Title: Stimulation of Cholesterol Excretion by the Liver X Receptor Agonist Requires ATP-binding Cassette Transporters G5 and G8
Abstract: Liver X receptor (LXR) is a nuclear receptor that plays a crucial role in orchestrating the trafficking of sterols between tissues. Treatment of mice with a potent and specific LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. Here we show that expression of two target genes of LXRα, the ATP-binding cassette (ABC) transportersAbcg5 and Abcg8, is required for both the increase in sterol excretion and the decrease in fractional cholesterol absorption associated with LXR agonist treatment. Mice expressing no ABCG5 and ABCG8 (G5G8 −/− mice) and their littermate controls were treated for 7 days with T0901317. In wild type animals, treatment with the LXR agonist resulted in a 3-fold increase in biliary cholesterol concentrations, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, the LXR agonist did not significantly affect biliary cholesterol levels, fractional cholesterol absorption, or neutral fecal sterol excretion in the G5G8 −/−mice. Thus Abcg5 and Abcg8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking. These results establish a central role for ABCG5 and ABCG8 in promoting cholesterol excretion in vivo. Liver X receptor (LXR) is a nuclear receptor that plays a crucial role in orchestrating the trafficking of sterols between tissues. Treatment of mice with a potent and specific LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. Here we show that expression of two target genes of LXRα, the ATP-binding cassette (ABC) transportersAbcg5 and Abcg8, is required for both the increase in sterol excretion and the decrease in fractional cholesterol absorption associated with LXR agonist treatment. Mice expressing no ABCG5 and ABCG8 (G5G8 −/− mice) and their littermate controls were treated for 7 days with T0901317. In wild type animals, treatment with the LXR agonist resulted in a 3-fold increase in biliary cholesterol concentrations, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, the LXR agonist did not significantly affect biliary cholesterol levels, fractional cholesterol absorption, or neutral fecal sterol excretion in the G5G8 −/−mice. Thus Abcg5 and Abcg8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking. These results establish a central role for ABCG5 and ABCG8 in promoting cholesterol excretion in vivo. ATP-binding cassette wild type forAbcg5 and Abcg8 allele homozygous for an allele with inactivated Abcg5 and Abcg8 liver X receptor high density lipoprotein low density lipoprotein sterol regulatory element-binding protein gas chromatography fast protein liquid chromatography Cholesterol is an important structural component of animal cell membranes. The cholesterol required to maintain membrane integrity can be synthesized de novo from acetyl-CoA or can be obtained from cholesterol-containing foods in the diet. The typical Western diet includes ∼400 mg of cholesterol per day, of which 40–50% is absorbed in the proximal small intestine. The major pathway by which cholesterol is eliminated from the body is by excretion into bile either as free cholesterol or after conversion to bile acids. A variety of noncholesterol sterols are also present in the diet. The most plentiful of these are the two plant sterols, sitosterol and campesterol. The levels of these sterols in tissues are very low, because plant sterols are poorly absorbed from the intestine and are preferentially secreted into the bile by hepatocytes (1Schoenheimer R. Science. 1931; 74: 579-584Crossref PubMed Scopus (43) Google Scholar, 2Gould R.G. Jones R.J. LeRoy G.V. Wissler R.W. Taylor C.B. Metabolism. 1969; 18: 652-662Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 3Salen G. Ahrens Jr., E.H. Grundy S.M. J. Clin. Invest. 1970; 49: 952-967Crossref PubMed Scopus (386) Google Scholar). One mechanism by which excess cholesterol and other sterols are eliminated from the body involves the action of two ATP-binding cassette (ABC)1half-transporters, ABCG5 and ABCG8 (4Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1351) Google Scholar, 5Lee M.H. Lu K. Hazard S. 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Invest. 1974; 53: 1033-1043Crossref PubMed Scopus (460) Google Scholar). The pivotal role of ABCG5 and ABCG8 in enterohepatic sterol transport has been demonstrated directly by manipulating the expression of these genes in mice (11Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar, 12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Transgenic mice containing ∼14 copies of a human genomic DNA fragment, including both human ABCG5 andABCG8 genes have a ∼50% reduction in the fractional absorption of dietary cholesterol, dramatically elevated levels of biliary cholesterol, and a 4.5-fold increase in fecal neutral sterol excretion (11Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar). Plant sterol levels are more than 50% lower in these mice than in their wild type littermates. Disruption of the mouseAbcg5 and Abcg8 genes has the opposite effect on dietary sterol trafficking. The G5G8 −/− mice have 30-fold higher plasma levels of sitosterol than do their wild type littermates due to increased fractional absorption of dietary plant sterols and impaired biliary sterol excretion (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Abcg5 and Abcg8 are expressed predominantly in the liver and small intestine (4Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1351) Google Scholar) and are coordinately up-regulated at the transcriptional level by dietary cholesterol. The response ofAbcg5 and Abcg8 to cholesterol requires the liver X receptor α (LXRα) (13Repa J.J. Berge K.E. Pomajzl C. Richardson J.A. Hobbs H. Mangelsdorf D.J. J. Biol. Chem. 2002; 277: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar), a nuclear receptor that regulates the expression of many key genes in lipid metabolism, includingABCA1 (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar, 15Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar), the gene mutated in Tangier disease (16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. 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Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1416) Google Scholar), an important transcription factor in the regulation of fatty acid biosynthesis (24Horton J.D. Goldstein J.L. Brown M. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3778) Google Scholar). By regulating the expression of these genes, LXRα coordinates the synthesis and trafficking of cholesterol and fatty acids between tissues. Mice lacking LXRα accumulate large amounts of cholesterol in the liver when fed a high cholesterol diet (21Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar), whereas wild type mice treated with an LXR agonist have decreased fractional absorption of dietary cholesterol (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar) and increased biliary cholesterol excretion (25Groen A.K. Bloks V.W. Bandsma R.H. Ottenhoff R. Chimini G. Kuipers F. J. Clin. Invest. 2001; 108: 843-850Crossref PubMed Scopus (143) Google Scholar). The mechanism by which LXRα prevents the accumulation of dietary cholesterol has not been fully defined. The decreased fractional absorption of dietary cholesterol associated with LXR agonist treatment was attributed initially to the action of ABCA1, because levels of ABCA1 mRNA increased dramatically in the small intestine of animals given the nonsteroidal synthetic LXR agonist:N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)phenyl]benzenesulfonamide (T0901317) (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar). Subsequent characterization of mice expressing no ABCA1 (Abca1 −/− mice) revealed no impairment in biliary cholesterol secretion or fecal neutral sterol excretion (25Groen A.K. Bloks V.W. Bandsma R.H. Ottenhoff R. Chimini G. Kuipers F. J. Clin. Invest. 2001; 108: 843-850Crossref PubMed Scopus (143) Google Scholar, 26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In the current study, we tested the hypothesis that ABCG5 and ABCG8 mediate the LXR agonist-associated increase in biliary and fecal excretion of cholesterol and reduction in cholesterol absorption. The synthetic LXR agonist T0901317 was purchased from Cayman Chemical Company (Ann Arbor, MI). Sterols were obtained either from Steraloids Inc. (Newport, RI) or from Sigma-Aldrich (St. Louis, MO). Mice homozygous for a disruptedAbcg5 and Abcg8 allele (G5G8 −/−) were generated as described previously (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). The mice used in these studies were offspring ofG5G8 +/− mice of mixed genetic background (129S6SvEv × C57BL/6J). The mice were housed in plastic cages in a temperature-controlled room (22 °C) with a daylight cycle from 6 am to 6 pm, and fed ad libitum a cereal-based rodent chow diet (Diet 7001, Harlan Teklad, Madison, WI) containing 0.02% cholesterol and 4% fat. All animal procedures were performed with approval of the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center. Diets containing 0.025% of T0901317 (T-diet) were made by mixing powdered chow diet (Diet 7001, Harlan Teklad) with pure T0901317. The T-diet was stored in aluminum foil-covered containers at 4 °C for no more than 3 days. Female mice were housed individually 1 week before initiation of the T-diet and then fed for 1 week with either the T-diet or the chow diet dispensed from a feeder jar. The dose and duration of T0901317 treatment were based on prior studies (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar, 27Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1395) Google Scholar). Sterol levels in plasma and liver were measured by gas chromatography (GC) as described (28Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar, 29Turley S.D. Herndon M.W. Dietschy J.M. J. Lipid Res. 1994; 35: 328-339Abstract Full Text PDF PubMed Google Scholar) with some modifications. Briefly, plasma and tissues were saponified in 3% potassium hydroxide/ethanol at 66 °C for 3 h after addition of 5α-cholestane as a quantitative recovery standard. Lipids were extracted using petroleum ether and dried under nitrogen. The residual lipids were re-dissolved in Tri-Sil reagent (product 48999, Pierce, Rockford, IL) for analysis by GC. Hepatic triglyceride levels were measured using Infinity triglycerides reagent (Sigma-Aldrich, St. Louis, MO). Lipoproteins were size-fractionated using fast protein liquid chromatography (FPLC), and the total sterol concentration in each fraction was measured using cholesterol/HP kits (catalog number 1127771, Roche Diagnostics Corp., Indianapolis, IN). Bile was collected from the gallbladder of anesthetized mice using a 30.5-gauge needle. The concentrations of cholesterol, phospholipids, and bile acids were measured as described previously (30Turley S.D. Daggy B.P. Dietschy J.M. Metabolism. 1991; 40: 1063-1073Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Mice were fed either the control or T-diet for 4 days prior to being moved to new cages containing fresh wood shavings. The diets were fed for an additional 3 days during which time the feces were collected. The feces were dried, weighed, and ground to a fine powder. An aliquot of 0.5 g of feces was used to determine fecal neutral and acidic sterol excretion (11Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar, 28Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Mice were fed either the control or T-diet for 4 days prior to administering an oil mixture containing deuterated cholesterol and sitostanol by gavage (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Mice were then housed individually in new cages containing fresh wood shavings. The diets were continued, and the feces were collected for 3 days and processed for sterol absorption as described (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Total RNA was extracted from tissues using the RNA Stat-60 kit (Tel-Test Inc., Friendswood, TX), and real-time PCR was performed to assay the relative amounts of selected mRNAs as described (31Yang J. Goldstein J.L. Hammer R.E. Moon Y.A. Brown M.S. Horton J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13607-13612Crossref PubMed Scopus (189) Google Scholar, 32Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). All data are reported as the mean ± S.E. The differences between the mean values were tested for statistical significance by the two-tailed Student's ttest. Plasma levels of sitosterol and campesterol were 30-fold higher inG5G8 −/− than in G5G8 +/+female mice fed a chow diet. Addition of T0901317 (0.025%) to the diet for 7 days resulted in a fall of plasma plant sterol levels to barely detectable levels in wild type mice. Mean plasma sitosterol levels increased from 20.6 to 39.6 mg/dl in the LXR agonist-treated knockout animals (Fig. 1); an increase of similar magnitude occurred in the plasma level of campesterol, the other major dietary plant sterol. Mean plasma levels of cholesterol were lower in chow-fedG5G8 −/− mice than in wild type mice, as previously described (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar) (Fig. 1). The levels of plasma cholesterol increased by 50% and by 90% in the wild type mice andG5G8 −/− mice with T0901317 treatment. FPLC analysis was performed to determine the distribution of sterols in the plasma of these mice. No significant difference was seen in the distribution of sterols in the knockout and wild type mice. The increase in plasma sterol levels was due to increases of HDL sterols in both strains of mice (Fig. 2). Treatment with T019101317 was associated with widening of the HDL sterol peaks and a shoulder extending into the larger size fractions, as has been reported previously (26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 27Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1395) Google Scholar). T0901317 treatment increased biliary cholesterol levels of wild type mice by almost 3-fold (from 7.13 to 20.12 μmol/ml), as was observed previously (26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) (Fig. 3). In contrast to wild type mice, no significant increase in mean biliary cholesterol level was seen in the G5G8 −/− mice after treatment with T0901317 (from 0.73 to 0.97 μmol/ml). These data are consistent with Abcg5 and Abcg8 being the target genes responsible for the increase in biliary cholesterol levels associated with LXR activation. Biliary phospholipid levels were significantly lower in the knockout than in the wild type mice. A similar difference in biliary phospholipid levels was observed previously in female, but not male,G5G8 −/− mice (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Biliary phospholipids and bile acid levels fell after T0901317 treatment in theG5G8 +/+ mice but not in theG5G8 −/− mice (Fig. 3). Similar reductions in biliary lipid levels were seen previously in wild type mice treated with an LXR agonist (26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Levels of plant sterols were significantly higher in chow-fed G5G8 −/− mice than in wild type animals. G5G8 −/− mice have increased fractional absorption of dietary plant sterols and a decrease in the biliary excretion of sterols, which contribute to the higher hepatic levels of plant sterols in these mice (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Treatment of theG5G8 −/− mice with T0901317 resulted in a modest, but significant reduction in hepatic sitosterol and campesterol levels (Fig. 4). The hepatic cholesterol level fell by 30% in the wild type mice after treatment with T0901317, presumably in part due to the increase in biliary cholesterol secretion (Fig. 4). In contrast to the wild type mice, no change in hepatic cholesterol levels was seen in the knockout mice after LXR agonist treatment, presumably resulting from the lack of increase in biliary cholesterol secretion in these mice (Fig. 4). Hepatic triglyceride levels were similar in the chow-fedG5G8 +/+ and G5G8 −/−mice. Treatment with T090137 was associated with an increase in hepatic triglycerides in both groups of mice (Fig. 4), as previously observed (27Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1395) Google Scholar). These results are attributed to the activation ofSREBP-1c, which stimulates the expression of multiple genes in the fatty acid biosynthetic pathway and is an LXR target gene (23Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1416) Google Scholar). The fractional absorption of dietary cholesterol was reduced from 72% to 52% in the wild type mice treated with T0901317, which is similar to that observed by Repa et al. (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar). In contrast to wild type mice, the fractional absorption of cholesterol increased slightly (from 76% to 81%) in T0901317-treated G5G8 −/− mice (Fig.5). Decreased fractional cholesterol absorption and increased biliary cholesterol secretion both may contribute to the dramatic increase in fecal neutral sterol excretion that occurs with LXR agonist treatment (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar, 26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The level of neutral sterols excreted into the feces over the 3-day collection period was ∼30% lower in the chow-fed G5G8 −/− mice than in the G5G8 +/+ mice, and this level failed to increase with T0901317 treatment (Fig.6). Thus, ABCG5 and ABCG8 are required for the stimulation of fecal neutral sterol excretion by the LXR agonist T0901317. Fecal bile acid excretion was similar in theG5G8 +/+ and G5G8 −/−mice and did not change significantly with T0901317 treatment (Fig.6). The mRNA levels of known LXR target genes, including Srebp-1c,Abca1, Abcg1, Abcg5, andAbcg8, were determined to confirm that T0901317 had the expected biological effects in the tissues of the treated mice. The expression levels of all selected LXR target genes were increased with T0901317 treatment in both strains of mice with the exceptions ofAbcg5 and Abcg8 inG5G8 −/− mice. The mRNA levels ofSrebp-2, which is not a target gene of LXR, did not increase significantly with T0901317 treatment in either wild type orG5G8 −/− mice (Fig.7). The major finding of this study is that ABCG5 and ABCG8 are required in mice for the stimulation of biliary and fecal cholesterol excretion by the synthetic LXR agonist, T0901317. Disruption ofAbcg5 and Abcg8 abolished the increase in biliary cholesterol levels, the reduction in fractional cholesterol absorption, and the increase in fecal neutral sterol excretion associated with LXR activation (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar, 26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). These data, taken together with the phenotypic characterization of mice expressing either no ABCG5 and ABCG8 or higher levels of both proteins (11Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar, 12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar), suggest that LXR activation promotes the excretion of sterols by increasing Abcg5 andAbcg8 expression. In wild type mice, treatment with the LXR agonist was associated with significantly lower plasma levels of both sitosterol and campesterol (Fig. 1). Similar reductions in plasma plant sterol levels were seen in transgenic mice containing 14 copies of the human ABCG5 andABCG8 transgenes (11Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar). In contrast to wild type mice, plasma levels of sitosterol and campesterol increased ∼2-fold with T0901317 treatment in the G5G8 −/− mice (Fig. 1). These data indicate that increased expression of Abcg5 andAbcg8 is both necessary and sufficient for the LXR agonist-associated reduction in plasma plant sterol levels observed in the wild type animals. In the absence of ABCG5 and ABCG8, plant sterols accumulate in the liver due to an inability to efficiently secrete sterols into the bile and an increased absorption of dietary plant sterols (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). The increased hepatic levels of plant sterols in the knockout animals likely result in an increased incorporation of these sterols into lipoproteins and secretion into plasma. Further studies are required to determine if LXR agonist treatment results in a greater increase in the incorporation of plant sterols into lipoproteins or an increased secretion of lipoproteins into the circulation inG5G8 −/− mice. The LXR agonist also may promote the transport of sterols from peripheral tissues into the circulation of G5G8 −/− mice. Plasma cholesterol levels also increased significantly with T0901317 treatment in G5G8 +/+ andG5G8 −/− mice (Fig. 1). The increase in plasma cholesterol was limited to the HDL fraction (Fig. 2) and was associated with an increase in the size of HDL particles, as reported previously (27Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1395) Google Scholar, 33Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. 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Chem. 2003; 278: 13356-13366Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The fall in hepatic cholesterol levels (Fig. 4) in theG5G8 +/+ mice with LXR activation may also in part be due to an ABCA1-mediated increase in the efflux of cholesterol from the liver into the circulation (14Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1150) Google Scholar, 26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) in addition to the increase in biliary cholesterol secretion. The most dramatic difference between G5G8 +/+ andG5G8 −/− mice in response to T0901317 treatment was in biliary cholesterol levels. Mean biliary cholesterol levels increased 3-fold in wild type mice but did not change significantly in knockout mice. Biliary phospholipid and bile acid levels were lower in the G5G8 −/− mice than in their wild type littermates, which is comparable to the reductions observed previously in female mice (12Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). Both the biliary phospholipid and bile acid levels fell in wild type mice treated with T0901317 (Fig. 3), as was seen previously in mice treated with LXR agonists (26Plosch T. Kok T. Bloks V.W. Smit M.J. Havinga R. Chimini G. Groen A.K. Kuipers F. J. Biol. Chem. 2002; 277: 33870-33877Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). No reduction in the levels of bile acids or phospholipids in the bile occurred in the T0901317-treated G5G8 −/− mice. No change in fecal bile acid excretion was seen in either the knockout or the wild type animals treated with the LXR agonist (Fig. 6). Therefore, the increased excretion of biliary cholesterol associated with LXR agonist treatment was not quantitatively coupled to biliary bile acid or phospholipid excretion. The results of these studies demonstrate that ABCG5 and ABCG8 mediate the effects of LXR agonists on the increase in fecal loss of cholesterol. These studies do not distinguish the relative contributions of the liver and the intestine to the increased fecal neutral sterol excretion. The stimulation of fecal cholesterol loss by the LXR agonist may result from an increase in biliary cholesterol secretion by hepatocytes and/or the decreased fractional absorption of dietary cholesterol by enterocytes. Tissue-specific disruptions ofAbcg5 and Abcg8 will be required to assess the function of these transporters in the liver and small intestine. Reverse cholesterol transport involves the efflux of cholesterol from peripheral tissues to the liver, the secretion of cholesterol into bile, and the excretion of sterols in feces. The molecular machinery that affects reverse cholesterol transport has not been fully characterized. The data in this report are consistent with the notion that two ABC half-transporters, ABCG5 and ABCG8, mediate the final step in this pathway. We thank Robert Guzman, Yinyan Ma, Anja Kerksiek, and Silvia Winnen for excellent technical assistance. We also thank Scott Clark, Anh Nguyen, Scott M. Grundy, and Gloria Vega for measuring lipids in bile, plasma, and tissues. We thank David W. Russell for helpful discussion.