubated for 15 min at 37°C. The reaction was stopped with 2.5 ml of chloroform/methanol (2:1) and [³H]cholesterol oleate (0.045 GBq/assay) was added as a recovery standard. An additional 2.5 ml of chloroform/methanol (2:1) and 1 ml of acidified water (0.05% H₂SO₄) were added to the samples. Samples were mixed and allowed to stand overnight. The aqueous phase was removed, and the samples were dried under nitrogen. Samples were resuspended in 0.150 ml of chloroform containing 30 µg of unlabeled cholesteryl oleate. Samples were applied to silica gel thin layer chromatography plates and developed with hexane/diethyl ether (9:1, v/v). Cholesteryl oleate was visualized with iodine vapors and scraped from the plate, and radioactivity was counted. Recoveries of [³H]cholesterol oleate were between 70 and 90%.

Cholesterol 7-hydroxylase (CYP7) Activity. CYP7 (EC 1.14.13.7) activity was assayed according to Jelinek et al. (1990). [¹⁴C]Cholesterol was used as a substrate and delivered as cholesterol-phosphatidylcholine liposomes (1:8 by weight). After preparation by sonification, an NADPH-regenerating system (glucose-6-phosphate dehydrogenase, NADP, and glucose 6-phosphate) was included as a source of NADPH. After addition of glucose-6-phosphate dehydrogenase (0.3 IU), samples were incubated for an additional 30 min. The reaction was stopped by the addition of 5 ml of chloroform/methanol (2:1) and 1 ml of acidified water (0.05% sulfuric acid). Tubes were mixed, the top layer was discarded, and samples were dried under nitrogen. Samples and 7- and 7-hydroxycholesterol standards were dissolved in 100 µl of chloroform, applied to silica gel thin layer chromatography plates, and developed with ethyl acetate/toluene (3:2). The plate was placed in iodine vapors to mark the 7- and 7-hydroxycholesterol standards and placed on XAR-5 film overnight. Using the film as a guide, the locations of the [¹⁴C]7-hydroxycholesterol spots were determined, scraped from the plate, and counted in a liquid scintillation counter.

Fecal Bile Acids. Fecal bile acids were assayed by a colorimetric method by Mashige et al. (1981). Feces were weighed, dried for 5 h at 37°C, pulverized, and weighed again. Four milliliters of T-butanol/water (1:1 by v/v) was added to 0.2 g of fecal samples and heated at 37°C for 15 min with continuous agitation. The samples were then centrifuged at 3000 rpm for 10 min (JA-20 rotor in a J2-21 centrifuge; Beckman Coulter, Inc.), and the supernatant was removed and the pellet was discarded. Each sample (200-µl aliquot) was analyzed in duplicate, and a blank reagent was added to a third aliquot to correct for the color provided by each sample. Samples were incubated at 37°C for 5 min. The reaction was stopped and color was read in a spectrophotometer at 530 nm. The amount of fecal bile acids was calculated as millimoles per kilogram per day after subtracting the blank.

Statistical Analysis. Data were analyzed by one-way analysis of variance (ANOVA). P values less than 0.05 were considered statistically significant. The Newman-Keuls test was used as post hoc analysis. Data are presented as means ± standard deviation.

	Results

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Plasma Lipids and Lipoproteins. The amount of food that the guinea pigs consumed was calculated by weighing food intake for 48 h 1 week before death. A subset of six guinea pigs per group was placed in individual cages and given 100 g of food per day. The food was then weighed on a daily basis to determine food consumption. Based on average food consumption and the percentage of the ASBT inhibitor known to be in each diet, the amount of drug consumed was calculated to be 0.0, 0.8, 3.7, and 13.4 mg/kg/day for guinea pigs fed 0, 0.0018, 0.009, or 0.036% ASBT diet as indicated in Table 2. There were no significant differences in weight gain or final weight of guinea pigs treated with the different drug doses. Final weights were 561 ± 101, 507 ± 47, 602 ± 55, and 547 ± 40 g for guinea pigs treated with 0, 0.8, 3.7, and 13.4 mg/kg/day, respectively.

Effects of ASBT Inhibitor on Hepatic Lipids. The highest dose (13.4 mg/kg/day) of ASBT inhibitor significantly reduced hepatic cholesterol ester concentration in guinea pigs by 70% (P < 0.01). Furthermore, hepatic cholesterol esters were significantly reduced by 43 and 56% with 0.8 and 3.7 mg/kg/day of ASBT inhibitor (P < 0.01), respectively (Table 4). There was no significant effect of the ASBT inhibitor on hepatic total and free cholesterol or hepatic TG.

Effects of ASBT Inhibitor on VLDL and LDL Composition. The composition of VLDL isolated from guinea pigs treated with the highest dose of ASBT inhibitor was significantly altered (Table 5). ASBT inhibitor treatment (13.4 mg/kg/day) resulted in a 41.8% increase in the number of VLDL TGs compared with the controls (P < 0.05). Furthermore, VLDL diameter was increased from 327.5 ± 70.9 Å in controls to 433.8 ± 52.9 Å in guinea pigs treated with the highest dose of ASBT inhibitor (P < 0.05). The number of molecules of the other VLDL components (cholesterol esters, free cholesterol, and phospholipids) was not significantly affected by the inhibitor (Table 5). In contrast to the effects observed in VLDL, the ASBT inhibitor did not affect LDL composition or diameter (Table 6).

Effects of ASBT Inhibitor on Hepatic Enzymes and Bile Acids. The enzymes associated with cholesterol metabolism were measured in microsomes isolated from guinea pigs treated with the ASBT inhibitor.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Fecal bile acid concentrations of guinea pigs fed diets containing 0 (control) and 13.4 mg/kg/day of the ASBT inhibitor. Bile acid excretion was approximately 2 times higher in the group treated with the ASBT inhibitor compared with the control. Values are significantly different as determined by paired t test (P < 0.05).

	Discussion

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Recently more attention has been focused on reducing the enterohepatic circulation of bile acids as an alternative means to treat hypercholesterolemia. In particular, the sodium-dependent ileal bile acid transporter ASBT has generated interest because of its critical role in the reabsorption of bile acids in the ileum. This target also is attractive because of its localization on the luminal surface of the ileal enterocytes and the potential to develop inhibitors that do not require systemic absorption to elicit a pharmacological response. In this study we observed a reduction of 44% in plasma LDL-C with 13.4 mg/kg/day (7.3 mg/day) of the ASBT inhibitor, which is consistent with other studies in animal models investigating drugs that lower plasma cholesterol (Ness et al., 1994; Conde et al., 1996). The lack of a dose response with 0.8- and 3.7-mg/kg/day doses may be related to the low concentrations of drug provided to these groups of guinea pigs. The findings from this study suggest that the reduction in LDL cholesterol concentrations in response to the ASBT inhibitor is a result of alterations in hepatic cholesterol metabolism due to reduction in the enterohepatic circulation of bile acids.

Effects of ASBT Inhibitor on Hepatic Cholesterol Homeostasis. Cholesterol plays a major role in regulatory enzymes for cholesterol homeostasis in the liver (Pandak et al., 2001). These key enzymes include 1) 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R), the rate-determining step in the cholesterol biosynthetic pathway; 2) ACAT, which esterifies cholesterol to maintain intracellular concentrations of free cholesterol and stores cholesterol in the form of cholesterol esters; and 3) CYP7, the initial enzyme in classic bile acid biosynthesis. The ability of each of these enzymes to respond to changes in the hepatic bile acid pool is the means by which cholesterol homeostasis is maintained (Makishima et al., 1999).

Effects of ASBT Inhibitor on Plasma Lipids and Lipoprotein Distribution. High plasma LDL-C levels are a major risk factor in the development of coronary heart disease and atherosclerosis (Stamler et al., 1986; McNamara, 1992). In the current study, the ASBT inhibitor's primary effect was a 44% decrease in LDL-C concentrations for guinea pigs treated with the highest dose of inhibitor (13.4 mg/kg/day). In contrast to other reports on the effects of ASBT inhibitor on plasma TG concentrations (Duane et al., 2000), we did not observe a statistically significant increase in plasma TG in the drug-treated compared with the vehicle-treated guinea pigs. The plasma LDL-cholesterol lowering could be related to alterations in the enterohepatic circulation of bile acids by the ASBT inhibitor, which in turn altered hepatic cholesterol and plasma lipoprotein metabolism.