Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2
2005; Elsevier BV; Volume: 46; Issue: 11 Linguagem: Inglês
10.1194/jlr.m500232-jlr200
ISSN1539-7262
AutoresRyan E. Temel, Richard Lee, Kathryn L. Kelley, Matthew A. Davis, Ramesh Shah, Janet K. Sawyer, Martha D. Wilson, Lawrence L. Rudel,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe process of cholesterol absorption has yet to be completely defined at the molecular level. Because of its ability to esterify cholesterol for packaging into nascent chylomicrons, ACAT2 plays an important role in cholesterol absorption. However, it has been found that cholesterol absorption is not completely inhibited in ACAT2-deficient (ACAT2 KO) mice. Because ABCA1 mRNA expression was increased 3-fold in the small intestine of ACAT2 KO mice, we hypothesized that ABCA1-dependent cholesterol efflux sustains cholesterol absorption in the absence of ACAT2. To test this hypothesis, cholesterol absorption was measured in mice deficient in both ABCA1 and ACAT2 (DKO). Compared with wild-type, ABCA1 KO, or ACAT2 KO mice, DKO mice displayed the lowest level of cholesterol absorption. The concentrations of hepatic free and esterified cholesterol and gallbladder bile cholesterol were significantly reduced in DKO compared with wild-type and ABCA1 KO mice, although these measures of hepatic cholesterol metabolism were very similar in DKO and ACAT2 KO mice.We conclude that ABCA1, especially in the absence of ACAT2, can have a significant effect on cholesterol absorption, although ACAT2 has a more substantial role in this process than ABCA1. The process of cholesterol absorption has yet to be completely defined at the molecular level. Because of its ability to esterify cholesterol for packaging into nascent chylomicrons, ACAT2 plays an important role in cholesterol absorption. However, it has been found that cholesterol absorption is not completely inhibited in ACAT2-deficient (ACAT2 KO) mice. Because ABCA1 mRNA expression was increased 3-fold in the small intestine of ACAT2 KO mice, we hypothesized that ABCA1-dependent cholesterol efflux sustains cholesterol absorption in the absence of ACAT2. To test this hypothesis, cholesterol absorption was measured in mice deficient in both ABCA1 and ACAT2 (DKO). Compared with wild-type, ABCA1 KO, or ACAT2 KO mice, DKO mice displayed the lowest level of cholesterol absorption. The concentrations of hepatic free and esterified cholesterol and gallbladder bile cholesterol were significantly reduced in DKO compared with wild-type and ABCA1 KO mice, although these measures of hepatic cholesterol metabolism were very similar in DKO and ACAT2 KO mice. We conclude that ABCA1, especially in the absence of ACAT2, can have a significant effect on cholesterol absorption, although ACAT2 has a more substantial role in this process than ABCA1. Because the level of cholesterol absorption can influence plasma cholesterol concentrations (1Grundy S.M. Absorption and metabolism of dietary cholesterol.Annu. Rev. Nutr. 1983; 3: 71-96Crossref PubMed Scopus (209) Google Scholar), disruption of intestinal cholesterol transport by pharmacological means has been proven to reduce the concentration of plasma LDL cholesterol, the latter a risk factor that is tightly linked to the development of coronary heart disease. For example, administration of ezetimibe, a cholesterol absorption inhibitor, caused LDL cholesterol concentrations to decrease by 20%, whereas a reduction of almost 60% was achieved when ezetimibe and a statin were given together (2Sudhop T. Bergmann K. Von Cholesterol absorption inhibitors for the treatment of hypercholesterolaemia.Drugs. 2002; 62: 2333-2347Crossref PubMed Scopus (83) Google Scholar). Thus, inhibition of intestinal cholesterol absorption may provide an effective means of preventing coronary heart disease, the single leading cause of death in the United States (3Heart Disease and Stroke Statistics—2004 Update. American Heart Association: Dallas, TXGoogle Scholar). Although it is recognized that cholesterol absorption can have a significant impact on plasma cholesterol levels, many of the molecular mechanisms controlling cholesterol absorption remain to be elucidated. One cellular component of cholesterol absorption appears to be ACAT2. ACAT2 is a transmembrane protein that is expressed in the rough endoplasmic reticulum of enterocytes (4Rudel L.L. Lee R.G. Cockman T.L. Acyl coenzyme A:cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis.Curr. Opin. Lipidol. 2001; 12: 121-127Crossref PubMed Scopus (208) Google Scholar, 5Chang T-Y. Chang C.C.Y. Lin S. Yu C. Li B-L. Miyazaki A. Roles of acyl-coenzyme A:cholesterol acyltransferase 1 and 2.Curr. Opin. Lipidol. 2001; 12: 289-296Crossref PubMed Scopus (211) Google Scholar) and esterifies free cholesterol that has been either synthesized de novo or internalized from the intestinal lumen (6Swell L. Trout E.C.J. Hopper J.R. Field H.J. Treadwell C.R. Mechanism of cholesterol absorption. I. Endogenous dilution and esterification of fed cholesterol-4-C14.J. Biol. Chem. 1958; 232: 1-8Abstract Full Text PDF PubMed Google Scholar, 7Klein R.L. Rudel L.L. Cholesterol absorption and transport in thoracic duct lymph lipoproteins of nonhuman primates. Effect of dietary cholesterol level.J. Lipid Res. 1983; 24: 343-356Abstract Full Text PDF PubMed Google Scholar). Because a limited amount of free cholesterol can associate with the phospholipid monolayer of chylomicrons, the production of cholesteryl ester by ACAT2 allows cholesterol to be more efficiently packaged into apolipoprotein B (apoB)-containing lipoproteins formed during lipid absorption. For example, after a meal, cholesteryl ester can represent up to 78% of the total cholesterol in chylomicrons (8Fielding C.J. Renston J.P. Fielding P.E. Metabolism of cholesterol-enriched chylomicrons. Catabolism of triglyceride by lipoprotein lipase of perfused heart and adipose tissues.J. Lipid Res. 1978; 19: 705-711Abstract Full Text PDF PubMed Google Scholar, 9Riley J.W. Glickman R.M. Green P.H.R. Tall A.R. The effect of chronic cholesterol feeding on intestinal lipoproteins in the rat.J. Lipid Res. 1980; 21: 942-952Abstract Full Text PDF PubMed Google Scholar, 10Redgrave T.G. Dunne K.B. Chylomicron formation and composition in unanesthetized rabbits.Atherosclerosis. 1975; 22: 389-400Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 11Zilversmit D.B. Partition of lipid between chylomicrons and chylomicron-free lymph of the dog fed corn oil with or without cholesterol.Proc. Soc. Exp. Biol. Med. 1968; 128: 1116-1121Crossref Scopus (4) Google Scholar, 12Klein R.L. Rudel L.L. Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates.J. Lipid Res. 1983; 24: 357-367Abstract Full Text PDF PubMed Google Scholar), which carry >85% of the total cholesterol secreted by the small intestine into lymph (7Klein R.L. Rudel L.L. Cholesterol absorption and transport in thoracic duct lymph lipoproteins of nonhuman primates. Effect of dietary cholesterol level.J. Lipid Res. 1983; 24: 343-356Abstract Full Text PDF PubMed Google Scholar, 9Riley J.W. Glickman R.M. Green P.H.R. Tall A.R. The effect of chronic cholesterol feeding on intestinal lipoproteins in the rat.J. Lipid Res. 1980; 21: 942-952Abstract Full Text PDF PubMed Google Scholar). Thus, under normal circumstances, ACAT2 esterifies the majority of cholesterol absorbed by the body. Direct evidence for the involvement of ACAT2 in cholesterol absorption comes from studies of ACAT2-deficient (ACAT2 KO) mice. In these studies, it was shown that as dietary cholesterol content was incrementally increased, ACAT2 KO mice absorbed proportionally less cholesterol than wild-type mice (13Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V.J. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar, 14Repa J.J. Buhman K.K. Farese Jr., R.B. Dietschy J.M. Turley S.D. ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: impact on hepatic cholesterol homeostasis.Hepatology. 2004; 40: 1088-1097Crossref PubMed Scopus (96) Google Scholar). Although this finding implicated ACAT2 as a significant factor in cholesterol absorption, the incomplete inhibition of this process in ACAT2 KO mice indicated that cholesterol absorption may be mediated by additional mechanisms. One of these pathways may be via ABCA1. Expressed in enterocytes and many other cell types in the body (15Wellington C.L. Walker E.K.Y. Suarez A. Kwok A. Bissada N. Singaraja R. Yang Y-Z. Zhang L-H. James E. Wilson J.E. et al.ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation.Lab. Invest. 2002; 82: 273-283Crossref PubMed Scopus (244) Google Scholar), ABCA1 is a transmembrane protein that mediates the efflux of phospholipids and free cholesterol to lipid-poor apolipoproteins, such as apoA-I and apoE (16Wang N. Tall A.R. Regulation and mechanisms of ATP-binding cassette transporter A-I-mediated cellular cholesterol efflux.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (218) Google Scholar, 17Oram J.F. HDL apolioproteins and ABCA1. Partners in the removal of excess cellular cholesterol.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 720-727Crossref PubMed Scopus (212) Google Scholar). Based on the current literature, the importance of ABCA1 in cholesterol absorption is unclear. Using CaCo-2 cells, an immortalized cell line with properties similar to those of enterocytes, it has been shown that free cholesterol can be effluxed, presumably via ABCA1, from the basolateral membrane of the cells (18Ohama T. Hirano K. Zhang Z.Y. Aoki R. Tsujii K. Nakagawa-Toyama Y. Tsukamoto K. Ikegami C. Matsuyama A. Ishigami M. et al.Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands.Biochem. Biophys. Res. Commun. 2002; 296: 625-630Crossref PubMed Scopus (58) Google Scholar, 19Murthy S. Born E. Mathur S.N. Field F.J. LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells.J. Lipid Res. 2002; 43: 1054-1064Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Iqbal J. Anwar K. Hussain M.M. Multiple, independently regulated pathways of cholesterol transport across the intestinal epithelial cells.J. Biol. Chem. 2003; 278: 31610-31620Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In animal models, cholesterol absorption has been shown to decrease by 13% in ABCA1 KO mice (21Drobnik W. Lindenthal B. Lieser B. Ritter M. Weber T.C. Kiebisch G. Giesa U. Igel M. Borsukova H. Büchler C. et al.ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and by 79% in ABCA1 KO chickens (22Mulligan J.D. Flowers M.T. Tebon A. Bitgood J.J. Wellington C. Hayden M.R. Attie A.D. ABCA1 is essential for efficient basolateral cholesterol efflux during the absorption of dietary cholesterol in chickens.J. Biol. Chem. 2003; 278: 13356-13366Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In contrast, other studies have reported that ABCA1 KO mice displayed either similar (23Groen A.K. Bloks V.W. Bandsma R.H.J. Ottenhoff R. Chimini G. Kuipers F. Hepatobiliary cholesterol transport is not impaired in Abca1-null mice lacking HDL.J. Clin. Invest. 2001; 108: 843-850Crossref PubMed Scopus (143) Google Scholar) or significantly increased (24McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. Wet J. de et al.High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.Proc. Natl. Acad. Sci. USA. 2000; 97: 4245-4250Crossref PubMed Scopus (482) Google Scholar) cholesterol absorption compared with wild-type mice. A patient with Tangier disease had a cholesterol absorption percentage similar to those of control subjects (25Schaefer E.J. Brousseau M.E. Diffenderfer M.R. Cohn J.S. Welty F.K. O'Connor J.J. Dolnikowski G.G. Wang J. Hegele R.A. Jones P.J. Cholesterol and apolipoprotein B metabolism in Tangier disease.Atherosclerosis. 2001; 159: 231-236Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, ABCA1 appears to mediate only a limited amount of cholesterol absorption. However, when other components in the process are not functioning normally, such as when ACAT2 is missing, ABCA1 might assume a more significant role. The main line of evidence supporting the hypothesis that cholesterol absorption is sustained by ABCA1 in the absence of ACAT2 comes from a study showing that ABCA1 mRNA expression was significantly greater in ACAT2 KO compared with wild-type small intestine (14Repa J.J. Buhman K.K. Farese Jr., R.B. Dietschy J.M. Turley S.D. ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: impact on hepatic cholesterol homeostasis.Hepatology. 2004; 40: 1088-1097Crossref PubMed Scopus (96) Google Scholar). Because the regulatory pool of free cholesterol may be increased in enterocytes of ACAT2 KO mice, the increase in ABCA1 expression is likely mediated by liver X receptor (LXR) activation (26Repa J.J. Turley S.D. Lobaccaro J-M.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Regulation of absorption of ABC1-mediated efflux of cholesterol by RXR heterodimers.Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar). In turn, an increase in ABCA1-mediated cholesterol efflux to lipid-poor apolipoproteins may serve as a means of cholesterol absorption in ACAT2 KO mice. To test the hypothesis that cholesterol absorption can be partially maintained by ABCA1 in the absence of ACAT2, the present study of intestinal cholesterol transport and hepatic cholesterol metabolism was conducted using mice deficient in both ABCA1 and ACAT2 (DKO mice). All mice were maintained in an American Association for Accreditation of Laboratory Animal Care-approved animal facility under protocols approved by the institutional animal care and use committee at Wake Forest University School of Medicine. Male wild-type mice (50% C57Bl/6, 50% 129Sv/Jae) were purchased from Jackson Laboratories, and male ACAT2 KO mice (50% C57Bl/6, 50% 129Sv/Jae) were provided by Dr. Robert J. Farese, Jr. (13Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V.J. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar). To generate DKO mice, ACAT2 KO mice were crossed with ABCA1 KO mice (50% FVB, 50% 129Sv/Jae) provided by CV Therapeutics. ABCA1 KO mice were created by Dr. Eddy Rubin at Berkeley National Laboratory and lack exons 17–22, which encode nucleotide binding domain 1. ABCA1+/− ACAT2+/− siblings were then mated to establish the following lines of mice: wild-type (ABCA1+/+ ACAT2+/+), ABCA1 KO (ABCA1−/− ACAT2+/+), ACAT2 KO (ABCA1+/+ ACAT2−/−), and DKO (ABCA1−/− ACAT2−/−). Male wild-type and ACAT2 KO mice at least 6 weeks of age and 6–7 week old female wild-type, ABCA1 KO, ACAT2 KO, and DKO mice were offered 10 g/day of a low-fat (20% of energy as palm-enriched fat), moderate-cholesterol (0.17%, w/w) diet. After feeding the diet to the males for 10 days and to the females for 4 weeks, the mice were gavaged with 0.1 μCi of [4-14C]cholesterol (Amersham Pharmacia Biotech) and 0.2 μCi of [22,23-3H]sitosterol (New England Nuclear) dissolved in 100 μl of soybean oil. Each mouse was individually housed in a cage with a wire bottom and was allowed free access to diet and water for 3 days. The feces were collected and homogenized in 95% ethanol using a Polytron PT 1200 homogenizer (Kinematica). An aliquot of the fecal slurry was saponified by adding 50% KOH to a final concentration of 5% (w/v) and heating at 65°C for 2 h. Neutral lipids were extracted by adding hexane and deionized water, vortexing, and centrifuging at 2,000 g for 10 min. The hexane phase was transferred to a scintillation vial and dried at 65°C under N2. In addition, aliquots of the initial [14C]cholesterol/[3H]sitosterol dose were placed into scintillation vials. Bio-Safe II scintillation fluid (Research Products International) was added to the vials, and the [14C]cholesterol and [3H]sitosterol counts were measured in a scintillation spectrometer. Percentage cholesterol absorption was calculated using the following equation: ( 14C/ 3H dose ratio- 14C/ 3H feces ratio) 14C/ 3H dose ratio×100 After being fed the low-fat, moderate-cholesterol diet for 5 weeks, female mice were singly housed as described above. After a 3 day fecal collection, the mice were weighed, and the feces were collected, dried in a 70°C vacuum oven, weighed, and crushed into a fine powder. A measured mass (50–100 mg) of feces was placed into a glass tube containing 103 μg of 5α-cholestane as an internal standard. The feces were saponified and the neutral lipids were extracted into hexane as described above. Mass analysis of the extracted neutral sterols was conducted by gas-liquid chromatography as described previously (27Temel R.E. Gebre A.K. Parks J.S. Rudel L.L. Compared with acyl-CoA:cholesterol O-acyltransferase (ACAT)1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol.J. Biol. Chem. 2003; 278: 47594-47601Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Fecal neutral sterol mass represents the sum of cholesterol, coprostanol, and coprostanone in each sample. Fecal neutral sterol excretion was expressed as mg sterol/day/100 g body weight. After being fed the low-fat, moderate-cholesterol diet for 6 weeks, mice were fasted for 4 h and euthanized. Blood was collected by heart puncture and was placed into a tube containing protease inhibitor cocktail (Sigma) dissolved in 5% EDTA, 5% NaN3. Gallbladder bile was collected, and then the liver was removed, weighed, and snap-frozen in liquid N2. The small intestine from the pyloric valve to the cecum was removed, cleaned of fat and luminal contents while on ice, and cut into either three sections of equal length for the male ACAT2 KO experiments or five pieces of equal length for the female DKO experiments. The intestinal sections were snap-frozen in liquid N2 and stored along with the liver and bile samples at −80°C. The blood was centrifuged at 12,000 g for 10 min at 4°C, and the plasma was analyzed for total and free cholesterol concentrations using the Cholesterol/HP (Roche) and the Free Cholesterol C (Wako) enzymatic assay kits, respectively. Plasma lipoprotein cholesterol distribution was determined after separation of lipoprotein classes from whole plasma by gel filtration chromatography as described previously (28Lee R.G. Kelley K.L. Sawyer J.K. Farese Jr., R.V. Parks J.S. Rudel L.L. Plasma cholesterol esters provided by lecithin:cholesterol acyltransferase and acyl-coenzyme A:cholesterol acyltransferase 2 have opposite atherosclerotic potential.Circ. Res. 2004; 95: 998-1004Crossref PubMed Scopus (100) Google Scholar). For analysis of the liver lipid composition, ∼100 mg of liver was thawed, minced, and weighed in a glass tube. Lipids were extracted in 2:1 CHCl3/methanol at room temperature overnight. The protein was quantitatively separated from the lipid extract, which was then dried down under N2 and redissolved in a measured volume of 2:1 CHCl3/methanol. Dilute H2SO4 was added to the sample, which was then vortexed and centrifuged to split the phases. The aqueous upper phase was aspirated and discarded, and an aliquot of the bottom phase was removed and dried down; 1% Triton X-100 in CHCl3 was then added, and the solvent was evaporated (29Carr T.P. Andresen C.J. Rudel L.L. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (484) Google Scholar). Deionized water was then added to each tube and vortexed until the solution was clear. Lipids were then quantified using the Triglycerides/GB kit (Roche) plus the enzymatic cholesterol assays described above. For analysis of biliary lipid concentrations, a measured volume (∼10 μl) of bile was placed into a glass tube and the neutral lipids were extracted and analyzed as described for liver. Aliquots of the aqueous phase of the extraction were analyzed for bile acid content using an enzymatic assay using hydroxysteroid dehydrogenase (30Turley S.D. Dietschy J.M. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1978; 19: 924-928Abstract Full Text PDF PubMed Google Scholar). Total mRNA was extracted from ∼100 mg of liver and proximal small intestine with Trizol (Invitrogen Life Technologies) using the protocol provided by the manufacturer. The mRNA was resuspended in 300 μl of diethyl pyrocarbonate water, and 1 μg of mRNA was reverse transcribed to cDNA using Omniscript reverse transcriptase (Qiagen) under the following conditions: 37°C for 1 h and 93°C for 5 min. The cDNA was diluted 1:10 using diethyl pyrocarbonate water, and real-time PCR was done in triplicate with 5 μl of cDNA, 12.5 μl of SYBR GREEN PCR master mix (Applied Biosystems), 5.5 μl of diethyl pyrocarbonate water, and 1 μl of forward and reverse primer (20 pmol) for a final reaction volume of 25 μl. The primer sequences are presented in Table 1. PCR was then run on the Sequence Detection System 7000 (Applied Biosystems) using the following conditions: 50°C for 2 min, 94°C for 10 min, and 40 cycles of 94°C for 10 s and 60°C for 1 min. The fluorescence measurement used to calculate threshold cycle (Ct) was made at the 60°C point. A dissociation curve was run at the end of the reaction to ensure a single amplification product. Ct values were entered into the following equation to determine the arbitrary unit value: 1 × 109 × e(−0.6931 × Ct). All values were then normalized to either GAPDH or cyclophilin mRNA concentration of the sample to take total mRNA concentration into account.TABLE 1Primer sequences used for real-time PCR analysisGenePrimer Sequence (5′→3′)ATP binding cassette transporter A1CGTTTCCGGGAAGTGTCCTAGCTAGAGATGACAAGGAGGATGGAATP binding cassette transporter G5TGGATCCAACACCTCTATGCTAAAGGCAGGTTTTCTCGATGAACTGCholesterol 7-α hydroxylaseAGCAACTAAACAACCTGCCAGTACTAGTCCGGATATTCAAGGATGCACyclophilinTGGAGAGCACCAAGACAGACATGCCGGAGTCGACAATGATGlyceraldehyde-3-phosphate dehydrogenaseTGTGTCCGTCGTGGATCTGACCTGCTTCACCACCTTCTTGAT3-Hydroxy-3-methylglutaryl-CoA synthase 1GCCGTGAACTGGGTCGAAGCATATATAGCAATGTCTCCTGCAANiemann-Pick C1-like protein 1ATCCTATCCCTGGACTTGCTGCTCAGTGAGGCTGGTGTTATGCGSterol-regulatory element binding protein 1cGGAGCCATGGATTGCACATTGGCCCGGGAAGTCACTGT Open table in a new tab To verify that cholesterol absorption was reduced in ACAT2 KO mice fed a semisynthetic diet without the fiber-rich properties of chow, male wild-type and ACAT2 KO mice were fed a low-fat (20% of energy as palm oil), moderate-cholesterol (0.17%, w/w) diet and fractional cholesterol absorption was measured using the fecal dual-isotope method. Similar to other studies (13Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V.J. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar, 14Repa J.J. Buhman K.K. Farese Jr., R.B. Dietschy J.M. Turley S.D. ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: impact on hepatic cholesterol homeostasis.Hepatology. 2004; 40: 1088-1097Crossref PubMed Scopus (96) Google Scholar), fractional cholesterol absorption was decreased from 53% in wild-type mice to 35% in ACAT2 KO mice (Fig. 1A). However, much of the cholesterol was still absorbed by the ACAT2 KO mice, suggesting that, in the absence of ACAT2, redundant pathways were allowing cholesterol absorption to be sustained. Because previous evidence indicated that ABCA1 could play a role in cholesterol absorption (14Repa J.J. Buhman K.K. Farese Jr., R.B. Dietschy J.M. Turley S.D. ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: impact on hepatic cholesterol homeostasis.Hepatology. 2004; 40: 1088-1097Crossref PubMed Scopus (96) Google Scholar, 21Drobnik W. Lindenthal B. Lieser B. Ritter M. Weber T.C. Kiebisch G. Giesa U. Igel M. Borsukova H. Büchler C. et al.ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), real-time PCR analysis of ABCA1 mRNA expression in the small intestine was conducted. Compared with wild-type mice, ACAT2 KO mice had an almost 3-fold increase in the expression of ABCA1 (Fig. 1B). This result supported the hypothesis that increased expression of ABCA1 in the absence of ACAT2 allowed cholesterol absorption to be partially maintained. To test this hypothesis, ABCA1 KO mice were mated with ACAT2 KO mice and the offspring were crossed to create DKO mice. Cholesterol absorption was then measured in female DKO mice along with wild-type, ABCA1 KO, and ACAT2 KO mice fed the low-fat, moderate-cholesterol diet. Using the fecal dual-isotope method, cholesterol absorption was decreased from 63% in wild-type mice to 25% in DKO mice (Fig. 2A). Fractional cholesterol absorption was also reduced to 55% in ABCA1 KO mice and to 35% in ACAT2 KO mice, and the sum of these decreases was similar to the decline displayed by the DKO mice. The extent of cholesterol absorption was also estimated by analyzing the excretion of neutral sterols in the feces. In agreement with the fractional cholesterol absorption data, sterol excretion was significantly increased from 11 mg/day/100 g body weight in the wild-type mice to 26 mg/day/100 g body weight in the DKO mice (Fig. 2B). The ACAT2 KO mice also showed a significant increase in neutral sterol excretion (18 mg/day/100 g body weight), whereas the mean value was not significantly higher in the ABCA1 KO mice (14 mg/day/100 g body weight). The data show that cholesterol absorption can be mediated by both ABCA1 and ACAT2 and that this process was greatly decreased when both of these proteins were absent. Real-time PCR analysis was used to determine whether any differences occurred in the expression of genes involved in the trafficking of cholesterol through the enterocytes. For this experiment, RNA was isolated from the duodenum and proximal jejunum. The mRNA of Niemann-Pick C1-like 1 (NPC1L1), which is the target of the cholesterol absorption inhibitor ezetimibe (31Altmann S.W. Davis H.R.J. Zhu L. Yao X. Hoos L.M. Tetzloff G. Iyer S.P.N. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1429) Google Scholar), was decreased to ∼60% of that of the wild type in the small intestine of DKO mice (Fig. 3A). A similar decrease in NPC1L1 mRNA was also observed for the small intestine of ACAT2 KO mice, although no significant change was seen in the intestine of ABCA1 KO mice. Because ABCG5, in concert with ABCG8, is believed to efflux excess cholesterol from enterocytes into the lumen of the intestine (32Yu L. Li-Hawkins J. Hammer R.E. Berge E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar, 33Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar), the mRNA level of this LXR-sensitive gene was also determined. Although a trend toward increased expression was evident for the ACAT2 KO mice, the amounts of ABCG5 mRNA were not significantly different in the small intestines of the four different genotypes of mice (Fig. 3B). It was hypothesized that because of significant decreases in cholesterol absorption, the livers of DKO mice would reflect the changes in intestinal cholesterol metabolism. Accordingly, hepatic lipid compositions were determined. Although no statistically significant differences were found between wild-type and ABCA1 KO mouse livers, the livers of DKO mice contained 30% less free cholesterol and 99% less cholesteryl ester (Fig. 4A, B). In addition, a reduction of 64% was observed for hepatic triglyceride content of DKO versus wild-type and ABCA1 KO mice (Fig. 4C). ACAT2 KO mice had similar reductions in hepatic concentrations of these three lipids as DKO mice. Thus, even though the DKO mice displayed lower fractional cholesterol absorption than the ACAT2 KO mice (Fig. 2), the livers from these two genotypes showed similarly decreased concentrations of free cholesterol, cholesteryl ester, and triglyceride. The lipid composition of gallbladder bile collected from fasted mice was also analyzed. No significant differences in cholesterol, bile salt, and phospholipid concentrations were found upon comparison of DKO and ACAT2 KO gallbladder bile (Table 2). However, the concentrations of cholesterol and phospholipids were significantly decreased in the DKO and ACAT2 KO mice compared with the wild-type and ABCA1 KO mice (Table 2). Because the bile can be concentrated while being collected in the gallbladder, the percentage molar composition of the bile was calculated. The bile from DKO and ACAT2 KO mice contained a lower molar percentage of cholesterol than that from wild-type mice, whereas ABCA1 KO mouse bile contained a significantly higher molar percentage of cholesterol. ACAT2 KO and DKO mice also had bile with lower molar percentages of ph
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