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• W2074426271 abstract "The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561–39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice. The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561–39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice. Epidemiological studies have firmly established that plasma HDL 1The abbreviations used are: HDLhigh density lipoproteinABCA1ATP binding cassette transporter A1CETPcholesterol ester transport proteinCETP-TgCETP transgenicapoEapolipoprotein EapoAIapolipoprotein AILXRliver X receptorPLTPphospholipid transfer proteinLDLlow density lipoproteinLDLRlow density lipoprotein receptorFPLCfast protein liquid chromatography.1The abbreviations used are: HDLhigh density lipoproteinABCA1ATP binding cassette transporter A1CETPcholesterol ester transport proteinCETP-TgCETP transgenicapoEapolipoprotein EapoAIapolipoprotein AILXRliver X receptorPLTPphospholipid transfer proteinLDLlow density lipoproteinLDLRlow density lipoprotein receptorFPLCfast protein liquid chromatography. cholesterol is inversely correlated to coronary artery events (1.Wierzbicki A.S. Mikhailidis D.P. Curr. Med. Res. Opin. 2002; 18: 36-44Crossref PubMed Scopus (37) Google Scholar). The biogenesis of HDL is thought to originate from the secretion of its major apolipoprotein component, apoAI, from the liver and the small intestine. ApoAI enters the circulation and interacts with and removes excessive free cholesterol from peripheral tissues, forming disc-like nascent HDL particles. ApoAI-dependent phospholipid and cholesterol efflux from peripheral tissues requires the protein ABCA1, an ATP binding cassette transporter (2.Attie A.D. Kastelein J.P. Hayden M.R. J. Lipid Res. 2001; 42: 1717-1726Abstract Full Text Full Text PDF PubMed Google Scholar). The maturation of HDL depends on both lecithin-cholesterol acyltransferase and phospholipid transfer protein (PLTP) activities. The former esterifies free cholesterol to form spherical HDL (3.Eisenberg S. J. Lipid Res. 1984; 25: 1017-1058Abstract Full Text PDF PubMed Google Scholar), and the latter transfers phospholipid from triglyceride-rich lipoproteins into the nascent HDL particles (4.Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O.L. Tall A.R. J. Clin. Invest. 1999; 103: 907-914Crossref PubMed Scopus (319) Google Scholar). Cholesteryl ester transfer protein (CETP) catalyzes transfer of cholesteryl ester from mature HDL into apoB-containing lipoproteins for catabolism through liver low density lipoprotein receptor (LDLR) (5.Tall A.R. Jiang X. Luo Y. Silver D. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1185-1188Crossref PubMed Scopus (116) Google Scholar). HDL cholesterol can also be delivered to the liver via scavenger receptor BI, the HDL receptor, through the process of selective cholesterol uptake (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1974) Google Scholar). It is conceivable that the regulation of ABCA1, lecithin-cholesterol acyltransferase, PLTP, CETP, and scavenger receptor BI would have an important impact on HDL metabolism, including particle catabolic rate, lipid composition, and particle size. high density lipoprotein ATP binding cassette transporter A1 cholesterol ester transport protein CETP transgenic apolipoprotein E apolipoprotein AI liver X receptor phospholipid transfer protein low density lipoprotein low density lipoprotein receptor fast protein liquid chromatography. high density lipoprotein ATP binding cassette transporter A1 cholesterol ester transport protein CETP transgenic apolipoprotein E apolipoprotein AI liver X receptor phospholipid transfer protein low density lipoprotein low density lipoprotein receptor fast protein liquid chromatography. Liver X receptors were initially isolated as orphan nuclear receptors. Two isoforms, LXRα and LXRβ, exist with different expression patterns (7.Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Oxysterols were identified as their native ligands, implicating their role in cholesterol metabolism (8.Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1428) Google Scholar). Disruption of LXRα in mice led to dramatic accumulation of hepatic cholesterol when the mice were fed a high cholesterol diet (9.Peet 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 (1225) Google Scholar). The resulting phenotype was largely from a failure to up-regulate cholesterol 7α-hydroxylase (Cyp7a), the rate-limiting step in the conversion of cholesterol to bile acids. Since then, multiple LXR target genes, including ABCA1 (10.Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar), apoE (11.Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (561) Google Scholar), CETP (12.Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (304) Google Scholar), and PLTP (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 14.Mak P.A. Kast-Woelbern H.R. Anisfeld A.M. Edwards P.A. J. Lipid Res. 2002; 43: 2037-2041Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), have been identified, indicating the critical role of LXR in regulating HDL metabolism. It has also been reported recently that LXRs also play a central role in regulating fatty acid biosynthesis (15.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. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar, 16.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. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1393) Google Scholar), glucose metabolism (17.Cao G. Liang Y. Broderick C.L. Oldham B.A. Beyer T.P. Schmidt R.J. Zhang Y. Stayrook K.R. Suen C. Otto K.A. Miller A.R. Dai J. Foxworthy P. Gao H. Ryan T.P. Jiang X.C. Burris T.P. Eacho P.I. Etgen G.J. J. Biol. Chem. 2003; 278: 1131-1136Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), and inflammation process (18.Fowler A.J. Sheu M.Y. Schmuth M. Kao J. Fluhr J.W. Rhein L. Collins J.L. Willson T.M. Mangelsdorf D.J. Elias P.M. Feingold K.R. J. Invest. Dermatol. 2003; 120: 246-255Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 19.Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Nat. Med. 2003; 9: 213-219Crossref PubMed Scopus (989) Google Scholar). Administration of LXR agonist increases HDL cholesterol (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 15.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. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar) and HDL size in mice (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The enlarged HDL particles are apoE-enriched (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This feature is very much similar to an HDL subpopulation, HDL1 or HDLC (20.Koo C. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1985; 260: 11934-11943Abstract Full Text PDF PubMed Google Scholar). The enlargement of HDL after LXR agonist administration to mice may largely involve the induction of ABCA1 and apoE, since both gene products are intimately involved in HDL metabolism and are regulated by LXRs (10.Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 11.Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (561) Google Scholar, 21.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. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar). ABCA1 deficiency causes hypoalphalipoproteinemia in humans (22.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. et al.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1481) Google Scholar, 23.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. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1328) Google Scholar, 24.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. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1249) Google Scholar, 25.Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (648) Google Scholar) and mice (26.Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. Spruss T. Luciani M.F. Rothe G. Lackner K.J. Chimini G. Schmitz G. Nat. Genet. 2000; 24: 192-196Crossref PubMed Scopus (426) Google Scholar, 27.McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Crossref PubMed Scopus (475) Google Scholar). ABCA1 overexpression in mice leads to an increased plasma HDL cholesterol level (28.Singaraja R.R. Bocher V. James E.R. Clee S.M. Zhang L.H. Leavitt B.R. Tan B. Brooks-Wilson A. Kwok A. Bissada N. Yang Y.Z. Liu G. Tafuri S.R. Fievet C. Wellington C.L. Staels B. Hayden M.R. J. Biol. Chem. 2001; 276: 33969-33979Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 29.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. J. Clin. Invest. 2001; 108: 303-309Crossref PubMed Scopus (219) Google Scholar). It has also been reported that apoE plays an obligatory role in large HDL formation (20.Koo C. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1985; 260: 11934-11943Abstract Full Text PDF PubMed Google Scholar). CETP may be another factor that can influence the formation of large HDL particles. In patients with complete CETP deficiency, HDL is increased in size and enriched in apoE and cholesteryl esters (30.Yamashita S. Sprecher D.L. Sakai N. Matsuzawa Y. Tarui S. Hui D.Y. J. Clin. Invest. 1990; 86: 688-695Crossref PubMed Scopus (148) Google Scholar). Conversely, in CETP transgenic rats, conversion of large HDL to small HDL particles was observed (31.Zak Z. Lagrost L. Gautier T. Masson D. Deckert V. Duverneuil L. De Barros J.P. Le Guern N. Dumont L. Schneider M. Risson V. Moulin P. Autran D. Brooker G. Sassard J. Bataillard A. J. Lipid Res. 2002; 43: 2164-2171Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In order to investigate the contribution of apoE and CETP in the LXR-mediated formation of enlarged HDL particles, we treated CETP transgenic (CETP-Tg), apoE-deficient (apoE–/–), and wild type mice with various doses of LXR agonist, T0901317. We show that in these mice, the enlargement of HDL particle size after administrating the LXR agonist requires apoE and is completely abolished by CETP expression. Animals—Seven-week-old male mice were purchased and acclimated for 1 week prior to the start of the study. C57BL/6 mice were purchased from Harlan (Indianapolis, IN). Human CETP-Tg and apoE-deficient (apoE–/–) mice were purchased from Taconic (Germantown, NY). Mice were provided Purina 5001 food ad libitum, and the compound was dosed once daily via oral gavage for 7 days. Animals were sacrificed by CO2 asphyxiation in the morning, 2 h after the eighth dose; blood samples were taken by cardiac puncture; and tissues were collected and frozen in liquid nitrogen. Lipoprotein Analysis by FPLC—Lipoprotein analysis was performed as described (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Briefly, plasma samples from animals were prepared and pooled. 50 μl of pooled sample was applied to Superose 6 size exclusion columns and eluted with phosphate-buffered saline, pH 7.4. Cholesterol content of different fractions was measured by commercial kit. Lipoprotein Analysis by Nondenaturing Gel Electrophoresis—Five microliters of mouse plasma was loaded on a 4–20% nondenaturing gel (Bio-Rad), which was prerun with TBE buffer (10 mm Tris, 10 mm boric acid, and 1 mm EDTA, pH 7.4) at 4 °C, 50 V, for 1 h. The electrophoresis was carried out at 4 °C and 50 V for 12 h. The gel then was stained with 1% of Oil Red O (in isopropyl alcohol) at 58 °C for 6 h. Analysis of Isolated HDL Particles by Native Gel Electrophoresis— Lipoproteins were purified by sequential ultracentrifugation using a TL 100.4 rotor with a tabletop TL 100 centrifuge (Beckman Instruments). HDL was purified between the densities of 1.063 and 1.210 g/ml in all animals. HDL particles were separated by gradient gel electrophoresis (4–20%; Bio-Rad) under nonreducing and nondenaturing conditions. The electrophoresis was carried out for 3 h, and the gel was stained with Coomassie Brilliant Blue R-250 and destained with a solution of methanol/acetic acid/water (30:58:12, v/v/v). Western Blot Analysis—Plasma samples were separated by FPLC size exclusion columns and different fractions were pooled (fractions 20–23, 24–27, 28–31, 32–36, and 37–41) for apolipoprotein analysis. Designated FPLC fraction samples were separated on Tris/glycine gels (Novex) under denaturing conditions. Protein was transferred to nitrocellulose membrane and then blotted with antibodies to apolipoprotein AI or E (Biodesign) or apolipoprotein B48/100 (U. S. Biological). Blots were developed with ECL Western blotting detection reagents (Amersham Biosciences) and documented using X-Omat film (Eastman Kodak Co.). Isolated HDL fractions were analyzed on a gradient gel (4–20%; Bio-Rad) under reducing and denaturing conditions, followed by a similar procedure as described above. SDS-PAGE Apolipoprotein Analysis—Plasma HDL (density = 1.063–1.21 g/ml) and LDL (density = 1.006–1.063 g/ml) were separated by preparative ultracentrifugation. SDS-PAGE was performed on 3–20% SDS-polyacrylamide gradient gel, and the aplipoproteins were stained by Coomassie Brilliant Blue as described (4.Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O.L. Tall A.R. J. Clin. Invest. 1999; 103: 907-914Crossref PubMed Scopus (319) Google Scholar). Electron Microscopy—Negative stain electron microscopy was done using a JEM-100C electron microscope (JEOL USA Inc., Cranford, NJ). Lipoprotein samples in 125 mm ammonium acetate, 2.6 mm ammonium carbonate, and 0.26 mm EDTA (pH 7.4) were mixed with an equal volume of 2% sodium phosphotungstate (pH 7.4) on a carbon/Formvar-coated copper grid (Electron Microscopy Sciences, Fort Washington, PA). After 30 s, the excess liquid was blotted, the rest was allowed to dry, and the grid was examined within 1 h. mRNA Analysis—Total RNAs were prepared from frozen tissue samples with TRIzol reagent (Invitrogen). Mouse ABCA1 and PLTP mRNA were measured by an RNase protection assay. A primer set was used to amplify a fragment of about 200 base pairs from mouse macrophage RAW cells for ABCA1 probe. The resulting 200-bp fragment was cloned into pGEM-T Easy (Promega) and sequenced. The resulting construct was linearized, and RNA probe was synthesized using the Promega T7/SP6 transcription kit. Specific activity was >108 dpm/μg. After column purification, the probe was used for RPA analysis by using a kit from Ambion. The probe for PLTP mRNA measurement was described previously (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The signal was quantified with an Amersham Biosciences PhosphorImager model 51. Previously, it was observed that treatment of C57BL/6 mice with a specific LXR agonist, T0901317, resulted in an increase of both HDL cholesterol and particle size (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). To investigate the effect of apoE on the formation of enlarged HDL particles, as well as on HDL cholesterol levels, we treated apoE–/– mice with T0901317 at 10 and 50 mg/kg for 7 days. Wild type C57BL/6 mice were treated in a similar scheme for comparison. As reported previously (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), treatment of C57BL/6 mice with 50 mg/kg T0901317 resulted in dramatic increase in HDL cholesterol, as determined by FPLC (Fig. 1A). The HDL fraction included a primary peak (fractions 32–41) as well as a second peak (fractions 28–31), which was largely absent in vehicle-treated C57BL/6 mice. This latter peak overlapped with that of the LDL fraction, based on its size and the presence of apolipoproteins B-48 and B-100 (Fig. 2A). The apoE content of these FPLC elutes was markedly increased by treatment of 50 mg/kg T0901317 (the overall plasma apoE content was increased about 80% as judged by the quantitative Western blots), whereas the apoB level was not significantly changed (Fig. 2A), which confirmed our previous findings (13.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. Eacho P.I. Jiang X.C. J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The apoAI content of both HDL peaks was increased by T0901317.Fig. 2Apolipoprotein analysis of FPLC fractions by Western blots. The fractions eluted from FPLC size exclusion columns were pooled (18–22, 23–28, 29–33, 34–39, and 40–44), and 10-μl samples were applied to Tris/glycine gels under denaturing conditions. Protein was transferred to nitrocellulose membrane and then blotted with antibodies to apolipoprotein AI or E (Biodesign) or apolipoprotein B48/100 (U. S. Biological). Blots were developed with ECL Western blotting detection reagents (Amersham Biosciences) and documented using X-Omat film (Kodak). Samples were from wild type (A), apoE–/– (B), and CETP transgenic mice (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In apoE–/– mice, VLDL/LDL represent major lipoprotein components and HDL represents a minor fraction of the FPLC profile. There was no apparent increase in HDL size or HDL cholesterol after the LXR agonist treatment (Fig. 1B). To further analyze the data, we expanded the FPLC plot that covered fractions 30–42 (Fig. 1B′). No HDL cholesterol increase was observed (fractions 36–42), and some elevation of cholesterol upon LXR agonist treatment (fractions 30–35) was closely associated with a large amount of apoB48 representing remnant lipoprotein particles. In VLDL/LDL fractions 24–27 and 28–31 of the apoE–/– mice, there was a dose-related increase in the cholesterol content (Fig. 1B) and an increase of apoB48 (Fig. 2B). Interestingly, apoAI was present in these fractions, as was observed previously in apoE–/– mice (32.Plump A.S. Breslow J.L. Annu. Rev. Nutr. 1995; 15: 495-518Crossref PubMed Scopus (124) Google Scholar) and was slightly increased by treatment with T0901317 (Fig. 2B). ApoAI level was not changed in fractions 32–36 and appeared to be slightly decreased in fractions 37–41 in response to LXR agonist treatment. Thus, administration of the LXR agonist to apoE–/– mice did not appear to cause HDL particle enlargement or an increase in HDL cholesterol levels. Instead, cholesterol in remnant particles was increased. To investigate the role of CETP in the LXR agonist-mediated changes in HDL cholesterol metabolism, we utilized a transgenic model in which the human CETP transgene was expressed under the control of the human apoAI promoter (33.Grass D.S. Saini U. Felkner R.H. Wallace R.E. Lago W.J. Young S.G. Swanson M.E. J. Lipid Res. 1995; 36: 1082-1091Abstract Full Text PDF PubMed Google Scholar). LXRs are not known to regulate apoAI (15.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. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar), and we found that treatment of these mice with T0901317 did not result in a change in plasma CETP activity (data not shown). Treatment of these mice with T0901317 did not lead to the formation of enlarged HDL particles (Fig. 1C), as was observed in C57BL/6 mice. A small increase in HDL cholesterol was only observed at the higher dose of the compound used. No significant increase of apoAI or apoE in the HDL fraction was observed (Fig. 2C). Thus, CETP expression greatly attenuated the LXR agonist-induced HDL cholesterol increase and abolished the increase in HDL particle size. To further confirm the HDL cholesterol and particle size changes in these different animal models after oral administration of the LXR agonist, we utilized nondenaturing gel electrophoresis to evaluate HDL particles. Mouse plasma samples were applied to the nondenaturing gels (4–20% gradient), which were then stained with Oil Red O after running. HDL particles were readily separated and stained on the gel, whereas VLDL/LDL particles could not get into the gel because of the size. In C57BL/6 mice, T0901317 treatment increased the intensity of Oil Red O staining of HDL in a dose-dependent manner, reflecting an increase in HDL lipid (Fig. 3A). A dose-dependent HDL particle size enlargement was also evident (Fig. 3A). In apoE–/– mice, there was reduced HDL lipid content but no change in particle size after LXR agonist treatment (Fig. 3B). In CETP transgenic mice, LXR agonist administration did not change HDL particle size, and the increase of HDL lipid contents was attenuated (Fig. 3C). Thus, the analysis of HDL cholesterol and particle size with nondenaturing gels confirmed the findings of our FPLC analysis. In order to unequivocally prove the results described above, we physically separated HDL particles from LDL particles by ultracentrifugation. The isolated HDL fractions were subjected to electron microscopic study (Fig. 4A). The HDL particle size from wild type animals was in the range of 10–12 nm in diameter. T0901317 treatment resulted in an obvious enlargement of HDL particles. The size of largest HDL particle reached about 20 nm in diameter. On the contrary, T0901317 treatment in either apoE-deficient mice or CETP transgenic mice did not induce any significant change in HDL particle size (Fig. 4A). The isolated HDL fractions were then analyzed by native gel electrophoresis and Coomassie Blue staining (Fig. 4B). Consistent with electron microscopic data, T0901317 treatment of wild type mice resulted in slower migration of HDL particles in the native gel under nondenaturing conditions, suggesting the increase in HDL particle size. Treatment of apoE-deficient mice or CETP transgenic mice, however, did not result in any observable changes in the mobility of HDL particles. The presence of apoAI and apoE in these HDL particles isolated from ultracentrifugation was further proved with Western blot analysis (Fig. 4C). Increases in both apoAI and apoE content in these HDL particles after T0901317 treatment of wild type mice was evident, whereas no change was observed in either apoE-deficient mice or CETP transgenic mice. We further performed SDS-PAGE apolipoprotein analysis on isolated LDL and HDL from wild type mouse plasma with or without LXR agonist treatment. As can be seen from Fig. 5, the isolated LDL contained significant amounts of apoB48 and apoB100. The isolated HDL particles had no detectable apoB, whereas the LXR agonist treatment greatly increased apoE and apoAI levels. Since HDL particles from LXR agonist-treated animal plasma contained normal HDL and enlarged HDL, the results indicated that the later did not contain any appreciable amount of apoB.Fig. 5SDS-PAGE analysis of apolipoproteins from ultracentrifugally isolated mouse plasma with or without LXR agonist treatment. Plasma HDL (density = 1.063–1.21 g/ml) and LDL (density = 1.006–1.063 g/ml) were separated by preparative ultracentrifugation as described. SDS-PAGE was performed on 3–20% SDS-polyacrylamide gradient gel, and the apolipoproteins were stained by Coomassie Brilliant Blue as described.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the molecular mechanisms of the above-mentioned observations, we analyzed mRNA regulation of two important LXR target genes, ABCA1 and PLTP, in the liver. Treatment of C57BL/6 mice at 50 mg/kg resulted in a 2.7-fold induction of ABCA1 and a 5.3-fold increase of PLTP. In apoE–/– mice, both ABCA1 and PLTP up-regulation was readily detectable (2.4- and 3.7-fold, respectively). In CETP transgenic mice, ABCA1 and PLTP were also up-regulated by LXR agonist (2.3- and 2.5-fold, respectively) (Fig" @default.
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• W2074426271 date "2003-12-01" @default.
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• W2074426271 title "Enlargement of High Density Lipoprotein in Mice via Liver X Receptor Activation Requires Apolipoprotein E and Is Abolished by Cholesteryl Ester Transfer Protein Expression" @default.
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