Inhibition of the Apical Sodium-Dependent Bile Acid Transporter Reduces LDL Cholesterol and ApoB by Enhanced Plasma Clearance of LDL ApoB
Objective— Cloning of the ileal apical sodium-dependent bile acid transporter (ASBT) has identified a new pharmacological target for the modulation of plasma lipoproteins. The objective of this study was to determine whether a novel, specific, minimally absorbed ASBT inhibitor (SC-435) decreases LDL cholesterol through the alteration of plasma apoB kinetics.
Methods and Results— Miniature pigs were treated for 21 days with 10 mg/kg/day of SC-435 or placebo. SC-435 decreased plasma cholesterol by 9% and LDL cholesterol by 20% with no effect on other lipids. Autologous 131I-VLDL, 125I-LDL, and [3H]-leucine were injected simultaneously to determine apoB kinetics. LDL apoB concentrations decreased significantly by 10% resulting entirely from an increase in LDL–apoB fractional catabolic rate. SC-435 had no effect on either total LDL apoB production or VLDL apoB converted to LDL. SC-435 increased VLDL apoB production by 22%; however, the concentration was unchanged as a result of increased VLDL apoB direct removal. SC-435 increased hepatic mRNA and enzymatic activity for both cholesterol 7α-hydroxylase and HMG-CoA reductase. Hepatic LDL receptor mRNA increased significantly, whereas apoB expression was unaffected.
Conclusions— A low dose of the ASBT inhibitor, SC-435, significantly reduces plasma LDL cholesterol through enhanced LDL receptor-mediated LDL apoB clearance, secondary to increased expression of cholesterol 7α-hydroxylase.
- apical sodium-dependent bile acid transporter
- bile acid reabsorption
- apoB kinetics
- cholesterol 7α-hydroxylase
- LDL receptor
Under normal conditions, bile acids form mixed micelles with dietary fat in the small intestine, thus allowing for fat absorption.1 Bile acids are mostly reabsorbed via an active transport mechanism in the terminal ileum, mediated by a specific sodium bile acid cotransporter, the apical sodium-dependent bile acid transporter (ASBT), formerly referred to as intestinal bile acid transporter (IBAT).1 ASBT was recently cloned and characterized.2–4⇓⇓ The ASBT gene product is a 48-kDa protein located on the apical surface of ileal enterocytes within the terminal 15% of the human small intestine1 and its specificity includes both conjugated and unconjugated bile salts. ASBT-mediated reabsorption of bile acids facilitates their return to the liver via the portal vein, thereby completing the enterohepatic circulation of bile acids.1 In humans, greater than 90% of bile acids are recycled, with about 75% being mediated by the ASBT. Interruption of this enterohepatic circulation by the prevention of bile acid reabsorption leads to a rapid upregulation of hepatic bile acid synthesis through activation of microsomal cholesterol 7α-hydroxylase, the rate-limiting enzyme for the conversion of cholesterol to bile acids.5 In turn, the concentration of cholesterol in a putative regulatory pool within the endoplasmic reticulum decreases, which can result in the activation of several compensatory reactions to maintain cellular cholesterol homeostasis: 1) LDL receptors are upregulated, resulting in increased expression at the hepatocyte cell surface, thereby increasing the clearance of plasma LDL; 2) the assembly and secretion of hepatic apoB-containing lipoproteins is reduced because of reduced cholesterol availability; and 3) the activity of HMG–CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, is increased.
Studies of the effects of bile acid sequestrants on apoB kinetics in humans6 and miniature pigs7 demonstrated that LDL cholesterol was reduced significantly primarily through compensating mechanisms 1) and 2) above. In patients with primary hypercholesterolemia, reduced LDL apoB production contributed significantly to the reduction in LDL cholesterol, whereas in patients heterozygous for familial hypercholesterolemia, increased LDL apoB clearance was the predominant mechanism.6,8⇓ Although resins significantly reduce LDL cholesterol,6 leading to a decrease in cardiovascular events,9,10⇓ these compounds are nonspecific with respect to anion binding, and their use is associated with side effects, including unpalatability; gastrointestinal symptoms; and altered absorption of vitamins, minerals, and co-administered drugs,11 thus limiting compliance. Therefore, specific inhibition of ileal bile acid active transport represents an alternative method for decreasing bile acid reabsorption.
The cloning and characterization of ASBT2–4,12⇓⇓⇓ greatly facilitated the development of specific inhibitors of bile acid reabsorption, potentially a new class of plasma cholesterol-lowering agents.13–15⇓⇓ In addition, recent advances in our understanding of the molecular basis of bile acid metabolism have allowed for the prediction of the consequences of ASBT inhibition.16,17⇓ Decreased ileal reuptake of bile acids and return to the liver via the enterohepatic circulation would block the transcription of the short heterodimer partner (SHP), which is normally activated by bile acids via a complex consisting of the farnesoid X receptor and the 9-cis-retinoic acid receptor. A reduction in SHP would allow for the induction of the liver receptor homolog-1, which is a positive transacting factor for the cholesterol 7α-hydroxylase gene (CYP7A1). In turn, increased bile acid synthesis would result in changes in hepatic cholesterol homeostasis, ultimately leading to decreased plasma cholesterol concentrations. ASBT inhibitors seem to be effective for lowering serum cholesterol in small animal models, which consist of animals who are fed diets high in fat and cholesterol.15,18,19⇓⇓ However, their efficacy and mechanism of action have not been established in large animals, such as the miniature pig, who are fed physiologically relevant diets.
In the present study, regulation of apoB metabolism was examined in miniature pigs after administration of a recently developed inhibitor of ASBT, SC-435.20 SC-435 significantly reduced LDL cholesterol through enhanced LDL apoB clearance, which was mediated through increased hepatic expression of LDL receptors, secondary to upregulation of hepatic cholesterol 7α-hydroxylase expression.
Please see online data supplement (which can be accessed at http:/www.atvb.ahajournals.org) for materials and methods concerning animals and diets, lipoprotein turnover studies, apoB kinetic analysis, hepatic total and microsomal lipids, activities of cholesterol 7α-hydroxylase and HMG-CoA reductase, oleate incorporation into hepatic and intestinal cholesteryl ester and triglyceride, hepatic mRNA abundance, plasma and lipoprotein lipid determinations, and statistical analysis.
Pigs were treated with SC-435 at a dose of 10 mg/kg/day or diet alone containing 34% of energy from fat and 400 mg/day of cholesterol. The SC-435 dose was determined in a pilot experiment in which fecal samples were collected over 48 hours from three pigs after 2 weeks of diet alone and again after 2 weeks of treatment with diet plus 10 mg/kg/day of SC-435. Fecal bile acid excretion in treated animals increased 1.8-fold (49±7 versus 27±8 μmoles/kg body weight/day), establishing the efficacy of the dose of SC-435 used in the lipoprotein turnover study.
The effect of SC-435 on plasma and lipoprotein lipid concentrations, after 21 days of treatment, is shown in Table 1. SC-435 significantly reduced total plasma cholesterol by 9% (P<0.03) and LDL cholesterol by 20%, (P<0.001). HDL cholesterol was unchanged. Total plasma triglycerides and VLDL triglycerides increased by 7% and 10%, respectively; however these changes were not significant. VLDL apoB increased by 17% (NS), whereas LDL apoB decreased by 10% (P<0.04) with SC-435 treatment.
Autologous 131I-VLDL and 125I-LDL and [3H]-leucine were simultaneously injected into each control and SC-435–treated pig. The kinetic parameters of apoB were determined from the simultaneous analysis of all the specific activity data using the models described previously.21 The kinetic parameters for VLDL apoB and LDL apoB are summarized in Tables 2 and 3. A fit of the model to the apoB-specific radioactivity curves for VLDL, IDL, and LDL after the injection of tracers for a representative pair of animals is shown in Figure 1.
SC-435 treatment increased the total VLDL apoB production rate by 22% (P<0.03). However, this was partially compensated for by an 8% (NS) increase in the fractional catabolic rate (FCR), such that the VLDL pool size increased by 17% (NS) (Table 2). The amount of VLDL apoB converted to LDL apoB was not affected by SC-435, but the flux of apoB cleared directly, without conversion to IDL or LDL, was significantly increased by 22% (P<0.03). Neither the percentage of VLDL apoB flux converted to LDL nor the percent cleared directly were affected by SC-435 treatment. The FCRs for VLDL apoB direct catabolism [k(0,1), k(0,2), and k(0,3)] and for IDL direct catabolism [k(0,4) and k(0,5)] were not significantly affected by SC-435 (data not shown). No changes were observed in the production or flux of VLDL apoB that was converted to LDL without being transported through the plasma IDL pool or in the amount of VLDL apoB converted to LDL via the IDL fraction (data not shown).
SC-435 treatment decreased the LDL apoB pool size by 10% (P<0.035) (Table 3). This was primarily due to a significant 18% (P<0.032) increase in the LDL apoB FCR. SC-435 had no effect on the total production rate of LDL apoB, the amount of LDL apoB derived from VLDL apoB catabolism, or LDL apoB direct synthesis. The latter is defined as LDL apoB entering the plasma compartment without initial metabolism through the plasma VLDL or IDL pools. Total production of apoB into plasma (VLDL plus LDL direct synthesis) increased by 20% (P<0.03). However, this was completely due to the increase in VLDL apoB production, and all of this increased production was cleared from the plasma compartment before conversion to IDL or LDL apoB.
Figure 2 summarizes the kinetic parameters calculated for apoB transport into plasma and through the metabolic cascade in SC-435–treated pigs as compared with controls. Although VLDL apoB secretion was increased, the VLDL apoB pool size was not changed as a result of a nonsignificant increase in the VLDL apoB FCR (not shown). The increased VLDL production rate did not result in enhanced conversion to LDL apoB because VLDL apoB direct catabolism was significantly increased with treatment. The LDL pool was significantly decreased, entirely the result of an increase in the LDL apoB FCR. LDL apoB direct synthesis and conversion from VLDL were not affected.
VLDL and LDL were analyzed for lipid and protein composition (please see online Table I, which can be accessed at http:/www.atvb.ahajournals.org). SC-435 treatment resulted in a 25% decrease (P<0.002) in the percentage of cholesteryl ester in VLDL. The percent composition of other VLDL lipids and protein were not significantly different. SC-435 increased the percent of triglyceride in LDL by 14% (P<0.035), whereas the percent composition of other lipids was unchanged. The ratio of triglyceride/cholesteryl ester (wt/wt) in VLDL increased by 40% (P<0.009) and that in LDL by 17% (P<0.04).
Approximately 24 hours after the last dose of SC-435 was administered, the pigs were sacrificed, and sections of liver and small intestine were removed and stored at −80°C before analyses. Hepatic lipids including triglyceride, free cholesterol, and cholesteryl ester were not significantly changed by SC-435 (Table 4). Intestinal total cholesterol, free cholesterol, and triglyceride concentrations were unaffected by SC-435; however, intestinal cholesteryl ester was decreased by 44% (P<0.045; data not shown). Liver microsomes were prepared and the concentrations of free cholesterol, cholesteryl ester, and triglyceride were determined; however, no significant changes were observed with treatment.
The activity of cholesterol 7α-hydroxylase in hepatic microsomes was increased 2.3-fold (P<0.003) by SC-435 treatment as compared with control animals (Table 4). Furthermore, the activity of HMG-CoA reductase was increased 3.8-fold (P<0.048) in hepatic microsomes of SC-435–treated animals. The incorporation of oleate into cholesteryl ester and triglyceride was determined in liver homogenates. No significant changes were observed for either parameter with SC-435 treatment.
The liver concentrations of mRNA for the LDL receptor, cholesterol 7α-hydroxylase, and HMG-CoA reductase were increased by SC-435 treatment (Table 4). The mRNA abundance for the LDL receptor increased 1.6-fold (P<0.03), for cholesterol 7α-hydroxylase by 1.3-fold (P<0.017), and for HMG-CoA reductase by 2.5-fold (P<0.0003). Hepatic apoB mRNA was unchanged.
Interruption of the enterohepatic circulation of bile acids through specific inhibition of ASBT represents an attractive alternative approach for the treatment of hypercholesterolemia. The aim of the present study was to assess the impact of ASBT inhibition on plasma lipoprotein concentrations in a large animal model of human lipoprotein metabolism and to determine the mechanism(s) underlying changes in the metabolism of the apoB-containing lipoproteins. The ASBT inhibitor, SC-435, significantly reduced the concentrations of LDL-C and LDL apoB. We have shown for the first time that the decrease in LDL apoB is due to enhanced clearance, which is mediated by increased hepatic LDL receptor expression, consequent to the upregulation of hepatic cholesterol 7α-hydroxylase expression and activity.
Administration of SC-435 to miniature pigs fed normal amounts of dietary fat and cholesterol reduced both the concentrations of total plasma and LDL-C, which was associated with an increase in the fecal excretion of bile acids. This indicates that SC-435 inhibits ASBT in vivo in this animal model, thereby blocking the reabsorption of bile acids. Because ASBT inhibition would reduce the amount of bile acid recycled to the liver, it was predicted that the conversion of cholesterol to bile acids and cholesterol synthesis would increase.4 To establish this, we determined the hepatic activities of cholesterol 7α-hydroxylase and HMG-CoA reductase, the rate-limiting enzymes catalyzing the synthesis of bile acids and cholesterol, respectively. The expression and activity of both of these microsomal enzymes increased significantly with SC-435 treatment, indicating activation, at the transcriptional level, of compensatory responses in hepatic cholesterol homeostasis.4
LDL receptor activity, an important determinant of plasma LDL cholesterol concentrations, is increased by HMG-CoA reductase inhibitors and bile acid sequestrants.22 In this study, we show that SC-435 significantly increased the hepatic LDL receptor expression as measured by LDL receptor mRNA abundance, thereby enhancing LDL apoB FCR. Because SC-435 only blocks ileal ASBT-mediated bile acid transport and not passive bile acid reuptake, our results indicate that inhibition of active transport is sufficient to induce alterations in liver enzymes and receptor activities as would be expected from the interruption of the enterohepatic circulation of bile acids. It is possible that if SC-435 also inhibited cholesterol absorption, this would contribute to the upregulation of hepatic LDL receptors and cholesterol biosynthesis. Although cholesterol absorption has not been measured in this or any other studies, in hamsters treated with a compound structurally similar to SC-435, neutral steroid excretion was unaffected (B. T. Keller, unpublished data, 2002).
The changes we observed in cholesterol 7α-hydroxylase expression as a result of ASBT inhibition are consistent with the molecular basis for the regulation of this enzyme as recently elucidated by Lu et al17 Depletion of bile acids returned to the liver via the enterohepatic circulation would reduce the farnesoid X receptor–mediated activation of the SHP gene. A reduction in SHP would allow for the induction of liver receptor homolog-1, a positive transacting factor for the cholesterol 7α-hydroxylase gene.
The reduction in LDL apoB was mediated entirely by an increase in LDL apoB clearance, as evidenced by a significant increase in FCR. This finding is consistent with the increased expression of hepatic LDL receptor mRNA. No significant changes in LDL apoB production were observed. This observation differs from our previous results in pigs treated with the resin cholestyramine.7 This suggests that the molecular events, subsequent to upregulation of bile acid synthesis with an inhibitor of ASBT, differ from those induced by cholestyramine. In studies in which pigs were treated with lovastatin23 or low-dose atorvastatin,21 the LDL FCR and hepatic LDL receptor mRNA were unchanged; the decreased LDL apoB was due entirely to decreased production. However, at a higher dose, atorvastatin (10 mg/kg/day) increased LDL FCR and hepatic concentrations of LDL receptor mRNA by 15% and 76%, respectively,24 values similar to those observed with SC-435 in the present study. In contrast to statin treatment, SC-435 had no effect on LDL production, which may be related to the increase in VLDL apoB production by SC-435, an effect not observed in pigs treated with cholestyramine or a statin.7,21,23,24⇓⇓⇓
Although depletion of hepatic cholesterol, subsequent to the increased activity of cholesterol 7α-hydroxylase, might be expected to decrease the availability of cholesterol for VLDL assembly,25 SC-435 treatment increased the VLDL apoB production rate by 22%. In contrast, depletion of hepatic cholesterol through inhibition of cholesterol synthesis by treatment of miniature pigs with statins21,24⇓ resulted in decreased VLDL production. Increased VLDL triglyceride production has been observed in patients treated with bile acid sequestrants,26 an effect apparently linked to the upregulation of cholesterol 7α-hydroxylase activity. This concept is supported by studies demonstrating that overexpression of the cholesterol 7α-hydroxylase gene (CYP7A1) in rat hepatoma cells27 and in transgenic mice28 stimulates VLDL apoB secretion. Overexpression of CYP7A1 resulted in increases in the mature nuclear forms of the sterol regulatory element binding proteins (SREBP)-1 and -2, through mechanisms that are not well understood.28 SREBP-1a is a strong transcriptional activator of genes that encode enzymes involved in the synthesis of cholesterol and fatty acids, as well as the LDL receptor. SREBP-1c activates genes that code for enzymes involved in the synthesis of unesterified fatty acids, whereas SREBP-2 is a potent activator of cholesterol synthesis and the LDL receptor, but a weaker activator of fatty acid biosynthesis.29 Our results are consistent with increased hepatic concentrations of mature SREBP-1 or -2 as a result of increased cholesterol 7α-hydroxylase activity, which in turn is secondary to the inhibition of ASBT by SC-435. Although neither of these transcription factors were measured in the present study, hepatic HMG-CoA reductase expression was increased 2.5-fold and LDL receptor expression 1.6-fold, suggesting an increase in the mature form of SREBP-2.
Although increases in SREBP-1a and -1c would be predicted to increase liver fatty acid and triglyceride synthesis and thus increase VLDL production, we observed no changes in liver triglyceride content or triglyceride synthesis subsequent to SC-435 treatment. This result is consistent with the results of Sheng et al,30 who found that treatment of hamsters with the bile acid sequestrant colestipol had no effect on hepatic mature SREBP-1. A plausible explanation for the increased VLDL production rate observed in the present study is an increase in the expression of the microsomal triglyceride transfer protein (MTP) mediated by SREBP-2. MTP activity is required for hepatic VLDL assembly and secretion,31 and increased hepatic MTP expression after injection of an adenovirus vector encoding MTP has been shown to raise VLDL production.32 Furthermore, the increased VLDL apoB secretion observed in transgenic mice overexpressing CYP7A1 was associated with increased hepatic MTP expression and protein content, which the authors ascribed to an increased hepatic expression of SREBP-2.28 Despite our observation that SC-435 increased VLDL production in miniature pigs, concentrations of VLDL triglyceride and apoB were increased only modestly, and these changes were not statistically significant. We attribute this to an increase in VLDL lipolysis or enhanced LDL receptor–mediated clearance of VLDL remnants, leading to an 8% increase in VLDL apoB FCR. Miyake et al28 reported similar conclusions in mice overexpressing CYP7A1 in which the increased VLDL production failed to change plasma triglyceride or apoB concentrations. Furthermore, the CYP7A1-transgenic mice exhibited a significant reduction in atherosclerosis, suggesting that the increased VLDL-production was not detrimental.33
In summary, this study clearly demonstrates in a large animal model of human lipoprotein metabolism that interruption of the enterohepatic circulation of bile acids through inhibition of the ileal ASBT results in significant decreases in LDL cholesterol and apoB. The primary mechanism for this effect is increased LDL clearance, which is mediated through enhanced hepatic expression of LDL receptors, secondary to upregulation of hepatic cholesterol 7α-hydroxylase. Based on our results, it is predicted that SC-435 would enhance the activity of statins because of their complimentary mechanisms of action. We suggest that inhibitors of ASBT, such as SC-435, represent a novel approach for the treatment of hypercholesterolemia.
We thank Kim Wood for performing the surgeries and Sara Lipson and Stefanie Bombardier for their technical assistance. This work is supported by grants from the Heart and Stroke Foundation of Ontario (T-3371), the National Institutes of Health (NCRR RR12609), and Pharmacia Corp. M.W.H. is a Career Investigator of the Heart and Stroke Foundation of Ontario and P.H.R.B. is a National Heart Foundation of Australia Career Development Fellow.
Received July 1, 2002; revision accepted July 29, 2002.
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- ↵Gaw A, Packard CJ, Lindsay GM, Murray EF, Griffin BA, Caslake MJ, Colquhoun I, Wheatley DJ, Lorimer AR, Shepherd J. Effects of colestipol alone and in combination with simvastatin on apolipoprotein B metabolism. Arterioscler Thromb Vasc Biol. 1996; 16: 236–249.
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- ↵Root C, Smith CD, Winegar DA, Brieaddy LE, Lewis MC. Inhibition of ileal sodium-dependent bile acid transport by 2164U90. J Lipid Res. 1995; 36: 1106–1115.
- ↵Hara SJ, Higaki K-J, Higashino K-I, Iwai M, Takasu N, Miyata K, Tonda K, Nagata K, Goh Y, Mizui T. S-8921, an ileal Na+ /bile acid cotransporter inhibitor decreases serum cholesterol in hamsters. Life Sci. 1997; 60: 365–370.
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- ↵Rapp SR, Bhat BG, Beaudry JA, Napawan N, Keller BT. SC-435 is a potent inhibitor of the apical sodium co-dependent bile acid transporter (ASBT) in mice, rats, hamsters and dogs and reduces atherosclerotic lesions in apoE−/− mice [abstract]. Abstract Book: XIV International Symposium on Drugs Affecting Lipid Metabolism. Milan, Italy: Giovanni Lorenzini Medical Science Foundation; 2001; 50.
- ↵Burnett JR, Wilcox LJ, Telford DE, Kleinstiver SJ, Barrett PHR, Newton RS, Huff MW. Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoproteinB production in miniature pigs. Arterioscler Thromb Vasc Biol. 1997; 17: 2589–2600.
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