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Stimulation of Cholesterol Excretion by the Liver X Receptor Agonist Requires ATP-binding Cassette Transporters G5 and G8*

  • Liqing Yu
    Affiliations
    ‡McDermott Center for Human Growth and Development, the Departments of Molecular Genetics and Internal Medicine, Dallas, Texas 75390-9046
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  • Jennifer York
    Affiliations
    ‡McDermott Center for Human Growth and Development, the Departments of Molecular Genetics and Internal Medicine, Dallas, Texas 75390-9046
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  • Klaus von Bergmann
    Affiliations
    §Department of Clinical Pharmacology, University of Bonn, Bonn 53105, Germany
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  • Dieter Lutjohann
    Affiliations
    §Department of Clinical Pharmacology, University of Bonn, Bonn 53105, Germany
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  • Jonathan C. Cohen
    Affiliations
    ¶Center for Human Nutrition, Dallas, Texas 75390-9046
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  • Helen H. Hobbs
    Correspondence
    Supported by the Howard Hughes Medical Institute, by National Institutes of Health Grants HL20948 and HL72304, by the Perot Family Foundation, by the W. M. Keck Foundation, by the Donald W. Reynolds Clinical Cardiovascular Research Center at Dallas, and by Bundesministerium für Bildung, Forschung, Wissenschaft und Technologie Grant 01EC9402. To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-6724; Fax: 214-648-7539;
    Affiliations
    ‡McDermott Center for Human Growth and Development, the Departments of Molecular Genetics and Internal Medicine, Dallas, Texas 75390-9046

    ‖the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:February 22, 2003DOI:https://doi.org/10.1074/jbc.M301311200
      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.
      ABC
      ATP-binding cassette
      G5G8 +/+
      wild type forAbcg5 and Abcg8 allele
      G5G8 −/−
      homozygous for an allele with inactivated Abcg5 and Abcg8
      LXR
      liver X receptor
      HDL
      high density lipoprotein
      LDL
      low density lipoprotein
      SREBP
      sterol regulatory element-binding protein
      GC
      gas chromatography
      FPLC
      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 (
      • Schoenheimer R.
      ,
      • Gould R.G.
      • Jones R.J.
      • LeRoy G.V.
      • Wissler R.W.
      • Taylor C.B.
      ,
      • Salen G.
      • Ahrens Jr., E.H.
      • Grundy S.M.
      ).
      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 (
      • Berge K.E.
      • Tian H.
      • Graf G.A.
      • Yu L.
      • Grishin N.V.
      • Schultz J.
      • Kwiterovich P.
      • Shan B.
      • Barnes R.
      • Hobbs H.H.
      ,
      • Lee 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.
      ). Mutations in either of these genes cause sitosterolemia, a rare autosomal recessive disorder of sterol trafficking (
      • Berge K.E.
      • Tian H.
      • Graf G.A.
      • Yu L.
      • Grishin N.V.
      • Schultz J.
      • Kwiterovich P.
      • Shan B.
      • Barnes R.
      • Hobbs H.H.
      ,
      • Lee 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.
      ). Subjects with sitosterolemia have increased fractional absorption of dietary noncholesterol sterols and decreased biliary secretion of plant- and animal-derived sterols (
      • Miettinen T.A.
      ,
      • Salen G.
      • Shore V.
      • Tint G.S.
      • Forte T.
      • Shefer S.
      • Horak I.
      • Horak E.
      • Dayal B.
      • Nguyen L.
      • Batta A.K.
      ). Consequently, these patients accumulate sitosterol, as well as other plant- and shellfish-derived neutral sterols, in the blood and tissues (
      • Salen G.
      • Horak I.
      • Rothkopf M.
      • Cohen J.L.
      • Speck J.
      • Tint G.S.
      • Shore V.
      • Dayal B.
      • Chen T.
      • Shefer S.
      ,
      • Gregg R.E.
      • Connor W.E.
      • Lin D.S.
      • Brewer Jr., H.B.
      ). Subjects with sitosterolemia also are frequently hypercholesterolemic and develop tendon xanthomas and premature coronary artery disease (
      • Salen G.
      • Horak I.
      • Rothkopf M.
      • Cohen J.L.
      • Speck J.
      • Tint G.S.
      • Shore V.
      • Dayal B.
      • Chen T.
      • Shefer S.
      ,
      • Bhattacharyya A.K.
      • Connor W.E.
      ).
      The pivotal role of ABCG5 and ABCG8 in enterohepatic sterol transport has been demonstrated directly by manipulating the expression of these genes in mice (
      • Yu L.
      • Li-Hawkins J.
      • Hammer R.E.
      • Berge K.E.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      ,
      • Yu L.
      • Hammer R.E.
      • Li-Hawkins J.
      • Von Bergmann K.
      • Lutjohann D.
      • Cohen J.C.
      • Hobbs H.H.
      ). 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 (
      • Yu L.
      • Li-Hawkins J.
      • Hammer R.E.
      • Berge K.E.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      ). 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 (
      • Yu L.
      • Hammer R.E.
      • Li-Hawkins J.
      • Von Bergmann K.
      • Lutjohann D.
      • Cohen J.C.
      • Hobbs H.H.
      ).
      Abcg5 and Abcg8 are expressed predominantly in the liver and small intestine (
      • Berge K.E.
      • Tian H.
      • Graf G.A.
      • Yu L.
      • Grishin N.V.
      • Schultz J.
      • Kwiterovich P.
      • Shan B.
      • Barnes R.
      • Hobbs H.H.
      ) 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) (
      • Repa J.J.
      • Berge K.E.
      • Pomajzl C.
      • Richardson J.A.
      • Hobbs H.
      • Mangelsdorf D.J.
      ), a nuclear receptor that regulates the expression of many key genes in lipid metabolism, includingABCA1 (
      • Repa 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.
      ,
      • Costet P.
      • Luo Y.
      • Wang N.
      • Tall A.R.
      ), the gene mutated in Tangier disease (
      • Brooks-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.
      • Koop B.
      • Pimstone S.
      • Kastelein J.J.
      • Hayden M.R.
      ,
      • Bodzioch M.
      • Orso E.
      • Klucken J.
      • Langmann T.
      • Bottcher A.
      • Diederich W.
      • Drobnik W.
      • Barlage S.
      • Buchler C.
      • Porsch-Ozcurumez M.
      • Kaminski W.E.
      • Hahmann H.W.
      • Oette K.
      • Rothe G.
      • Aslanidis C.
      • Lackner K.J.
      • Schmitz G.
      ,
      • Rust S.
      • Rosier M.
      • Funke H.
      • Real J.
      • Amoura Z.
      • Piette J.C.
      • Deleuze J.F.
      • Brewer H.B.
      • Duverger N.
      • Denefle P.
      • Assmann G.
      ,
      • Marcil M.
      • Brooks-Wilson A.
      • Clee S.M.
      • Roomp K.
      • Zhang L.H.
      • Yu L.
      • Collins J.A.
      • van Dam M.
      • Molhuizen H.O.
      • Loubster O.
      • Ouellette B.F.
      • Sensen C.W.
      • Fichter K.
      • Mott S.
      • Denis M.
      • Boucher B.
      • Pimstone S.
      • Genest Jr., J.
      • Kastelein J.J.
      • Hayden M.R.
      ), murine (but not human) cholesterol 7α-hydroxylase (Cyp7A1) (
      • Lehmann J.M.
      • Kliewer S.
      • Moore L.
      • Smith-Oliver T.
      • Oliver B.
      • Su J.-L.
      • Sundseth S.
      • Winegar D.
      • Blanchard D.
      • Spencer T.
      • Willson T.M.
      ,
      • Peet D.J.
      • Turley S.D.
      • Ma W.
      • Janowski B.A.
      • Lobaccaro J.M.
      • Hammer R.E.
      • Mangelsdorf D.J.
      ,
      • Chen J.Y.
      • Levy-Wilson B.
      • Goodart S.
      • Cooper A.D.
      ), the rate-limiting enzyme in bile acid synthesis, and sterol regulatory element-binding protein 1c (SREBP-1c) (
      • Repa J.J.
      • Liang G.
      • Ou J.
      • Bashmakov Y.
      • Lobaccaro J.M.
      • Shimomura I.
      • Shan B.
      • Brown M.S.
      • Goldstein J.L.
      • Mangelsdorf D.J.
      ), an important transcription factor in the regulation of fatty acid biosynthesis (
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.
      ). 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 (
      • Peet D.J.
      • Turley S.D.
      • Ma W.
      • Janowski B.A.
      • Lobaccaro J.M.
      • Hammer R.E.
      • Mangelsdorf D.J.
      ), whereas wild type mice treated with an LXR agonist have decreased fractional absorption of dietary cholesterol (
      • Repa 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.
      ) and increased biliary cholesterol excretion (
      • Groen A.K.
      • Bloks V.W.
      • Bandsma R.H.
      • Ottenhoff R.
      • Chimini G.
      • Kuipers F.
      ).
      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) (
      • Repa 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.
      ). Subsequent characterization of mice expressing no ABCA1 (Abca1 −/− mice) revealed no impairment in biliary cholesterol secretion or fecal neutral sterol excretion (
      • Groen A.K.
      • Bloks V.W.
      • Bandsma R.H.
      • Ottenhoff R.
      • Chimini G.
      • Kuipers F.
      ,
      • Plosch T.
      • Kok T.
      • Bloks V.W.
      • Smit M.J.
      • Havinga R.
      • Chimini G.
      • Groen A.K.
      • Kuipers F.
      ). 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.

      DISCUSSION

      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 (
      • Repa 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.
      ,
      • Plosch T.
      • Kok T.
      • Bloks V.W.
      • Smit M.J.
      • Havinga R.
      • Chimini G.
      • Groen A.K.
      • Kuipers F.
      ). These data, taken together with the phenotypic characterization of mice expressing either no ABCG5 and ABCG8 or higher levels of both proteins (
      • Yu L.
      • Li-Hawkins J.
      • Hammer R.E.
      • Berge K.E.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      ,
      • Yu L.
      • Hammer R.E.
      • Li-Hawkins J.
      • Von Bergmann K.
      • Lutjohann D.
      • Cohen J.C.
      • Hobbs H.H.
      ), 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 (
      • Yu L.
      • Li-Hawkins J.
      • Hammer R.E.
      • Berge K.E.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      ). 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 (
      • Yu L.
      • Hammer R.E.
      • Li-Hawkins J.
      • Von Bergmann K.
      • Lutjohann D.
      • Cohen J.C.
      • Hobbs H.H.
      ). 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 (
      • Schultz 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.
      ,
      • Joseph S.B.
      • Laffitte B.A.
      • Patel P.H.
      • Watson M.A.
      • Matsukuma K.E.
      • Walczak R.
      • Collins J.L.
      • Osborne T.F.
      • Tontonoz P.
      ), which may contribute to LXR agonist-induced expression ofAbca1 in the intestine, liver (Fig. 7), and macrophages (
      • Repa 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.
      ). ABCA1 participates in the efflux of excess cholesterol from peripheral cells to HDL (
      • Brooks-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.
      • Koop B.
      • Pimstone S.
      • Kastelein J.J.
      • Hayden M.R.
      ,
      • Bodzioch M.
      • Orso E.
      • Klucken J.
      • Langmann T.
      • Bottcher A.
      • Diederich W.
      • Drobnik W.
      • Barlage S.
      • Buchler C.
      • Porsch-Ozcurumez M.
      • Kaminski W.E.
      • Hahmann H.W.
      • Oette K.
      • Rothe G.
      • Aslanidis C.
      • Lackner K.J.
      • Schmitz G.
      ,
      • Rust S.
      • Rosier M.
      • Funke H.
      • Real J.
      • Amoura Z.
      • Piette J.C.
      • Deleuze J.F.
      • Brewer H.B.
      • Duverger N.
      • Denefle P.
      • Assmann G.
      ,
      • Bortnick A.E.
      • Rothblat G.H.
      • Stoudt G.
      • Hoppe K.L.
      • Royer L.J.
      • McNeish J.
      • Francone O.L.
      ) and promotes formation of pre-β-HDL particles by hepatocytes (
      • Vaisman B.L.
      • Lambert G.
      • Amar M.
      • Joyce C.
      • Ito T.
      • Shamburek R.D.
      • Cain W.J.
      • Fruchart-Najib J.
      • Neufeld E.D.
      • Remaley A.T.
      • Brewer Jr., H.B.
      • Santamarina-Fojo S.
      ) and possibly enterocytes (
      • Mulligan J.D.
      • Flowers M.T.
      • Tebon A.
      • Bitgood J.J.
      • Wellington C.
      • Hayden M.R.
      • Attie A.D.
      ). 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 (
      • Repa 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.
      ,
      • Plosch T.
      • Kok T.
      • Bloks V.W.
      • Smit M.J.
      • Havinga R.
      • Chimini G.
      • Groen A.K.
      • Kuipers F.
      ) 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 (
      • Yu L.
      • Hammer R.E.
      • Li-Hawkins J.
      • Von Bergmann K.
      • Lutjohann D.
      • Cohen J.C.
      • Hobbs H.H.
      ). 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 (
      • Plosch T.
      • Kok T.
      • Bloks V.W.
      • Smit M.J.
      • Havinga R.
      • Chimini G.
      • Groen A.K.
      • Kuipers F.
      ). 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.

      Acknowledgments

      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.

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