Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL
2008; Elsevier BV; Volume: 49; Issue: 11 Linguagem: Inglês
10.1194/jlr.m800241-jlr200
ISSN1539-7262
AutoresAnny Mulya, Ji‐Young Lee, Abraham K. Gebre, Elena Boudyguina, Soon-Kyu Chung, Thomas L. Smith, Perry L. Colvin, Xian-Cheng Jiang, John S. Parks,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoWe investigated the in vivo metabolic fate of pre-β HDL particles in human apolipoprotein A-I transgenic (hA-ITg) mice. Pre-β HDL tracers were assembled by incubation of [125I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-β HDLs of increasing size (pre-β1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-ITg-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-β2, -3, and -4 had similar plasma die-away rates, whereas pre-β1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-β HDL size increased. In plasma, pre-β1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-β3 and -4 were remodeled into smaller HDLs. Pre-β HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-β HDL particle size increases. We investigated the in vivo metabolic fate of pre-β HDL particles in human apolipoprotein A-I transgenic (hA-ITg) mice. Pre-β HDL tracers were assembled by incubation of [125I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-β HDLs of increasing size (pre-β1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-ITg-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-β2, -3, and -4 had similar plasma die-away rates, whereas pre-β1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-β HDL size increased. In plasma, pre-β1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-β3 and -4 were remodeled into smaller HDLs. Pre-β HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-β HDL particle size increases. HDLs are the smallest lipoprotein (7–12 nm in diameter) and the most dense (1.063–1.21 g/ml) of the plasma lipoprotein particles and consist of a surface monolayer of protein, phospholipid (PL), and free cholesterol surrounding a hydrophobic core of triglyceride (TG) and cholesteryl ester (CE) (1Atkinson D. Small D.M. Recombinant lipoproteins: implications for structure and assembly of native lipoproteins.Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 403-456Crossref PubMed Scopus (136) Google Scholar). Apolipoprotein A-I (apoA-I) is the predominant apolipoprotein on HDLs, representing 80–90% of the total protein (2Eisenberg S. High density lipoprotein metabolism.J. Lipid Res. 1984; 25: 1017-1058Abstract Full Text PDF PubMed Google Scholar). Interest in understanding HDL metabolism stems from the well-documented fact that plasma HDL cholesterol concentrations are inversely associated with coronary heart disease risk (3Gordon D.J. Probstfield J.L. Garrison R.J. Neaton J.D. Castelli W.P. Knoke J.D. Jacobs Jr., D.R. Bangdiwala S. Tyroler H.A. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2660) Google Scholar). This inverse association is most likely due to the role of HDLs in reverse cholesterol transport (RCT), a process in which HDLs transport excess cholesterol from peripheral tissue to the liver for secretion into bile and, ultimately, for excretion in the feces (4Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). However, other functions of HDL may result in protection against development of coronary heart disease, including protecting plasma LDLs from oxidation (5Nofer J.R. Kehrel B. Fobker M. Levkau B. Assmann G. von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport.Atherosclerosis. 2002; 161: 1-16Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar), decreasing inflammation (6Xia P. Vadas M.A. Rye K.A. Barter P.J. Gamble J.R. High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. A possible mechanism for protection against atherosclerosis by HDL.J. Biol. Chem. 1999; 274: 33143-33147Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and improving vascular function (7Yuhanna I.S. Zhu Y. Cox B.E. Hahner L.D. Osborne-Lawrence S. Lu P. Marcel Y.L. Anderson R.G. Mendelsohn M.E. Hobbs H.H. et al.High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.Nat. Med. 2001; 7: 853-857Crossref PubMed Scopus (645) Google Scholar). HDLs are a heterogeneous mixture of discrete-sized particles that can be separated by density (8Anderson D.W. Nichols A.V. Forte T.M. Lindgren F.T. Particle distribution of human serum high density lipoproteins.Biochim. Biophys. Acta. 1977; 493: 55-68Crossref PubMed Scopus (157) Google Scholar), size (9Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (443) Google Scholar), electrophoretic mobility (10Kunitake S.T. La Sala K.J. Kane J.P. Apolipoprotein A-I-containing lipoproteins with pre-beta electrophoretic mobility.J. Lipid Res. 1985; 26: 549-555Abstract Full Text PDF PubMed Google Scholar), and apolipoprotein content (11Cheung M.C. Albers J.J. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II.J. Biol. Chem. 1984; 259: 12201-12209Abstract Full Text PDF PubMed Google Scholar). Analysis of plasma HDL particles by agarose gel electrophoresis has shown that most plasma HDLs migrate in the α position (90–95% of total HDL in normal human plasma), whereas only 5–10% migrate in the pre-β position (designated as pre-β HDL) (12O'Connor P.M. Zysow B.R. Schoenhaus S.A. Ishida B.Y. Kunitake S.T. Naya-Vigne J.M. Duchateau P.N. Redberg R.F. Spencer S.J. Mark S. et al.Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender.J. Lipid Res. 1998; 39: 670-678Abstract Full Text Full Text PDF PubMed Google Scholar). The term pre-β HDL has evolved to describe any lipoprotein that migrates in the pre-β position on agarose gel electrophoresis, including lipid-free apoA-I, lipid-poor HDL (containing only a few molecules of lipid), and discoidal HDLs (containing PL, cholesterol, and apoA-I, but no core lipid) (13Rye K.A. Barter P.J. Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 421-428Crossref PubMed Scopus (262) Google Scholar, 14Mulya A. Lee J.Y. Gebre A.K. Thomas M.J. Colvin P.L. Parks J.S. Minimal lipidation of pre-beta HDL by ABCA1 results in reduced ability to interact with ABCA1.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1828-1836Crossref PubMed Scopus (106) Google Scholar). Lipid-poor and discoidal HDLs are referred to as nascent HDLs because they must undergo maturation processes that ultimately result in their conversion into mature, spherical plasma HDLs containing a core of hydrophobic lipid (i.e., CE and TG). Although pre-β HDLs are minor constituents of plasma HDL, there is an intense interest in how these particles are formed and catabolized, inasmuch as several studies have suggested that pre-β HDLs are the initial acceptors of peripheral tissue cholesterol in RCT (15Castro G.R. Fielding C.J. Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein.Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar, 16Huang Y. von Eckardstein A. Assmann G. Cell-derived unesterified cholesterol cycles between different HDLs and LDL for its effective esterification in plasma.Arterioscler. Thromb. 1993; 13: 445-458Crossref PubMed Scopus (111) Google Scholar). Despite the potential importance of pre-β HDLs in RCT, little is known about their in vivo metabolism. We previously isolated a pre-β HDL fraction from plasma of human apoA-I transgenic (hA-ITg) mice using a combination of anti-human apoA-I immunoaffinity and size-exclusion chromatography and investigated its in vivo metabolism in hA-ITg mice (17Lee J.Y. Lanningham-Foster L. Boudyguina E.Y. Smith T.L. Young E.R. Colvin P.L. Thomas M.J. Parks J.S. Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice.J. Lipid Res. 2004; 45: 716-728Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whose plasma HDL size heterogeneity resembles that in human plasma (18Chajek-Shaul T. Hayek T. Walsh A. Breslow J.L. Expression of the human apolipoprotein A-I gene in transgenic mice alters high density lipoprotein (HDL) particle size distribution and diminishes selective uptake of HDL cholesteryl esters.Proc. Natl. Acad. Sci. USA. 1991; 88: 6731-6735Crossref PubMed Scopus (73) Google Scholar, 19Rubin E.M. Ishida B.Y. Clift S.M. Krauss R.M. Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses.Proc. Natl. Acad. Sci. USA. 1991; 88: 434-438Crossref PubMed Scopus (242) Google Scholar). The pre-β HDL tracer isolated from hA-ITg mouse or human plasma, which was <7.1 nm in diameter, exhibited two metabolic fates in vivo. About 60% of the pre-β HDL tracer was rapidly removed from plasma and catabolized by the kidney, whereas the remainder was rapidly transferred to medium-sized (8.6 nm-diameter) plasma HDL particles, resulting in a slower removal rate from plasma and a preferential uptake and catabolism by the liver instead of the kidney. These results suggested that most pre-β HDLs circulating in plasma might not be nascent particles that are undergoing maturation in plasma but rather are terminal particles ready to be catabolized and incapable of mediating RCT. This concept was further supported by the absence of radiolabeled pre-β HDL in plasma after injection of small or large plasma HDL tracers into nonhuman primates or hA-ITg mice (17Lee J.Y. Lanningham-Foster L. Boudyguina E.Y. Smith T.L. Young E.R. Colvin P.L. Thomas M.J. Parks J.S. Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice.J. Lipid Res. 2004; 45: 716-728Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Colvin P.L. Moriguchi E. Barrett P.H.R. Parks J.S. Rudel L.L. Small HDL particles containing two apoA-I molecules are precursors in vivo to medium and large HDL particles containing three and four apoA-I molecules in nonhuman primates.J. Lipid Res. 1999; 40: 1782-1792Abstract Full Text Full Text PDF PubMed Google Scholar). The initial and obligatory step in HDL particle assembly is the addition of lipid to apoA-I by ABCA1, a member of the ATP binding cassette transporter family (21Yokoyama S. Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body.Biochim. Biophys. Acta. 2000; 1529: 231-244Crossref PubMed Scopus (117) Google Scholar). The critical nature of this step for nascent HDL biogenesis and for maintaining plasma HDL cholesterol levels is demonstrated in Tangier disease subjects and ABCA1 knockout mice, both characterized by a near absence of plasma HDL (22Fredrickson D.S. The inheritance of high density lipoprotein deficiency (Tangier disease).J. Clin. Invest. 1964; 43: 228-236Crossref PubMed Scopus (80) Google Scholar, 23McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. 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). In a recent study, we showed that incubation of apoA-I with human embryonic kidney 293 cells expressing ABCA1 (HEK293-ABCA1) is necessary and sufficient to produce at least four discrete-sized pre-β HDLs that can be isolated to apparent homogeneity (14Mulya A. Lee J.Y. Gebre A.K. Thomas M.J. Colvin P.L. Parks J.S. Minimal lipidation of pre-beta HDL by ABCA1 results in reduced ability to interact with ABCA1.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1828-1836Crossref PubMed Scopus (106) Google Scholar). These pre-β HDL particles bound poorly with ABCA1 when added back to ABCA1-expressing cells, suggesting that other non-ABCA1-mediated pathways must function to add more lipids and complete the maturation process. The lack of other HDL-modifying proteins [i.e., ATP binding cassette transporter G1 (ABCG1), phospholipid transfer protein (PLTP), LCAT, apoM, or scavenger receptor class B type I (SR-BI)] in the HEK293 cells or conditioned medium of these cells suggested that the pre-β HDLs were nascent, rudimentary HDLs poised for maturation in vivo. The purpose of the present study was to determine the in vivo plasma decay, interconversion, and tissue sites of catabolism for the ABCA1-generated nascent pre-β HDLs. Our results suggest a novel finding that the initial interaction of apoA-I with ABCA1 in vitro determines, in part, the plasma remodeling and tissue site of catabolism of these pre-β HDL particles in vivo. hA-ITg mice (line 427) (24Walsh A. Ito Y. Breslow J.L. High levels of human apolipoprotein A-I in transgenic mice result in increased plasma levels of small high density lipoprotein (HDL) particles comparable to human HDL3.J. Biol. Chem. 1989; 264: 6488-6494Abstract Full Text PDF PubMed Google Scholar) were obtained from Charles River Laboratories (Wilmington, MA). The mice were housed in the Wake Forest University Health Sciences transgenic facility and maintained on a chow diet. All protocols and procedures were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences. Human HDLs were isolated by sequential ultracentrifugation of human plasma, and apoA-I was isolated from HDL by GndHCl denaturation (25Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Anderson D.W. Effects of guanidine hydrochloride on human plasma high density lipoproteins.Biochim. Biophys. Acta. 1976; 446: 226-239Crossref PubMed Scopus (51) Google Scholar, 26Parks J.S. Rudel L.L. Isolation and characterization of high density lipoprotein apoproteins in the non-human primate (vervet).J. Biol. Chem. 1979; 254: 6716-6723Abstract Full Text PDF PubMed Google Scholar). The purity of the apoA-I and phosphorus content of lipid extract from 1 mg of purified apoA-I were confirmed by SDS-PAGE and the method of Fiske and Subbarow (27Fiske C.H. Subbarow Y. Colorimetric determination of phosphorus.J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar), respectively. ApoA-I preparations contained less than one molecule of PL per molecule of apoA-I. ApoA-I was coupled to 125I-radiolabeled tyramine cellobiose (TC) (a generous gift from Dr. Steve Adelman, Wyeth-Ayerst) as previously described (17Lee J.Y. Lanningham-Foster L. Boudyguina E.Y. Smith T.L. Young E.R. Colvin P.L. Thomas M.J. Parks J.S. Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice.J. Lipid Res. 2004; 45: 716-728Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28Pittman R.C. Carew T.E. Glass C.K. Green S.R. Taylor Jr., C.A. Attie A.D. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.Biochem. J. 1983; 212: 791-800Crossref PubMed Scopus (222) Google Scholar, 29Glass C.K. Pittman R.C. Keller G.A. Steinberg D. Tissue sites of degradation of apoprotein A-I in the rat.J. Biol. Chem. 1983; 258: 7161-7167Abstract Full Text PDF PubMed Google Scholar, 30Huggins K.W. Burleson E.R. Sawyer J.K. Kelly K. Rudel L.L. Parks J.S. Determination of the tissue sites responsible for the catabolism of large high density lipoprotein in the African green monkey.J. Lipid Res. 2000; 41: 384-394Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, 0.01 μmol TC/mg apoA-I protein was incubated for 10 min with 5 mCi of 125I (carrier-free) in a microreaction vessel coated with 20 μg Iodogen (1,3,4,6-tetrachloro-3α,6α-dephenylglycouril; Pierce Chemical Co.). The reaction was stopped by transferring the 125I-radiolabeled TC to a second (iodogen-free) reaction vessel containing 10 μl of 0.1 M NaHSO3 and 5 μl of 0.1 M NaI. ApoA-I was coupled to the [125I]TC with cyanuric chloride (1:1 protein to TC molar ratio) by incubation at room temperature for 30 min. The [125I]TC-apoA-I was then passed over a desalting column (Bio-Rad) to remove free iodine and dialyzed overnight in 0.15 M NaCl, 0.01% EDTA, pH 7.4. After removal from dialysis, the tracers were assayed for protein concentration using absorbance at 280 nm (ε = 1.13 ml/mg), and an aliquot was taken for radioactivity quantification. Human embryonic kidney (HEK)-293 ABCA1 (HEK293-ABCA1) (31See R.H. Caday-Malcolm R.A. Singaraja R.R. Zhou S. Silverston A. Huber M.T. Moran J. James E.R. Janoo R. Savill J.M. et al.Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux.J. Biol. Chem. 2002; 277: 41835-41842Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) cells (gift from Dr. Michael Hayden, University of British Columbia) were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml hygromycin B (Invitrogen). HEK293-FlpIn cells were used as negative controls and cultured in DMEM complete media in the presence of 50 μg/ml Zeocin™. Cells from a rat hepatoma cell line (McA-RH7777) were maintained in DMEM media supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Mouse peritoneal macrophages (MPMs) were isolated from the peritoneal cavity of C57BL/6 mice 4 days after intraperitoneal injection of 1 ml of 10% thioglycolate. The cells obtained were washed with Media A (MEM + 10 mM HEPES) (Cellgro; Mediatech, Inc.), spun at 100 g for 20 min, and plated into 6-well plates at a density of 1 × 106 cells/well in MEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1% MEM vitamin solution 100× (Mediatech, Inc.), and 2 mM l-glutamine. Cells were washed 2 h later and incubated overnight before use in experiments. ABCA1 expression among the different cell types (HEK293, HEK293-ABCA1, McA-RH7777, MPM) was determined using Western blot analysis. Total cell protein was harvested using RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% nonidet P-40, 10% sodium cholate, 1 mM EDTA) containing protease inhibitors (1 mM PMSF, 1 μg/ml pepstain, 1 μg/ml leupeptin, 1 μg/ml aprotinin). Cellular proteins (25 μg) were separated on 4–16% SDS-PAGE gels, transferred to nitrocellulose membranes (Schleicher and Schuell BioScience), and incubated for 2 h at room temperature with rabbit anti-human/mouse ABCA1 (1:1,000 dilution) (32Timmins J.M. Lee J.Y. Boudyguina E. Kluckman K.D. Brunham L.R. Mulya A. Gebre A.K. Coutinho J.M. Colvin P.L. Smith T.L. et al.Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.J. Clin. Invest. 2005; 115: 1333-1342Crossref PubMed Scopus (426) Google Scholar) or anti-GAPDH monoclonal antibody (1:2,000; Santa Cruz 32233), which cross-reacts with human, rat, and mouse GAPDH. The blots were then incubated with HRP-linked anti-rabbit IgG or anti-mouse IgG (Amersham) (1:5,000 dilution) at room temperature for 1 h. Immunoblots were visualized with a chemiluminescent reagent (Pierce), and the chemiluminescence was captured with an LAS-3000 imaging system (Fujifilm Life Science) and quantified using Multi Gauge™ software. Control (HEK293-FlpIn) and HEK293-ABCA1 cells were plated in 5 × 150 mm dishes, and McA-RH7777 cells were plated in 6-well plates and grown until they reached 95% confluence. MPMs were cultured for 2 days after isolation before experiments were initiated. All cells (HEK293, McA-RH7777, and MPMs) were washed three times with serum-free medium and then incubated with 10 μg/ml of [125I]TC-apoA-I (specific activity = 5 × 104 cpm/μg) in serum-free medium for 24 h. The conditioned medium from control, ABCA1, McA-RH7777 cells, and MPMs was harvested and aliquots analyzed on 4–30% nondenaturing gradient gel electrophoresis (NDGGE). The remainder of the conditioned media was concentrated using Amicon Ultra-10 concentrators and fractionated on three Superdex 200 HR fast-protein liquid chromatography (FPLC) columns (Amersham-Biosciences) connected in series and equilibrated with 0.15 M NaCl, 0.01% EDTA, pH 7.4 (column buffer). The particles were eluted at a flow rate of 0.3 ml/min. Individual fractions were analyzed for 125I radioactivity and the 125I elution profile was plotted. An aliquot of each fraction of HEK293-ABCA1 cell conditioned media was analyzed by NDGGE to determine which fractions contained homogeneous-sized nascent HDL particles, after which the homogeneous-sized fractions were pooled for subsequent analyses. In vivo turnover studies were performed with [125I]TC-pre-β HDL particles as previously described (17Lee J.Y. Lanningham-Foster L. Boudyguina E.Y. Smith T.L. Young E.R. Colvin P.L. Thomas M.J. Parks J.S. Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice.J. Lipid Res. 2004; 45: 716-728Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) with modifications. Briefly, 1.5–3 × 105 cpm of the radiolabeled tracer was injected into the jugular vein of anesthetized recipient hA-ITg mice. Blood samples were obtained by retro-orbital bleeding at 5 min, 30 min, and 1, 2, 3, 5, and 24 h after dose injection to determine the plasma decay of radiolabeled doses. Radioactivity in a 15 μl sample of plasma was quantified using a γ counter. Aliquots of plasma from the various time points were fractionated on NDGGE for 1,100 V-h at 10°C to determine the fractional distribution of apoA-I radioactivity. After electrophoresis, gels were exposed in a phosphorimager cassette and the images were developed and quantified using a Typhoon 8600 (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (version 5.2). Twenty-four hours after tracer injection, animals were euthanized, tissues were harvested and digested with 1 N NaOH overnight at 60°C, and 125I radioactivity was quantified. The remaining carcasses were digested with 10 g KOH in 150 ml of ethanol for 2–3 days; the digested carcasses were boiled in a water bath until the volume of ethanol reached about 30 ml. All remaining ethanol was counted for 125I radioactivity. Plasma volume was estimated as 3.5% of body weight, and the total amount of radiolabel in plasma at each time point was determined by multiplying the 125I cpm/ml by plasma volume. For the data presented in this study, percentage of injected dose remaining in plasma at each time point was determined by dividing the amount of total radioactivity in plasma by the dose injected × 100%. Percentage of injected dose trapped in the tissue was calculated by dividing the 125I radioactivity in a particular tissue by the dose injected × 100%. Fractional catabolic rate (FCR) values for HDL tracer decay from whole plasma and uptake by tissues were performed using Simulation, Analysis, and Modeling (SAAM) software, as described previously (17Lee J.Y. Lanningham-Foster L. Boudyguina E.Y. Smith T.L. Young E.R. Colvin P.L. Thomas M.J. Parks J.S. Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice.J. Lipid Res. 2004; 45: 716-728Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To investigate the role of LCAT and PLTP in remodeling pre-β HDL particles, we performed an in vitro incubation study using C57BL/6 mouse plasma that had been immunodepleted of LCAT or PLTP−/− mouse plasma. LCAT-immunodepleted plasma was prepared as follows. Preimmune serum and rabbit anti-mouse LCAT antisera were incubated at 56°C for 20 min to inactivate LCAT. After inactivation, either preimmune serum or rabbit anti-mouse LCAT antiserum (0.6 of plasma volume) was added to C57Bl/6 mouse plasma and incubated overnight at 4°C with rotation. LCAT bound to IgG was pelleted by adding Protein A beads to the incubation mixture, followed by a 2 h incubation at 4°C and low-speed centrifugation. The supernatant was recovered and had 1% of the LCAT activity (33Parks J.S. Gebre A.K. Furbee J.W. Lecithin-cholesterol acyltransferase. Assay of cholesterol esterification and phospholipase A2 activities.Methods Mol. Biol. 1999; 109: 123-131PubMed Google Scholar) measured in preimmune serum-treated plasma. Twenty microliters of LCAT immunodepleted, preimmune-treated plasma, or plasma from wild-type (PLTP+/+) or PLTP−/− mice was incubated with [125I]TC radiolabeled pre-β1, -2, -3 and -4 HDL particles (20,000 cpm) at 4°C for 1 h or 37°C for 5 or 60 min. Subsequently, incubated plasma samples were resolved on 4–30% NDGGE and gels were analyzed by phosphorimager analysis. To determine the reactivity of individual pre-β HDL particles with LCAT, pre-β1, -2, -3 and -4 HDLs were prepared using the same protocol as described above, except that cells were radiolabeled with [3H]cholesterol for 24 h before incubation with apoA-I. Aliquots of pre-β1, -2, -3, and -4 HDL (104 dpm of [3H]cholesterol) were then incubated with 50 ng of purified human recombinant LCAT in 0.5 ml of buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 0.01% EDTA, 0.07% NaN3, 0.6% BSA, 2 mM β-mercaptoethanol) at 37°C for 1 h as described previously (33Parks J.S. Gebre A.K. Furbee J.W. Lecithin-cholesterol acyltransferase. Assay of cholesterol esterification and phospholipase A2 activities.Methods Mol. Biol. 1999; 109: 123-131PubMed Google Scholar, 34Chisholm J.W. Gebre A.K. Parks J.S. Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase.J. Lipid Res. 1999; 40: 1512-1519Abstract Full Text Full Text PDF PubMed Google Scholar). After incubation, the samples were lipid-extracted and free cholesterol and CE radiolabels were separated and quantified. Differences among the genotypes of mice were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test to identify individual differences. All statistical analyses were performed using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA). We previously reported that several heterogeneous-sized nascent pre-β HDL particles were generated by incubation of apoA-I with HEK293 cells stably transfected with ABCA1 (14Mulya A. Lee J.Y. Gebre A.K. Thomas M.J. Colvin P.L. Parks J.S. Minimal lipidation of pre-beta HDL by ABCA1 results in reduced ability to interact with ABCA1.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1828-1836Crossref PubMed Scopus (106) Google Scholar). To evaluate whether the formation of these heterogeneous-sized nascent pre-β HDL particles was uniquely related to the level of ABCA1 overexpression in HEK293 cells, we compared ABCA1 expression and HDL particle formation using HEK293 cells stably transfected with human ABCA1, McA-RH7777 cells, and MPMs. Western blot analysis demonstrated that HEK293-ABCA1 cells expressed approximately 1.5- and 2.5-fold more ABCA1 protein compared with McA-RH7777 cells and elicited MPMs, respectively, whereas HEK293 cells had only background levels of ABCA1 expression (Fig. 1A). Despite the relatively modest increased level of ABCA1 protein expression in ABCA1-expressing HEK293 cells, the size distribution of nascent HDL particles was similar among the three cell types, as demonstrated by NDGGE (Fig. 1B) and high-resolution FPLC (Fig. 1C). However, the relative distribution of particles did change among cell types, with McA-RH 7777 cell medium containing a relative enrichment of larger pre-β HDL (i.e., pre-β3 and -4) (Fig. 1B). These results suggested that nascent HDLs assembled by human ABCA1 expressed in HEK293 cells were similar to those assembled by hepatoma cells and macrophages. Nascent pre-β HDL tracers for in vivo turnover studies were generated by incubating [125I]tyramine cellobiose-apoA-I ([125I]TC-apoA-I) with HEK293-ABCA1 cells for 24 h. ApoA-I was coupled with [125I]TC, a tissue-residualizing compound, allowing us to determine tissue sites of catabolism of the injected doses. Following incubation of [125I]TC-apoA-I with HEK293-ABCA-expressing cells, the conditioned medium was fractionated by FPLC, and pre-β HDL elution from the column was monitored by γ counting. As shown in Fig. 1C (black circles), five distinct peaks were eluted from post-fractionation of ABCA1 cell-conditioned medium after FPLC. Fractions for each peak were pooled, as indicated by the vertical dashed lines in Fig. 1C, and designated as pre-β1, -2, -3, -4, and -5, from the smallest to the largest particle size. Aliquots of each pool were subjected to NDGGE, and gels were developed using a phosphorimager (Fig. 1D). These results show that five discrete-sized pre-β HDLs of apparent homogeneity were isolated by the FPLC
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