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• W2115366657 abstract "Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL33333391) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053.We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane. Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL33333391) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane. Elevated plasma levels of LDL cholesterol (LDL-C) constitute a major risk factor for the development of atherosclerosis (1The Expert PanelExecutive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel of Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III).JAMA. 2001; 285: 2486-2497Google Scholar). The cholesterol carried in LDL, like all the other cholesterol in the body, is derived from de novo synthesis and absorption from the diet (2Turley S.D. Dietschy J.M. The metabolism and excretion of cholesterol by the liver.in: Arias I.M. Jakoby W.B. Popper H. Schachter D. Shafritz D.A. The Liver: Biology and Pathobiology. Raven Press, New York1988: 617-641Google Scholar). In humans, the plasma LDL-C concentration often correlates positively with the level of intestinal cholesterol absorption (3McMurry M.P. Connor W.E. Lin D.S. Cerqueira M.T. Connor S.L. The absorption of cholesterol and the sterol balance in the Tarahumara Indians of Mexico fed cholesterol-free and high cholesterol diets.Am. J. Clin. Nutr. 1985; 41: 1289-1298Google Scholar, 4Kesäniemi Y.A. Miettinen T.A. Cholesterol absorption efficiency regulates plasma cholesterol level in the Finnish population.Eur. J. Clin. Invest. 1987; 17: 391-395Google Scholar). This association is also seen in various primate models that have been identified as being hypo- or hyperresponsive to a dietary cholesterol challenge (5Bhattacharyya A.K. Eggen D.A. Cholesterol absorption and turnover in rhesus monkeys as measured by two methods.J. Lipid Res. 1980; 21: 518-524Google Scholar, 6St. Clair R.W. Wood L.L. Clarkson T.B. Effect of sucrose polyester on plasma lipids and cholesterol absorption in African green monkeys with variable hypercholesterolemic response to dietary cholesterol.Metabolism. 1981; 30: 176-183Google Scholar, 7Kushwaha R.S. Rice K.S. Lewis D.S. McGill Jr., H.C. Carey K.D. The role of cholesterol absorption and hepatic cholesterol content in high and low responses to dietary cholesterol and fat in pedigreed baboons (Papio species).Metabolism. 1993; 42: 714-722Google Scholar, 8Turley S.D. Spady D.K. Dietschy J.M. Identification of a metabolic difference accounting for the hyper- and hyporesponder phenotypes of cynomolgus monkey.J. Lipid Res. 1997; 38: 1598-1611Google Scholar). The wide individual variation in cholesterol absorption seen within most species, including humans (9Bosner M.S. Lange L.G. Stenson W.F. Ostlund Jr., R.E. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.J. Lipid Res. 1999; 40: 302-308Google Scholar), has been well characterized in different strains of mice (10Carter C.P. Howles P.N. Hui D.Y. Genetic variation in cholesterol absorption efficiency among inbred strains of mice.J. Nutr. 1997; 127: 1344-1348Google Scholar, 11Jolley C.D. Dietschy J.M. Turley S.D. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: Effect on cholesterol responsiveness.Am. J. Physiol. 1999; 276: G1117-G1124Google Scholar, 12Schwarz M. Davis D.L. Vick B.R. Russell D.W. Genetic analysis of intestinal cholesterol absorption in inbred mice.J. Lipid Res. 2001; 42: 1801-1811Google Scholar). Recent studies in this species demonstrate that such variability results from the interplay of multiple genes (12Schwarz M. Davis D.L. Vick B.R. Russell D.W. Genetic analysis of intestinal cholesterol absorption in inbred mice.J. Lipid Res. 2001; 42: 1801-1811Google Scholar, 13Schwarz M. Davis D.L. Vick B.R. Russell D.W. Genetic analysis of cholesterol accumulation in inbred mice.J. Lipid Res. 2001; 42: 1812-1819Google Scholar).The major cellular and biochemical steps involved in the translocation of cholesterol from the intestinal lumen to the lymph have been described in detail (14Tso P. Intestinal lipid absorption.in: Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, New York1994: 1867-1907Google Scholar, 15Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol.J. Lipid Res. 1994; 35: 943-955Google Scholar). The efficiency with which cholesterol is absorbed can be changed dramatically by manipulating many of these steps. It is well documented, for example, that shifts in either the size or composition of the intestinal bile acid pool, or in the amount and species of biliary phospholipid entering the lumen, can result in profound changes in the level of cholesterol absorption (16Homan R. Krause B.R. Established and emerging strategies for inhibition of cholesterol absorption.Curr. Pharm. Des. 1997; 3: 29-44Google Scholar, 17Wang D.Q-H. Lammert F. Paigen B. Carey M.C. Hyposecretion of biliary phospholipids (PL) significantly decreases the intestinal absorption of cholesterol (Ch) in Mdr2(−/−) and (+/−) mice.Gastroenterology. 1998; 114: G3744Google Scholar, 18Wang D.Q-H. Tazuma S. Cohen D.E. Carey M.C. Natural hydrophilic bile acids profoundly inhibit intestinal cholesterol absorption in mice.Hepatology. 1999; 30: 395AGoogle Scholar, 19Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Alternate pathways of bile acid synthesis in the cholesterol 7α-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding.J. Lipid Res. 2001; 42: 1594-1603Google Scholar). Deleting the gene responsible for apoB synthesis in the intestine or inhibiting the activity of acylCoA:cholesterol acyltransferase (ACAT) can also result in dramatic changes in the amount of cholesterol reaching the lymph from the intestinal lumen (16Homan R. Krause B.R. Established and emerging strategies for inhibition of cholesterol absorption.Curr. Pharm. Des. 1997; 3: 29-44Google Scholar, 20Turley S.D. Herndon M.W. Dietschy J.M. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster.J. Lipid Res. 1994; 35: 328-339Google Scholar, 21Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. A genetic model for absent chylomicron formation: mice producing apolipoprotein B in the liver, but not in the intestine.J. Clin. Invest. 1995; 96: 2932-2946Google Scholar).Although the rate limiting step in cholesterol absorption remains unknown, several recent studies have provided new insights into the mechanism(s) that may ultimately dictate how much cholesterol gets absorbed. One group of studies describes the search for a sterol permease in the microvillus membrane of the enterocyte that facilitates the uptake of cholesterol and potentially other sterols after their release from mixed micelles (22Kramer W. Glombik H. Petry S. Heuer H. Schäfer H-L. Wendler W. Corsiero D. Girbig F. Weyland C. Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter.FEBS Lett. 2000; 487: 293-297Google Scholar, 23Hernandez M. Montenegro J. Steiner M. Kim D. Sparrow C. Detmers P.A. Wright S.D. Chao Y-S. Intestinal absorption of cholesterol is mediated by a saturable, inhibitable transporter.Biochim. Biophys. Acta. 2000; 1486: 232-242Google Scholar). Initially, there was some evidence that scavenger receptor class B, type 1 (SR-BI) might be the putative sterol transporter (24Hauser H. Dyer J.H. Nandy A. Vega M.A. Werder M. Bieliauskaite E. Weber F.E. Compassi S. Gemperli A. Boffelli D. Wehrli E. Schulthess G. Phillips M.C. Identification of a receptor mediating absorption of dietary cholesterol in the intestine.Biochemistry. 1998; 37: 17843-17850Google Scholar). However, in mice lacking SR-BI, sterol absorption is not reduced (25Mardones P. Quiñones V. Amigo L. Moreno M. Miquel J.F. Schwarz M. Miettinen H.E. Trigatti B. Krieger M. VanPatten S. Cohen D.E. Rigotti A. Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type 1-deficient mice.J. Lipid Res. 2001; 42: 170-180Google Scholar, 26Altmann S.W. Davis Jr., H.R. Yao X. Laverty M. Compton D.S. Zhu L.-j. Crona J.H. Caplen M.A. Hoos L.M. Tetzloff G. Priestley T. Burnett D.A. Strader C.D. Graziano M.P. The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption.Biochim. Biophys. Acta. 2002; 1580: 77-93Google Scholar). If it can be demonstrated unequivocally that the uptake of cholesterol by the enterocyte is protein facilitated, then clearly such a transporter would make an attractive target for pharmacologic intervention.The other key discovery relates to the role that ABC transporter G5 (ABCG5) and ABC transporter G8 (ABCG8) play in preventing or impeding the absorption of plant sterols and stanols (27Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Google Scholar, 28Lee M.-H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption.Nat. Genet. 2001; 27: 79-83Google Scholar). Mutations in the genes for these two proteins result in sitosterolemia, a rare autosomal recessive disorder characterized by hyperabsorption of sitosterol and other plant sterols (29Salen G. Horak I. Rothkopf M. Cohen J.L. Speck J. Tint G.S. Shore V. Dayal B. Chen T. Shefer S. Lethal atherosclerosis associated with abnomal plasma and tissue sterol composition in sitosterolemia with xanthomatosis.J. Lipid Res. 1985; 26: 1126-1133Google Scholar, 30Lütjohann D. Björkhem I. Beil U.F. von Bergmann K. Sterol absorption and sterol balance in phytosterolemia evaluated by deuterium-labeled sterols: effect of sitostanol treatment.J. Lipid Res. 1995; 36: 1763-1773Google Scholar). Sitosterolemic individuals also absorb cholesterol more efficiently and are often hypercholesterolemic, implying a role of ABCG5 and ABCG8 in dictating the efficiency of cholesterol absorption.The quest to learn more about the role of specific proteins in regulating the flux of sterols into and out of the enterocyte is timely given the recent development of a new class of novel, selective, and potent cholesterol absorption inhibitors. Ezetimibe (SCH 58235) and an analog, SCH 48461, inhibit cholesterol absorption at very low doses and exert a marked hypocholesterolemic effect in humans (31Bays H.E. Moore P.B. Drehobl M.A. Rosenblatt S. Toth P.D. Dujovne C.A. Knopp R.H. Lipka L.J. LeBeaut A.P. Yang B. Mellars L.E. Cuffie-Jackson C. Veltri E.P. Effectiveness and tolerability of ezetimibe in patients with primary hypercholesterolemia: pooled analysis of two phase II studies.Clin. Ther. 2001; 23: 1209-1230Google Scholar, 32Dujovne C.A. Bays H. Davidson M.H. Knopp R. Hunninghake D.B. Stein E.A. Goldberg A.C. Jones P. Lipka L.J. Cuffie-Jackson C. Reduction of LDL cholesterol in patients with primary hypercholesterolemia by SCH 48461: Results of a multicenter dose-ranging study.J. Clin. Pharmacol. 2001; 41: 70-78Google Scholar, 33Gagné C. Gaudet D. Bruckert E. Efficacy and safety of ezetimibe coadministered with atorvastatin or simvastatin in patients with homozygous familial hypercholesterolemia.Circulation. 2002; 105: 2469-2475Google Scholar, 34Sudhop T. Lutjohann D. Kodal A. Tribble D. Shah S. Perevozskaya I. von Bergmann K. Inhibition of intestinal cholesterol absorption by ezetimibe in humans.Atheroscler. Suppl. 2002; 3: 213Google Scholar) and an array of different animal models (26Altmann S.W. Davis Jr., H.R. Yao X. Laverty M. Compton D.S. Zhu L.-j. Crona J.H. Caplen M.A. Hoos L.M. Tetzloff G. Priestley T. Burnett D.A. Strader C.D. Graziano M.P. The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption.Biochim. Biophys. Acta. 2002; 1580: 77-93Google Scholar, 35Salisbury B.G. Davis H.R. Burrier R.E. Burnett D.A. Boykow G. Caplen M.A. Clemmons A.L. Compton D.S. Hoos L.M. McGregor D.G. Schnitzer-Plokoff R. Smith A.A. Weig B.C. Zilli D.L. Clader J.W. Sybertz E.J. Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461.Atherosclerosis. 1995; 115: 45-63Google Scholar, 36Van Heek M. France C.F. Compton D.S. McLeod R.L. Yumibe N.P. Alton K.B. Sybertz E.J. Davis Jr., H.R. In vivo metabolism-based discovery of a potent cholesterol absorption inhibitor, SCH58235, in the rat and rhesus monkey through the identification of the active metabolites of SCH48461.J. Pharmacol. Exp. Ther. 1997; 283: 157-163Google Scholar, 37Van Heek M. Austin T.M. Farley C. Cook J.A. Tetzloff G.G. Davis H.R. Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters.Diabetes. 2001; 50: 1330-1335Google Scholar, 38Davis Jr., H.R. Pula K.K. Alton K.B. Burrier R.E. Watkins R.W. The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in dogs.Metabolism. 2001; 50: 1234-1241Google Scholar, 39Van Heek M. Compton D.S. Davis H.R. The cholesterol absorption inhibitor, ezetimibe, decreases diet-induced hypercholesterolemia in monkeys.Eur. J. Pharmacol. 2001; 415: 79-84Google Scholar, 40Davis Jr., H.R. Compton D.S. Hoos L. Tetzloff G. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in apoE knockout mice.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 2032-2038Google Scholar). Ezetimibe is glucuronidated in the enterocyte during its first pass. Both ezetimibe and its glucuronide are circulated enterohepatically, repeatedly delivering the agent back to the site of action on the luminal surface of the enterocyte (41Van Heek M. Farley C. Compton D.S. Hoos L. Alton K.B. Sybertz E.J. Davis Jr., H.R. Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663.Br. J. Pharmacol. 2000; 129: 1748-1754Google Scholar). The molecular mechanism by which this agent inhibits cholesterol absorption is, however, unknown.In the present studies, we used the 129/Sv mouse, a strain distinguished by its inherently high levels of cholesterol absorption (10Carter C.P. Howles P.N. Hui D.Y. Genetic variation in cholesterol absorption efficiency among inbred strains of mice.J. Nutr. 1997; 127: 1344-1348Google Scholar, 11Jolley C.D. Dietschy J.M. Turley S.D. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: Effect on cholesterol responsiveness.Am. J. Physiol. 1999; 276: G1117-G1124Google Scholar, 12Schwarz M. Davis D.L. Vick B.R. Russell D.W. Genetic analysis of intestinal cholesterol absorption in inbred mice.J. Lipid Res. 2001; 42: 1801-1811Google Scholar), to learn more about how another one of the ezetimibe analogs, SCH 58053, mediates it cholesterol lowering effect. The data show that this agent does not change the physicochemical nature of the intraluminal environment, nor does it increase the expression of proteins that drive sterol efflux from the enterocyte. We speculate that, instead, it mediates its inhibitory action at the level of a putative sterol permease that facilitates the movement of cholesterol into the intestinal cell.MATERIALS AND METHODSAnimals and dietsThe female 129/Sv mice used in these studies were generated in our own colony from 129/SvEvBrd-Hprtb-m2 breeding stock as described earlier (11Jolley C.D. Dietschy J.M. Turley S.D. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: Effect on cholesterol responsiveness.Am. J. Physiol. 1999; 276: G1117-G1124Google Scholar). All experiments with mice used this strain except one study that utilized female LDL-receptor (LDLR) knockout mice. Those mice were of a mixed 129/Sv:C57BL/6 background, and were also bred in our own colony. All mice were housed either as groups or individually in plastic colony cages containing wood shavings in a temperature controlled room (22°C) with light cycling. At the time of study, the mice were about 13- to 20-weeks-old. They had access to drinking water at all times and were fed ad libitum a pelleted cereal-based rodent diet (Wayne Lab Blox, No. 8604; Harlan Teklad, Madison, WI). This formulation (basal diet) had an inherent cholesterol content of 0.02% (w/w) and a fatty acid composition as described elsewhere (20Turley S.D. Herndon M.W. Dietschy J.M. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster.J. Lipid Res. 1994; 35: 328-339Google Scholar). The meal form of this diet was used to prepare the experimental diets which, in some of the studies, also contained additional cholesterol (final levels of either 0.20 or 1.00% w/w). These diets were prepared without or with SCH 58053 at a level of 0.021% (w/w). Based on their daily food consumption of approximately 165 g diet/kg body weight, this dietary level of SCH 58053 provided the mice with a dose of ∼35 mg/day/kg body weight. The experimental diets were fed from 10 to 23 days, depending on the types of measurements that were made. All experiments were performed toward the end of the 12-h dark phase of the lighting cycle, and all animals were in the fed state at the time of study. Experiments were approved by the Institutional Animal Care and Research Advisory Committee.SCH 58053The SCH 58053 used in these studies was supplied by the Schering-Plough Research Institute (Kenilworth, NJ). SCH 58053 ((+)-7-(4-chlorophenyl)-2-(4fluorophenyl)-7-hydroxy-3(R)- (4-hydroxyphenyl)-2-azaspiro[3.5]nonan-1-one) is an analog of ezetimibe (SCH 58235) ((-)-1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4-hydroxyphenyl)-2-azetidinone). Figure 1shows the respective structures of SCH 58053 (A) and SCH 58235 (B).Sterol synthesis in liver and extrahepatic organsThe rate of sterol synthesis in all major organs was measured in vivo as described (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). Mice were given an ip injection of 40 mCi of [3H]water (NEN Life Science Products, Boston, MA) and after 1 h were anesthetized and exsanguinated. Aliquots of liver, the entire small intestine, and the remaining carcass were saponified and their content of radiolabeled digitonin-precipitable sterols (DPS) was measured. The rate of sterol synthesis in each organ was expressed as the nmol of [3H]water incorporated into DPS/h/g of tissue, while whole animal synthesis was calculated as μmol of [3H]water incorporated/h/100 g body weight.Intestinal cholesterol and lipid absorptionCholesterol absorption was measured by a fecal dual-isotope ratio method. Mice were dosed ig with a mixture of 2 μCi [5,6-3H]sitostanol (American Radiolabeled Chemicals, Inc., St. Louis, MO) and 1 μCi [4-14C]cholesterol (NEN Life Science Products, Boston, MA). They were then housed individually in fresh cages and stools were collected over the following 3 days. Aliquots of stool and the dosing mixture were extracted, and the ratio of 14C to 3H in each was determined. The percent cholesterol absorption was calculated from these data as described (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). To determine the level of total lipid absorption, the lipid content of the diet and stools was determined gravimetrically. These data, together with the amount of diet consumed and stool excreted (both expressed as g/day/100 g body weight), were used to calculate the fraction of lipid consumed that was absorbed.Fecal bile acid and neutral sterol excretionStools collected from individually housed mice over 3 days were dried, weighed, and ground to a fine powder. A 1-g aliquot of this material was used to determine total bile acid content by an enzymatic method previously described (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). A second 1-g aliquot was used to quantitate the amounts of cholesterol, coprostanol, epicoprostanol, and cholestanone by GC as described in detail elsewhere (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). The excretion rates of both bile acids and neutral sterols were expressed as μmol/day/100 g body weight.Bile acid pool size and compositionPool size was determined as the total bile acid content of the small intestine, gallbladder, and liver, which were extracted in ethanol in the presence of an internal standard ([24-14C]taurocholic acid, NEN Life Science Products) and analyzed by high performance liquid chromatography (HPLC) (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). Bile acids were detected by measurement of the refractive index and identified by comparison to authentic standards. Pool size was expressed as μmol/100 g body weight.Biliary lipid compositionGallbladder bile was harvested from mice in the fed state. The gallbladder with contents was carefully excised and placed in a small microfuge tube (0.5 ml capacity). The gallbladder was punctured with a 23 gauge needle and the tube was then centrifuged at 2,000 rpm for 5 min in a tabletop centrifuge (Sorvall RT7), Dupont Co., Newtown, CT) fitted with a swinging bucket rotor (RTH-750). An aliquot of bile (10 μl to 20 μl) was then extracted in 1.0 ml of methanol containing 50 μg of stigmastanol. The respective concentrations of bile acid, phospholipids, and cholesterol in the methanolic extract were determined as described (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar), and these data were used to calculate the molar percent of cholesterol in the bile.Relative cholesterol and bile acid concentrations in liquid phase of intestinal contentsThe proximal half of the small intestine was cut into four sections. The contents of each section were removed by gentle finger stripping and combined in a single microfuge tube The tubes were centrifuged at 4,000 rpm for 15 min at room temperature in a tabletop centrifuge (Sorvall RT7, Dupont, Newtown, CT). A 25 μl aliquot of the resulting supernatant was extracted in 1 ml of methanol. The absolute concentrations of cholesterol and bile acid in each extract were measured as described for the bile samples.Plasma and tissue cholesterol levelsPlasma and tissue total cholesterol concentrations were determined by enzymatic or gas chromatographic methods as described (42Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). The latter used stigmastanol as an internal standard. In the studies with mice fed cholesterol enriched diets, the cholesterol concentration was measured in the plasma and liver, but not in the small intestine because the entire jejunal mucosa from each mouse was needed for the mRNA analyses as described below.RNA analysisFollowing exsanguination of the mice under ether anesthesia, aliquots of liver and the entire jejunal mucosa from each animal were frozen in liquid nitrogen. Total RNA was prepared from these tissues, and mRNA was further purified using oligo-dT resin from individual livers or a pool composed of equal amounts of jejunal RNA from the mice of each group. Five micrograms of poly(A)+ RNA was fractionated by electrophoresis, transferred to membrane, and subjected to Northern analysis using the 32P-labeled probes indicated. The amount of radioactivity in each band was quantified by phosphorimager, normalized to the signal generated by β-actin, and mathematically adjusted to establish a unit of 1 for the group fed the basal diet (0.02% w/w cholesterol) without SCH 58053 (43Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J-M.A. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ.Genes Dev. 2000; 14: 2819-2830Google Scholar). In the mouse, two major RNA transcripts are detected for ABCG5 (2.4 and 3.3 kb) and ABCG8 (2.5 and 3.9 kb) (27Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Google Scholar).RNA levels were also measured for jejunum using a quantitative real-time PCR assay. Total RNA was treated with DNase I (RNase-free, Roche), and reverse-transcribed with random hexamers using SuperScript II RNase H-reverse transcriptase to generate cDNA. Primer Express Software (PerkinElmer Life Sciences) was used to design the following primers: cyclophilin forward 5′-TGGAGAGCACCAAGACAGACA, reverse 5′-TGCCGGAGTCGACAATGAT; adenosine triphosphate-binding cassette transporter A1 (ABCA1) forward 5′-CGTTTCCGGGAAGTGTCCTA, reverse 5′-GCTAGAGATGACAAGGAGGATGGA; ABCG5 forward 5′-TGGATCCAACACCTCTATGCTAAA, reverse 5′-GGC-AGGTTTTCTCGATGAACTG; ABCG8 forward 5′-TGCCCACCTTCCACATGTC, reverse 5′-ATGAAGCCGGCAGTAAGGTAGA.Primers were validated by analysis of template titration and dissociation curves. PCR assays were performed on an Applied Biosystems Prism 7000 sequence detection system. The PCR reaction contained (final volume of 20 μl): 50 ng of reverse-transcribed RNA, a 150 nM concentration of" @default.
• W2115366657 created "2016-06-24" @default.
• W2115366657 creator A5000275357 @default.
• W2115366657 creator A5022438526 @default.
• W2115366657 creator A5056039594 @default.
• W2115366657 date "2002-11-01" @default.
• W2115366657 modified "2023-09-23" @default.
• W2115366657 title "Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte" @default.
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