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• W2075952712 abstract "The severe depletion of cholesteryl ester (CE) in adrenocortical cells of apoA-I−/−mice suggests that apolipoprotein (apo) A-I plays an important role in the high density lipoprotein (HDL) CE selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. A recent study showed that apoA-I−/− HDL binds to SR-BI with the same affinity as apoA-I+/+ HDL, butapoA-I−/− HDL has a decreasedV max for CE transfer from the HDL particle to adrenal cells. The present study was designed to determine the basis for the reduced selective uptake of CE fromapoA-I−/− HDL. Variations inapoA-I−/− HDL particle diameter, free cholesterol or phospholipid content, or the apoE or apoA-II content ofapoA-I−/− HDL had little effect on HDL CE selective uptake into Y1-BS1 adrenal cells. Lecithin cholesterol acyltransferase treatment alone or addition of apoA-I toapoA-I−/− HDL alone also had little effect. However, addition of apoA-I to apoA-I−/− HDL in the presence of lecithin cholesterol acyltransferase reorganized the large heterogeneous apoA-I−/− HDL to a more discrete particle with enhanced CE selective uptake activity. These results show a unique role for apoA-I in HDL CE selective uptake that is distinct from its role as a ligand for HDL binding to SR-BI. These data suggest that the conformation of apoA-I at the HDL surface is important for the efficient transfer of CE to the cell. The severe depletion of cholesteryl ester (CE) in adrenocortical cells of apoA-I−/−mice suggests that apolipoprotein (apo) A-I plays an important role in the high density lipoprotein (HDL) CE selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. A recent study showed that apoA-I−/− HDL binds to SR-BI with the same affinity as apoA-I+/+ HDL, butapoA-I−/− HDL has a decreasedV max for CE transfer from the HDL particle to adrenal cells. The present study was designed to determine the basis for the reduced selective uptake of CE fromapoA-I−/− HDL. Variations inapoA-I−/− HDL particle diameter, free cholesterol or phospholipid content, or the apoE or apoA-II content ofapoA-I−/− HDL had little effect on HDL CE selective uptake into Y1-BS1 adrenal cells. Lecithin cholesterol acyltransferase treatment alone or addition of apoA-I toapoA-I−/− HDL alone also had little effect. However, addition of apoA-I to apoA-I−/− HDL in the presence of lecithin cholesterol acyltransferase reorganized the large heterogeneous apoA-I−/− HDL to a more discrete particle with enhanced CE selective uptake activity. These results show a unique role for apoA-I in HDL CE selective uptake that is distinct from its role as a ligand for HDL binding to SR-BI. These data suggest that the conformation of apoA-I at the HDL surface is important for the efficient transfer of CE to the cell. It is well established that the risk of developing coronary heart disease is inversely proportional to plasma HDL 1The abbreviations used are: HDL, high density lipoprotein; apo, apolipoprotein; LCAT, lecithin cholesterol acyltransferase; SR-BI, scavenger receptor class B, type I; CE, cholesteryl ester; FC, free cholesterol; PL, phospholipid; NDGGE, nondenaturing gradient gel electrophoresis; ACTH, adrenocorticotropic hormone; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); PBS-E, NaCl, potassium phosphate, and EDTA; COE, cholesteryl oleoyl ether; DLT, dilactitol tyramine; BSA, bovine serum albumin; TC, total cholesterol cholesterol levels (1Castelli W.P. Garrison R.J. Wilson P.W. Abbott R.D. Kalousdian S. Kannel W.B. JAMA. 1986; 256: 2835-2838Crossref PubMed Scopus (2091) Google Scholar). Decreased levels of apoA-I, the major protein of HDL, are also associated with an increased risk for coronary heart disease (2Miller N.E. Am. Heart J. 1987; 113: 589-597Crossref PubMed Scopus (404) Google Scholar). The most widely accepted model explaining the anti-atherogenic properties of apoA-I is reverse cholesterol transport (3Glomset J.A. J. Lipid Res. 1968; 9: 155-167Abstract Full Text PDF PubMed Google Scholar). In this process, poorly lipidated apoA-I first removes excess free cholesterol (FC) from peripheral cells through a mechanism dependent on ABCA1 (4Oram J.F. Biochim. Biophys. Acta. 2000; 1529: 321-330Crossref PubMed Scopus (205) Google Scholar). The apoA-I then acts as a co-factor for LCAT, which transforms the FC to cholesteryl ester (CE). This event initiates the conversion of the poorly lipidated apoA-I to a spherical HDL particle. After remodeling by plasma enzymes including cholesteryl ester transfer protein, hepatic lipase, and phospholipid transfer protein, the HDL finally delivers its CE either to the liver, where it can be excreted or repackaged into new lipoproteins, or to ovaries, testes, and adrenal glands, where it can be used in the production of steroid hormones. ApoA-I has a number of important roles in HDL metabolism including activation of LCAT, determination of plasma HDL cholesterol levels, and interaction with the ABCA1 transporter and the HDL receptor, SR-BI. Mice deficient in apoA-I have a 70% reduction in total plasma cholesterol and HDL cholesterol (5Li H. Reddick R.L. Maeda N. Arterioscler. Thromb. 1993; 13: 1814-1821Crossref PubMed Scopus (163) Google Scholar, 6Williamson R. Lee D. Hagaman J. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar, 7Plump A.S. Azrolan N. Odaka H. Wu L. Jiang X. Tall A. Eisenberg S. Breslow J.L. J. Lipid Res. 1997; 38: 1033-1047Abstract Full Text PDF PubMed Google Scholar), a 75% reduction in LCAT activity (8Parks J.S. Li H. Gebre A.K. Smith T.L. Maeda N. J. Lipid Res. 1995; 36: 349-355Abstract Full Text PDF PubMed Google Scholar), and a severe depletion of cholesteryl ester stores in steroidogenic tissues (9Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar). In these animals, the cortical cells of the adrenal gland, the luteal and interstitial cells of the ovary, and the Leydig cells of the testis all display diminished CE content, indicating that apoA-I is important for the SR-BI-mediated HDL CE selective uptake process (10Glass C. Pittman R.C. Civen M. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar, 11Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor Jr., C.A. J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar). This deficiency appears to be directly attributable to the absence of apoA-I because apoA-II-deficient mice have a similar reduction in HDL cholesterol but do not show reduced CE reserves in their adrenals, ovaries, and testes (9Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar). In a recent study we addressed the role of apoA-I in HDL CE selective uptake by analyzing the structural, chemical, and functional properties of apoA-I+/+ andapoA-I−/− HDL. Compared with theapoA-I+/+ HDL, apoA-I−/− particles were larger, more heterogeneous in size, and enriched in apoA-II, apoCs, apoE, FC, and CE (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Compared withapoA-I+/+ HDL, CE selective uptake fromapoA-I−/− HDL was significantly reduced into Y1-BS1 adrenal cells and Fu5AH hepatoma cells, which naturally express SR-BI, and into ldlA[SR-BI] cells, a Chinese hamster ovary cell line expressing SR-BI from a transfected cDNA. In Y1-BS1 and ldlA[SR-BI] cells, the reduction in HDL CE selective uptake was attributed to a reduced V max for CE transfer to the cell. Interestingly, in both cell types,apoA-I−/− HDL showed a lowerK D for HDL cell association, indicating that the absence of apoA-I did not reduce the affinity of HDL for SR-BI. These findings illustrate that HDL properties necessary for HDL binding to SR-BI are distinct from those properties necessary for the transfer of HDL CE to the cell membrane. Additionally, theV max for endocytic uptake and degradation of HDL did not differ betweenapoA-I+/+ and apoA-I−/−HDL in either cell type. Thus, the absence of apoA-I on HDL particles selectively affected the SR-BI-mediated HDL CE selective uptake pathway. In the current report we have explored the basis for the reduced selective uptake of CE from apoA-I−/− HDL. Variation in HDL particle size, cholesterol to phospholipid ratios, and apolipoprotein compositions had little effect on HDL CE selective uptake into Y1-BS1 adrenal cells. Addition of apoA-I toapoA-I−/− HDL also had little effect. However, addition of apoA-I to apoA-I−/− HDL in the presence of LCAT reorganized HDL structure and produced an HDL particle with enhanced CE selective uptake activity. These data suggest that the conformation of apoA-I at the HDL surface is important for the efficient transfer of CE to the cell. The following reagents used for culturing Y1-BS1 cells were purchased from the listed vendors: poly-d-lysine (Becton Dickinson), heat-denatured fetal bovine serum (Atlanta Biologicals), 100× penicillin/streptomycin/glutamine (Invitrogen), six-well plates (Costar), Cortrosyn (Organon), Ham's F-10 medium, and heat-denatured horse serum (Sigma). Sodium[125I]iodide and [3H]cholesteryl oleoyl ether were acquired from PerkinElmer Life Sciences and Amersham Biosciences, respectively. apoA-I−/−C57BL/6J-Apoa1tm1Unc (6Williamson R. Lee D. Hagaman J. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar) andapoA-I+/+ C57BL/6J mice, andapoA-I−/− and apoA-I+/+mice on an 8:1 FVB/N:C57BL/6 background, were obtained and maintained on a 12-h light/12-h dark cycle with standard rodent chow and waterad libitum (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Housing and experimental procedures were approved by the State University of New York at Stony Brook Committee on Laboratory Animal Resources and the Scripps Research Institute Institutional Animal Care and Use Committee. After an overnight fast, mice were anesthetized and exsanguinated by heart puncture, and blood cells were removed by centrifugation at 2,000 × g for 30 min at 4 °C. If not used immediately, plasma was frozen at −80 °C after adding sucrose to a final concentration of 10%. Plasma was adjusted to 0.05% NaN3, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm EDTA, and lipoproteins in the 1.02–1.21 g/ml density range were isolated by sequential density ultracentrifugation at 1.02 and 1.21 g/ml, respectively. Following dialysis against PBS-E (150 mm NaCl, 10 mmpotassium phosphate, 1 mm EDTA, pH 7.4), lipoproteins were overlaid with argon and stored at 4 °C. Where specified,d 1.02–1.21 g/ml lipoproteins were fractionated on a Superose 6 size exclusion column (Amersham Biosciences) to obtainapoA-I+/+ and apoA-I−/−HDL as described (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Apolipoproteins were examined by separating 7.5 μg of protein on a 4–20% SDS-polyacrylamide gradient gel (Bio-Rad) and visualizing the proteins with 0.1% Coomassie Brilliant Blue R-250. Total cholesterol, free cholesterol, and phospholipid concentrations were measured using commercially available enzymatic kits (Wako). A modified Lowry assay using horse IgG as a standard (Pierce) was employed to determine HDL protein concentration (13Markwell M.A. Hass S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5347) Google Scholar). HDL was double radiolabeled with [3H]cholesteryl oleoyl ether ([3H]COE) and [125I]dilactitol tyramine ([125I]DLT) (14Azhar S. Stewart D. Reaven E. J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar) to yield specific activities of 4–20 cpm/ng of protein for [3H] and 90–200 cpm/ng of protein for [125I] as described (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). HDL was analyzed by nondenaturing 4–25% polyacrylamide gradient gel electrophoresis followed by staining with 0.1% Coomassie Brilliant Blue R-250 (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Alternatively, HDL apolipoproteins were transferred to a nitrocellulose membrane using a Trans-Blot Cell (Bio-Rad) run at 100 V for 90 min. The membrane was blocked for 1 h at room temperature with 5% nonfat dried milk in 150 mm NaCl, 20 mm Tris, pH 7.4, 0.05% Tween 20, and incubated overnight at 4 °C with either rabbit anti-mouse apoA-I (Biodesign) or rabbit anti-mouse apoE antiserum (15Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Crossref PubMed Scopus (109) Google Scholar) diluted 1:5,000 in 150 mmNaCl, 20 mm Tris, pH 7.4, 0.2% Tween 20. The blot was washed with 150 mm NaCl, 20 mm Tris, pH 7.4, 0.05% Tween 20, probed with 1:10,000 goat anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature, and washed again. Immunoreactive proteins were visualized by exposing X-Omat Blue XB-1 film (Eastman Kodak Co.) to the membrane, which had been treated with SuperSignal-West Pico chemiluminescent substrate (Pierce). Lipid-free mouse apoA-I was isolated with a modification of a previously published protocol (16Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Biochim. Biophys. Acta. 1983; 750: 353-364Crossref PubMed Scopus (76) Google Scholar). HDL (1.063–1.21 g/ml) was isolated fromapoA-I+/+ FVB/N mouse plasma using sequential density ultracentrifugation. Following dialysis against PBS-E, HDL was incubated in 3 m guanidine HCl for 2 h at 37 °C, dialyzed against PBS-E, and subjected to ultracentrifugation (39,000 rpm, 19 h, 15 °C) at d = 1.21 g/ml in a SW60 Ti rotor. HDL remnants (d < 1.21 g/ml) were separated from the lipid-free apoA-I (d > 1.21) using a tube slicer. ApoA-I was dialyzed against PBS-E and stored under argon at 4 °C. Because of the tendency of the protein to form multimers at concentrations above 1 mg/ml, the apoA-I was not concentrated after the final dialysis step (17Gong E.L. Tan C.S. Shoukry M.I. Rubin E.M. Nichols A.V. Biochim. Biophys. Acta. 1994; 1213: 335-342Crossref PubMed Scopus (13) Google Scholar). Lipoproteins (1.02–1.21 g/ml) radiolabeled with [3H]COE were separated using a Superose 6 column run at a flow rate of 0.4 ml/min. Following the initial injection of the sample, fractions were collected from 33–35.6, 35.6–37, and 37–40 min for apoA-I+/+ HDL and from 31–33.3, 33.3–35.3, and 35.3–40 min forapoA-I−/− HDL. Each fraction (10 μg of total cholesterol) was analyzed on a nondenaturing 4–25% polyacrylamide gradient gel as described above. The HDL apolipoproteins were then visualized using 0.1% Coomassie Brilliant Blue R-250. After concentrating the HDL fractions using a Centricon 50 (Millipore), HDL apolipoproteins were radiolabeled using [125I]DLT as described above. Using 12% SDS-PAGE, the apolipoprotein complement of the [3H]COE-[125I]DLT HDL was analyzed by separating an equal number of [125I] counts from each fraction. Following fixation of the gel with 40% methanol, 10% acetic acid, the [125I]DLT-labeled apolipoproteins were visualized by PhosphorImager analysis (AmershamBiosciences). Plasma was isolated from FVB/N mice as described above and subjected to ultracentrifugation (39,000 rpm, 24 h, 15 °C) atd = 1.21 g/ml in a SW41 Ti rotor. The d> 1.21 fraction was washed by ultracentrifugation (39,000 rpm, 24 h, 15 °C) at d = 1.21 g/ml and dialyzed against PBS-E supplemented with 0.02% NaN3. [3H] COE apoA-I+/+ andapoA-I−/− HDL (1.5 mg of protein) were incubated at 37 °C for 16 h in 5.5 ml of PBS-E containing 50% by volume d > 1.21 g/ml plasma or d > 1.21 g/ml plasma plus 0.3 mg/ml lipid-free apoA-I, respectively. For controls, HDL (1.5 mg of protein) was incubated under similar conditions in PBS-E alone. The density of the samples was adjusted to 1.21 g/ml with KBr, and HDL was isolated by ultracentrifugation (39,000 rpm, 19 h, 15 °C) in a SW41 Ti rotor. The HDL was dialyzed against PBS-E, and apolipoproteins were radiolabeled using [125I]DLT as described above. Recombinant human LCAT was purified as previously described and stored at −70 °C in 50 mm imidazole, 10% glycerol (18Chisholm J.W. Gebre A.K. Parks J.S. J. Lipid Res. 1999; 40: 1512-1519Abstract Full Text Full Text PDF PubMed Google Scholar). LCAT in the presence of fatty acid-free bovine serum albumin (BSA; Intergen) was concentrated at 4 °C using an Ultrafree-15 centrifugal filter device with a 50,000 M r cut-off (Millipore). The final concentrations of the LCAT and the BSA were 1.1–1.4 × 105 units (nmol of CE formed/h/μg of enzyme)/ml and 60 mg/ml, respectively. [3H]COEapoA-I−/− HDL (1.2 mg of protein) was incubated at 37 °C for 24 h in 2.3 ml of reaction buffer (150 mm NaCl, 10 mm potassium phosphate, 1 mm EDTA, 0.05% NaN3, 5 mmimidazole, 1% glycerol, pH 7.4) containing 12,000 LCAT units/ml plus or minus 0.45 mg/ml lipid-free apoA-I. For controls, [3H]COE apoA-I+/+ andapoA-I−/− HDL (1.2 mg of protein) were incubated under the same conditions in reaction buffer supplemented with 6 mg/ml BSA. The sample was adjusted to 1.21 g/ml with KBr, and HDL was isolated by ultracentrifugation (39,000 rpm, 19 h, 15 °C) in a SW60 Ti rotor. Following dialysis against PBS-E, HDL were then radiolabeled using [125I]DLT as described above. Using particles that were only [3H]COE-radiolabeled, the total cholesterol concentrations of the HDL samples were determined using the Cholesterol CII enzymatic assay (Wako). An equal amount of each sample was analyzed by 12% SDS-polyacrylamide electrophoresis (5 μg of total cholesterol) and nondenaturing 4–25% polyacrylamide gradient gels (5 and 10 μg of total cholesterol) as described above. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 or evaluated by Western blot analysis as described above. Similar conditions were used to modify and analyze other HDL particles with the following exceptions. For one study, the [3H]COE apoA-I−/− HDL were incubated with 0.45 mg/ml lipid-free apoA-I in the absence or presence of 12,000 LCAT units/ml. Y1-BS1 cells were maintained in a 37 °C humidified 95% air, 5% CO2atmosphere in Ham's F-10 complete medium (12.5% heat-denatured horse serum, 2.5% heat-denatured fetal bovine serum, 2 mmglutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin). For experiments, the cells were seeded at a density of 1.5 × 106 cells/well into six-well plates, which had been treated with 100 μg/ml poly-d-lysine. After 48 h, medium was replaced with Ham's F-10 complete medium plus 100 nmCortrosyn, a 1–24ACTH synthetic analogue. All studies were conducted following a 24-h exposure to ACTH. After being seeded and treated as listed above, Y1-BS1 cells were washed and the medium replaced with serum-free Ham's F-10 medium. [3H]COE-[125I]DLTapoA-I+/+ or apoA-I−/−HDL was added to the final concentration specified in the figure legends. Following a 4-h incubation at 37 °C, the cells were washed three times with phosphate-buffered saline plus 0.1% BSA, pH 7.4; one time with phosphate-buffered saline, pH 7.4; lysed with 1.25 ml of 0.1n NaOH; and passed five times through a 28.5-gauge needle. The lysate was then processed to determine trichloroacetic acid-soluble and -insoluble 125I radioactivity and organic solvent-extractable 3H radioactivity as described (14Azhar S. Stewart D. Reaven E. J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 19Azhar S. Tsai L. Reaven E. Biochim. Biophys. Acta. 1990; 1047: 148-160Crossref PubMed Scopus (62) Google Scholar). Trichloroacetic acid-insoluble 125I radioactivity represents cell-associated HDL apolipoprotein that is the sum of cell surface-bound apolipoprotein and endocytosed apolipoprotein that is not yet degraded. Trichloroacetic acid-soluble 125I radioactivity represents endocytosed and degraded apolipoprotein that is trapped in lysosomes as a result of the dilactitol tyramine label (14Azhar S. Stewart D. Reaven E. J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 20Glass C.K. Pittman R.C. Keller G.A. Steinberg D. J. Biol. Chem. 1983; 258: 7161-7167Abstract Full Text PDF PubMed Google Scholar). The sum of the 125I-degraded and125I-cell-associated undegraded apolipoprotein expressed as CE equivalents was subtracted from the CE measured as extractable3H radioactivity to give the selective uptake of HDL-CE (14Azhar S. Stewart D. Reaven E. J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 19Azhar S. Tsai L. Reaven E. Biochim. Biophys. Acta. 1990; 1047: 148-160Crossref PubMed Scopus (62) Google Scholar). Values for these parameters are expressed as nanograms of HDL-CE/mg of cell protein. Previous studies with reconstituted or modified HDL suggest that specific apolipoproteins, particularly apoA-II and apoE, may alter the efficiency of SR-BI-mediated HDL CE selective uptake. However, there is no clear consensus in the literature as to whether these proteins have inhibitory or stimulatory effects (21Richard B.M. Pittman R.C. J. Lipid Res. 1993; 34: 571-579Abstract Full Text PDF PubMed Google Scholar, 22Rinninger F. Kaiser T. Windler E. Greten H. Fruchart J.C. Castro G. Biochim. Biophys. Acta. 1998; 1393: 277-291Crossref PubMed Scopus (33) Google Scholar, 23Pilon A. Briand O. Lestavel S. Copin C. Majd Z. Fruchart J.C. Castro G. Clavey V. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1074-1081Crossref PubMed Scopus (44) Google Scholar, 24Arai T. Rinninger F. Varban L. Fairchild-Huntress V. Liang C.P. Chen W. Seo T. Deckelbaum R. Huszar D. Tall A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12050-12055Crossref PubMed Scopus (74) Google Scholar, 25De Beer M.C. Durbin D.M. Cai L. Mirocha N. Jonas A. Webb N.R. De Beer F.C. van der Westhuyzen D.R. J. Biol. Chem. 2001; 276: 15832-15839Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To compareapoA-I−/− HDL with different apoA-II and apoE contents, HDL were isolated from mice on FVB/N or C57BL/6 genetic backgrounds. Previous studies showed thatapoA-I−/−HDL from C57BL/6 mice are enriched in apoE (5Li H. Reddick R.L. Maeda N. Arterioscler. Thromb. 1993; 13: 1814-1821Crossref PubMed Scopus (163) Google Scholar, 7Plump A.S. Azrolan N. Odaka H. Wu L. Jiang X. Tall A. Eisenberg S. Breslow J.L. J. Lipid Res. 1997; 38: 1033-1047Abstract Full Text PDF PubMed Google Scholar), and we noted that HDL from FVB/N mice are enriched in apoA-II. The SDS-PAGE analysis in Fig. 1 A shows the relative enrichment of FVB/N apoA-I−/− HDL in apoA-II in comparison to the enrichment of C57BL/6apoA-I−/− HDL in apoE. Each of these HDLs along with the respective apoA-I+/+ HDLs were labeled with [3H]COE and [125I]DLT and tested in a standard selective uptake assay using ACTH-treated Y1-BS1 adrenocortical cells in which HDL CE selective uptake is primarily the result of SR-BI (26Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13600-13605Crossref PubMed Scopus (210) Google Scholar). Comparing particles isolated from mice of the same genetic strain, ∼2-fold more selective CE uptake was observed from apoA-I+/+ than fromapoA-I−/− HDL (Fig. 1 D). Additionally, HDL CE selective uptake was similar when comparingapoA-I−/− HDL of both strains. This result indicates that apoA-I−/− HDL, regardless of its apoA-II or apoE content, is less efficient than wild type HDL at selectively transferring its CE to the Y1-BS1 cells. Interestingly, the Y1-BS1 cells displayed similar HDL-CE cell association and degradation of the different particles with the exception of the C57BL/6apoA-I−/− HDL (Fig. 1, B andC). The 2-fold increase in these parameters for the C57BL/6apoA-I−/− HDL is likely a result of this apoE-rich HDL being bound and internalized by proteoglycans or members of the LDL receptor family (27Mahley R.W. Ji Z.-S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast, differences in the apoA-II and apoE content of the apoA-I−/− HDL did not alter the ability of the Y1-BS1 cells to internalize CE via SR-BI-dependent selective uptake. Several studies have shown that HDL particle size affects the ability of cells to selectively internalize HDL-CE (28Pittman R.C. Glass C.K. Atkinson D. Small D.M. J. Biol. Chem. 1987; 262: 2435-2442Abstract Full Text PDF PubMed Google Scholar, 29Liadaki K.N. Liu T. Xu S. Ishida B.Y. Duchateaux P.N. Krieger J.P. Kane J. Krieger M. Zannis V.I. J. Biol. Chem. 2000; 275: 21262-21271Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 30De Beer M.C. Durbin D.M. Cai L. Jonas A. De Beer F.C. van der Westhuyzen D.R. J. Lipid Res. 2001; 42: 309-313Abstract Full Text Full Text PDF PubMed Google Scholar). To test whether the larger size and heterogeneity of theapoA-I−/− HDL (5Li H. Reddick R.L. Maeda N. Arterioscler. Thromb. 1993; 13: 1814-1821Crossref PubMed Scopus (163) Google Scholar, 12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) may explain its reduced selective uptake activity, [3H]COE-labeledapoA-I+/+ and apoA-I−/−HDL were separated into three size fractions by gel exclusion chromatography (Fig. 2 A). Analysis by nondenaturing gradient gel electrophoresis indicated that the HDL was separated into fractions with different mean particle diameters (Fig. 2 B). Following radiolabeling of the HDL with [125I]DLT, the apolipoprotein complement of each fraction was determined by SDS-PAGE (Fig. 2 C). As observed previously (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), apoA-II was enriched on smaller particles and apoE on larger particles of apoA-I−/− HDL. The functional properties of the size-fractionated HDL were tested on ACTH-treated Y1-BS1 cells. Although the two types of unfractionated particles were bound and degraded to similar extents (Fig. 3, A and B, T columns), more selective CE uptake was seen withapoA-I+/+ compared with theapoA-I−/− total HDL fraction (Fig. 3 C, T columns)). In contrast, cell association and degradation of the fractionated particles increased in proportion to their diameter for both apoA-I+/+ andapoA-I−/− HDL (Fig. 3, A andB). These differences among the size-fractionated particles likely reflect the larger, apoE-rich HDL interacting with LDL receptor family members or proteoglycans. In contrast, little difference was seen for HDL CE selective uptake among the size-fractionated particles for either apoA-I+/+ orapoA-I−/− HDL (Fig. 3 C). Additionally, more selective CE uptake was seen fromapoA-I+/+ thanapoA-I−/− HDL when particles of similar diameter were compared. For instance, the Y1-BS1 cells selectively internalized more HDL CE from apoA-I+/+ fraction 2 than from the similar sized apoA-I−/−fraction 3. Similar results were obtained with size-fractionated particles from two independent HDL preparations. Several conclusions can be drawn. First, size subpopulations within theapoA-I+/+ and apoA-I−/−HDL do not differentially transfer CE to the Y1-BS1 cells by selective uptake. Second, in agreement with the experiments comparing FVB/N and C57BL/6 apoA-I−/− HDL (Fig. 1), selective CE uptake from apoA-I−/− HDL is not significantly affected by the apoA-II and apoE content. Third, the larger size and heterogeneity of apoA-I−/− HDL are not responsible for the diminished SR-BI-mediated selective CE uptake. In a previous analysis, we noted that apoA-I−/− HDL has a significantly higher FC content than apoA-I+/+HDL, a factor that may reduce the fluidity of the PL monolayer and hinder SR-BI-mediated transfer of CE from the HDL core (12Temel R.E. Walzem R.L. Banka C.L. Williams D.L. J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To test the importance of the HDL FC content for CE selective uptake, [3H]COE apoA-I−/− HDL was incubated with apoA-I+/+ d > 1.21 g/ml plasma, which acted as a source of LCAT, and lipid-free mouse apoA-I. Radiolabeled apoA-I+/+ HDL was treated in a similar fashion but without the addition of apoA-I. Incubation with d > 1.21 g/ml plasma resulted in significant reductions in the FC content of both apoA-I+/+and apoA-I−/− HDL (Table I). In addition, apoA-I associated with the apoA-I−/− particles (data not shown). After radiolabeling with [125I]DLT, the HDL were incubated with ACTH-treated Y1-BS1 cells. The selective CE uptake from the particles exposed to the d > 1.21 g/ml plasma was significantly increased compared with their respective mock-treated controls (Table I). This result suggested that the high FC content of the apoA-I−/− HDL may impede selective transfer of CE to Y1-BS1 cells. However, the functional properties of the apoA-I−/− HDL may also may have been altered by the acquisition of apoA-I or modification by enzymes other than LCAT in the d > 1.21 g/ml plasma.Table IImpact of d > 1.21 g/ml mouse plasma on apoA-I+/+ and apoA-I−/− HDLHDLTreatmentFC/PTSelective CE uptakeng HDL-CE/mg cell proteinapoA-I+/+Mock0.1241170 ± 33apoA-I+/+d > 1.21 g/ml0.0581410 ± 531-ap < 0.01 compared to respective mock control.apoA-I−/−Mock0.27432 ± 68apoA-I−/−d > 1.21 g/ml + apoA-I0.185871 ± 331-ap < 0.01 compared to respective mock control.ACTH-treated Y1-BS1 cells were incubated with 25 μg of CE/ml of [3H]COE-[125I]DLT apoA-I +/+ andapoA-I −/− HDL for 4 h at 37 °C, and the amount of selective CE uptake was determined as" @default.
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• W2075952712 title "Enhancement of Scavenger Receptor Class B Type I-mediated Selective Cholesteryl Ester Uptake fromapoA-I High Density Lipoprotein (HDL) by Apolipoprotein A-I Requires HDL Reorganization by Lecithin Cholesterol Acyltransferase" @default.
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• W2075952712 doi "https://doi.org/10.1074/jbc.m208160200" @default.
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