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• W2113602208 abstract "The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [3H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but3H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1. The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [3H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but3H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1. high density lipoprotein ATP binding cassette transporter A1 adenovirus serotype 5 Ad5 adenoviral construct expressing human apolipoprotein A-I Ad5 adenoviral construct expressing firefly luciferase apolipoprotein endogenously synthesized exogenously added fast protein liquid chromatography human apolipoprotein A-I low density lipoprotein polyacrylamide gradient gel electrophoresis phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine retinoid X receptor peroxisome proliferator-activated receptor sphingomyelin thin layer chromatography very high density lipoprotein very low density lipoprotein. The hepatic and intestinal origins of the major high density lipoprotein (HDL)1apolipoproteins, apolipoprotein (apo)A-I and apoA-II, are well defined (1Eisenberg S. J. Lipid Res. 1984; 25: 1017-1058Abstract Full Text PDF PubMed Google Scholar, 2Zannis V.I. Kardassis D. Cardot P. Hadzopoulou-Cladaras M. Zanni E.E. Cladaras C. Curr. Opin. Lipidol. 1992; 3: 96-113Crossref Scopus (16) Google Scholar). In contrast, HDL lipid constituents have complex and multiple origins that include secretion as nascent lipoproteins containing apoA-I (3McCall M.R. Forte T.M. Shore V.G. J. Lipid Res. 1988; 29: 1127-1137Abstract Full Text PDF PubMed Google Scholar, 4Castle C.K. Pape M.E. Marotti K.R. Melchior G.W. J. Lipid Res. 1991; 32: 439-447Abstract Full Text PDF PubMed Google Scholar), acquisition of lipids from remnant lipoproteins arising from lipolysis of triglyceride-rich lipoproteins (5Patsch J.R. Gotto A.M.J. Olivercrona T. Eisenberg S. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4519-4523Crossref PubMed Scopus (402) Google Scholar, 6Tall A.R. Small D.M. N. Engl. J. Med. 1978; 299: 1232-1236Crossref PubMed Scopus (256) Google Scholar, 7Tam S.P. Breckenridge W.C. J. Lipid Res. 1983; 24: 1343-1357Abstract Full Text PDF PubMed Google Scholar), and from cellular lipid efflux (8Rothblat G.H. Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar). The relative contributions of the different pathways are not well understood, particularly the secretion of nascent lipoproteins and the contribution of the efflux pathway. The ATP-binding cassette transporter, ABCA1, was recently shown to control the efflux of cellular phospholipids and cholesterol (9Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 10Wang N. Silver D.L. Costet P. Tall A.R. J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 11Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 12Fielding P.E. Nagao K. Hakamata H. Chimini G. Fielding C.J. Biochemistry. 2000; 39: 14113-14120Crossref PubMed Scopus (183) Google Scholar) and through this pathway to maintain HDL in the circulation. Impairment of ABCA1, as in Tangier disease, leads to extremely low levels of HDL (13Brooks-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.F. Loubser O. Ouelette B.F.F. Fichter K. Ashbourne-Excoffon K.J.D. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar, 14Bodzioch M. Orsó E. Klucken T. Langmann T. Böttcher L. Diederich W. Drobnik W. Barlage S. Büchler C. Porsch-Özcürümez 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 (1349) Google Scholar, 15Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denèfle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1269) Google Scholar, 16Lawn R.M. Wade D.P. Garvin M.R. Wang X.B. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar). The major tissues affected in this disease are rich in macrophages, which express high levels of ABCA1 (17Lawn R.M. Wade D.P. Couse T.L. Wilcox J.N. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 378-385Crossref PubMed Scopus (104) Google Scholar, 18Singaraja 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.Q. 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 (173) Google Scholar, 19Cavelier L.B. Qiu Y. Bielicki J.K. Afzal V. Cheng J.F. Rubin E.M. J. Biol. Chem. 2001; 276: 18046-18051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This was first interpreted as evidence that excess lipids accumulated in scavenger receptor-expressing cells were a major source of HDL lipids (20Hayden M.R. Clee S.M. Brooks-Wilson A. Genest Jr., J. Attie A. Kastelein J.J.P. Curr. Opin. Lipidol. 2000; 11: 117-122Crossref PubMed Scopus (109) Google Scholar, 21Oram J.F. Vaughan A.M. Curr. Opin. Lipidol. 2000; 11: 253-260Crossref PubMed Scopus (243) Google Scholar), but recent evidence shows that macrophage contribution to HDL-cholesterol concentrations is minor (22Haghpassand M. Bourassa P.A. Francone O.L. Aiello R.J. J. Clin. Invest. 2001; 108: 1315-1320Crossref PubMed Scopus (234) Google Scholar). ABCA1 is expressed in many tissues and at high levels in liver, brain, and small intestine, but also testis, lung, spleen, and kidney (17Lawn R.M. Wade D.P. Couse T.L. Wilcox J.N. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 378-385Crossref PubMed Scopus (104) Google Scholar, 18Singaraja 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.Q. 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 (173) Google Scholar, 19Cavelier L.B. Qiu Y. Bielicki J.K. Afzal V. Cheng J.F. Rubin E.M. J. Biol. Chem. 2001; 276: 18046-18051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This suggests that in both liver and intestine, where apoA-I synthesis is also high, ABCA1 may contribute to the lipidation of newly secreted or nascent lipoproteins. Previously, work with hepatocytes from chicken (23Banerjee D. Redman C.M. J. Cell Biol. 1983; 96: 651-660Crossref PubMed Scopus (30) Google Scholar, 24Banerjee D. Redman C.M. J. Cell Biol. 1984; 99: 1917-1926Crossref PubMed Scopus (27) Google Scholar) or rat (25Howell K.E. Palade G.E. J. Cell Biol. 1982; 92: 833-845Crossref PubMed Scopus (46) Google Scholar) had suggested that apoA-I was lipidated intracellularly. However, Hamilton et al. (26Hamilton R.L. Moorehouse A. Havel R.J. J. Lipid Res. 1991; 32: 529-543Abstract Full Text PDF PubMed Google Scholar), using electron microscopy, failed to identify any lipidated apoA-I particles in hepatocytes, putting the intracellular lipidation hypothesis in dispute. Recently, Chisholm et al. (27Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. J. Lipid Res. 2002; 43: 36-44Abstract Full Text Full Text PDF PubMed Google Scholar) investigated the secretion and lipidation of apoA-I from HepG2 cells. They concluded that some apoA-I acquired lipid intracellularly and was then secreted along with lipid-poor apoA-I. Subsequently, the secreted apoA-I could acquire lipids extracellularly to form buoyant HDL particles. In those studies, which support the model of intracellular lipidation of apoA-I, HDL particles were obtained by carbonate extraction of cell homogenates. Despite careful quantitation and inclusion of controls, mixing of cell contents and artificial lipidation of apoA-I may occur. Here we have characterized the hepatic lipidation of apoA-I by using adenoviral expression of human apoA-I (hapoA-I) (28McManus D.C. Scott B.R. Frank P.G. Franklin V. Schultz J.R. Marcel Y.L. J. Biol. Chem. 2000; 275: 5043-5051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29McManus D.C. Scott B.R. Franklin V. Sparks D.L. Marcel Y.L. J. Biol. Chem. 2001; 276: 21292-21302Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) in primary hepatocytes of apoA-I-deficient and ABCA1-deficient mice. We also characterized the nascent lipoproteins formed by primary hepatocytes cultured in lipoprotein-free medium compared with those formed by interaction of exogenous apoA-I with the same cells. In both conditions hepatocytes generate a lipidated pool of apoA-I-containing lipoproteins via a pathway dependent on ABCA1. However, the lipidation of apoA-I is reduced but not abolished in experiments with ABCA1-deficient hepatocytes, suggesting the existence of alternate lipidation pathways. ApoA-I-deficient (Apoa1tm1Unc) C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). ABCA1-deficient mice were generated according to Hamon et al. (30Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar). The mice were maintained on a 12 h light/12 h dark schedule on a normal chow diet. Primary hepatocytes were prepared from these mice according to established protocols (31Subrahmanyan L. Kisilevsky R. Scand. J. Immunol. 1988; 27: 251-260Crossref PubMed Scopus (23) Google Scholar, 32Thomas S.S. Plenkiewicz J. Ison E.R. Bols M. Zou W. Szarek W.A. Kisilevsky R. Biochim. Biophys. Acta. 1995; 1272: 37-48Crossref PubMed Scopus (35) Google Scholar). Briefly, the cells were seeded in fibronectin-coated (25 μg/well) 6-well plates at an initial density of 1–2 × 106 cells per well in William's medium containing penicillin (100 units/ml), streptomycin sulfate (100 units/ml), Fungizone® (250 ng/ml; Invitrogen) and 10% fetal bovine serum (Sigma). Six h following the initial plating, the cells were washed in William's medium without fetal bovine serum (2 × 2 ml) and incubated with Hepatozyme® medium (Invitrogen) containing 10 μCi/well of [3H]choline (PerkinElmer Life Sciences). The following day (24 h) the labeled medium was removed and the cells were infected for 1 h with either the recombinant hapoA-I encoding Ad5 adenovirus (AdAI) or luciferase adenovirus (AdLuc) at a multiplicity of infection of 75:1 plaque-forming units per cell in William's medium without fetal bovine serum (28McManus D.C. Scott B.R. Frank P.G. Franklin V. Schultz J.R. Marcel Y.L. J. Biol. Chem. 2000; 275: 5043-5051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29McManus D.C. Scott B.R. Franklin V. Sparks D.L. Marcel Y.L. J. Biol. Chem. 2001; 276: 21292-21302Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). After the 1 h infection, the hepatocytes were incubated for an additional 24 h with fresh labeling medium as described above. The third day (18–24 h after adenovirus infection), following 2 × 2 ml washes in non-radioactive medium, the cells were incubated with unlabeled Hepatozyme® medium (1 ml per well) in the absence or presence of 5 μg of hapoA-I. The cells were returned to the 37 °C incubator (5% CO2) for 3.5 h, and the medium was subsequently collected and spun down to pellet any cell debris. The medium with newly secreted apoA-I (AdAI-infected cells) or with exogenously added hapoA-I (AdLuc-infected cells) was analyzed as described below. In some experiments, 10 μm 9-cis-retinoic acid, a retinoid X receptor (RXR) ligand, was added to the hepatocytes 12 h prior to and during the 3.5 h incubation. Glyburide (100 μm), a known inhibitor of ABCA1-mediated lipid efflux, was added only during the 3.5 h incubation. The medium from four 6-well plates (24 wells) were pooled and concentrated down to 2 ml (12-fold concentrated with Amicon 10K filter units). The samples were immediately loaded on two calibrated Superdex 200 columns connected in a series similar to that described previously for isolation of lipoproteins from plasma samples (28McManus D.C. Scott B.R. Frank P.G. Franklin V. Schultz J.R. Marcel Y.L. J. Biol. Chem. 2000; 275: 5043-5051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-sized species elute in the void volume on these columns. HDL2/3particles and smaller very high density lipoprotein (VHDL) fractions containing albumin (≤ 7.1 nm diameter) were carefully separated. Aliquots (200 μl) from each fraction were analyzed for apoA-I by Western blot analysis following transfer to nitrocellulose with a slot blot apparatus (BioRad Bio-Dot SF unit) as described previously (28McManus D.C. Scott B.R. Frank P.G. Franklin V. Schultz J.R. Marcel Y.L. J. Biol. Chem. 2000; 275: 5043-5051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). No background signal could be detected as indicated by analysis of medium collected from hepatocytes infected with the AdLuc. The relative distribution of apoA-I in the VLDL, HDL2/3, and VHDL pools was determined by densitometric scanning (BioRad software, Quantity One, version 4.11). For comparison, the relative distribution of murine apoB (apoB48 and apoB100) was also measured using a polyclonal anti-mouse apoB antibody (BIODESIGN International, Kennebunk, ME) and visualized by chemiluminescence (Pierce West Pico SuperSignal substrate, Pierce) after incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences). The charge and size of apoA-I secreted from the primary hepatocytes was determined by agarose gel (Beckman Lipogel, Beckman Coulter, Fullerton, CA) and 4–20% non-denaturing polyacrylamide gradient gel electrophoresis (PAGGE) (Novex, Invitrogen), respectively, as described previously (28McManus D.C. Scott B.R. Frank P.G. Franklin V. Schultz J.R. Marcel Y.L. J. Biol. Chem. 2000; 275: 5043-5051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29McManus D.C. Scott B.R. Franklin V. Sparks D.L. Marcel Y.L. J. Biol. Chem. 2001; 276: 21292-21302Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Briefly, following transfer of proteins from the gels to nitrocellulose, the membranes were probed with biotinylated monoclonal antibodies directed against human apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)) (33Marcel Y.L. Provost P.R. Koa H. Raffai E. Dac N.V. Fruchart J.C. Rassart E. J. Biol. Chem. 1991; 266: 3644-3653Abstract Full Text PDF PubMed Google Scholar). The antibodies were biotinylated with Sulfo-NHS-Biotin (Pierce) and visualized by chemiluminescence following treatment with Streptavidin-conjugated horseradish peroxidase (Amersham Biosciences). The size of the apoA-I species were compared with biotinylated molecular weight markers of known hydrodynamic diameter, and the charge of secreted apoA-I was compared with lipid-free apoA-I and HDL both isolated from human plasma. ApoA-I secreted from hepatocytes was immunoprecipitated under native conditions either directly from the medium or from lipoprotein fractions isolated by FPLC as follows. The immunoprecipitations were carried out with a polyclonal anti-human apoA-I antiserum from sheep (Roche Molecular Biochemicals) and protein G-Sepharose (Amersham Biosciences). An equal volume of an anti-human apoB antiserum from sheep, which does not cross-react with murine apoB, was used as a control where indicated. The immunoprecipitates were collected following centrifugation (10 min at 3000 ×g) and washed three times with 10 ml of phosphate-buffered saline (no detergents) and resuspended in a final volume of 1 ml of phosphate-buffered saline. These immunoprecipitates were either subjected directly to scintillation counting or were further analyzed by Bligh and Dyer lipid extraction (34Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar) and thin layer chromatography (TLC). TLC separation was performed on silica gel plates and a solvent system (chloroform/methanol/acetic acid/formic acid/water, 70:30:12:4:2) for separation of phosphatidylcholine and sphingomyelin. The TLC bands corresponding to phosphatidylcholine and sphingomyelin were excised and counted for radioactivity. Alternatively, cells were labeled with 32P-phosphate (Amersham Biosciences) to label all cellular phospholipids. Cells were treated as with labeling with [3H]choline, except that32P-phosphate in Hepatozyme medium was only added after adenoviral infection on the second day (not also on the first day as for [3H]choline). On the third day, the hepatocytes were washed as before and incubated in fresh Hepatozyme in the absence or presence of apoA-I for 3.5 h. The hapoA-I-containing lipoproteins were immunoprecipitated as described above, and then phospholipids were extracted by the method of Bligh and Dyer (34Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). The phospholipids were separated by TLC in the solvent system of chloroform, methanol, acetic acid, formic acid, water (at a volume ratio of 70:30:12:4:2). The TLC plate was exposed to a phosphorimaging plate and the relative amounts of phospholipids were determined by densitometry scanning (BioRad software, Quantity One, version 4.11). Results are expressed as the average of at least three replicates to control for variable loading and extraction efficiency. Lipidation of a newly synthesized apoA-I can occur both intracellularly during transport from the endoplasmic reticulum to the cell surface and peri-cellularly through lipid efflux. To document the relative contribution to the lipidation observed, we compared endogenously synthesized apoA-I and exogenously added apoA-I. Primary hepatocytes were isolated from 4–6-month-old mice by liver collagenase perfusion, and isolated cells were cultured on fibronectin-coated plates in the presence of serum-free media (Hepatozyme). The following day, cells were infected with either an adenoviral construct encoding human apoA-I (AdAI) or luciferase (AdLuc) for 1 h, washed, and then returned to Hepatozyme media. The next day, the cells were washed and then incubated with fresh Hepatozyme media in the absence or presence of exogenous hapoA-I for 3.5 h. This time period was chosen to allow sufficient secretion of hapoA-I for analysis, and yet minimize peri-cellular interactions. We chose a concentration of exo apoA-I that approximated the amount of hapoA-I secreted during the same time period. The hapoA-I-containing lipoproteins in the media were then analyzed by a number of methods. The adenoviral vector was selected specifically to ensure apoA-I synthesis and secretion independent of experimental factors. The electrophoretic migration on agarose gels of hapoA-I newly secreted from primary hepatocytes (hereafter referred to as endogenously synthesized apoA-I or “endo apoA-I”) or exogenously added hapoA-I (referred to as “exo apoA-I”) was assessed (Fig.1). Endo apoA-I and exo apoA-I from apoA-I-deficient mice were both found to have exclusively pre-β migration, and no α-migrating immunoreactive apoA-I band appeared even with prolonged exposure. This is in contrast with a previous study in monkey hepatocytes (4Castle C.K. Pape M.E. Marotti K.R. Melchior G.W. J. Lipid Res. 1991; 32: 439-447Abstract Full Text PDF PubMed Google Scholar), where apoA-I-containing lipoproteins were found to segregate into two pre-β- and one α-migrating fractions. Similarly, lipoproteins formed by hepatocytes from ABCA1−/− mice endo apoA-I or exo apoA-I (Fig. 1) possessed only pre-β migration. This result demonstrates that all HDL formed, whatever the source, have similar pre-β electrophoretic mobility, and thereby lack a significant hydrophobic core. To evaluate the lipoprotein size distribution of apoA-I secreted by the hepatocytes expressing hapoA-I, the medium was concentrated and immediately fractionated by FPLC and analyzed. The distribution of immunoreactive human apoA-I and murine apoB (both apoB100 and apoB48) in the FPLC fractions were analyzed by slot blot. Immunoreactive hapoA-I segregated into 3 well separated peaks (Fig.2 A). The largest apoA-I-containing lipoproteins eluted at a position previously calibrated for VLDL (fractions 10–14). This fraction also overlapped with the largest peak of murine apoB-containing lipoproteins (fractions 9–13; data not shown). The second peak of immunoreactive apoA-I-containing lipoproteins eluted at the position of HDL2/3 (fractions 19–23) and the third corresponded to lipid-poor VHDL and apoA-I (fractions 25–29). The results presented are representative of three separate experiments. The distribution of immunoreactive apoA-I after FPLC separation of medium lipoproteins shows that endo apoA-I and exo apoA-I form lipoproteins of similar sizes ranging from VLDL/LDL to VHDL (see Fig.2, A and B, and the distribution obtained with control hepatocytes in Fig. 3,A and B). Furthermore, when exo apoA-I was added to the medium of hepatocytes infected with AdAI, the amount of label associated with apoA-I was additive (data not shown). This result clearly indicates that lipidation occurs both peri-cellularly and intracellularly. ABCA1+/+ control and ABCA1−/− mouse hepatocytes were also infected with AdAI or AdLuc and then analyzed by FPLC for size separation of the endo apoA-I- and exo apoA-I-containing lipoproteins. Comparing the FPLC profiles of hapoA-I-containing lipoproteins from control and ABCA1-deficient hepatocytes, a reduction can be seen in the proportion of hapoA-I found in the buoyant VLDL and HDL fractions for both endo apoA-I (Fig. 3 A) and exo apoA-I (Fig. 3 B). These results demonstrate the important contribution of ABCA1 to both intracellular and peri-cellular lipidation of apoA-I. The different apoA-I-containing lipoprotein populations generated by the primary hepatocytes and separated by FPLC (VLDL, HDL, and VHDL) were further analyzed by non-denaturing PAGGE and Western blot analysis (Fig. 4). Similar amounts of immunoreactive hapoA-I were loaded from each lipoprotein pool. The same lipoprotein pool for different hepatocyte samples was similarly concentrated and loaded. The endo apoA-I present in VLDL (lane 1), HDL2/3 (lane 2), and VHDL fractions (lane 3) are well separated from one another. The HDL2/3 and lipid-poor apoA-I yield distinct bands, which are compatible with the known formation of lipoproteins with varying numbers of apoA-I and with varying degrees of lipidation. A large amount of hapoA-I is secreted as HDL2/3-sized species (Fig.2 A), with a significant size heterogeneity, which in this pool can reach 10.4 nm (Fig. 4, lane 2). The three lipoprotein fractions from the media of AdLuc-infected hepatocytes incubated with exo apoA-I (Fig. 2 B) were also analyzed by non-denaturing PAGGE: VLDL (lane 4), HDL (lane 5), and VHDL (lane 6). Interestingly, in comparison to endo apoA-I, lipidation of exo apoA-I produced profiles of similarly as well as differently sized hapoA-I-containing lipoproteins (comparinglanes 2 and 5 and 3 and 6). This suggests differences in how endo apoA-I and exo apoA-I lipoproteins are speciated and lipidated; it also indicates that our experiments distinguish between lipidation associated with secretion and efflux. Furthermore, when hepatocytes infected with AdLuc and incubated with exo apoA-I were incubated with 9-cis-retinoic acid, a retinoid X receptor ligand, the resulting hapoA-I-containing lipoproteins, VLDL (lane 7), HDL (lane 8), and VHDL (lane 9), were similar to control exo apoA-I fractions. An increased amount of larger-sized HDL particles is evident, suggesting that 9-cis-retinoic acid can enhance lipidation of exogenous hapoA-I. The 9-cis-retinoic acid effect was not observed with endo apoA-I (data not shown). Non-denaturing PAGGE and Western blot analysis were performed on the hapoA-I-containing lipoprotein fractions generated by ABCA1+/+ control and ABCA1−/− hepatocytes (Fig. 5). VLDL (lane 1), HDL (lane 2), and VHDL (lane 3) from control hepatocytes incubated with exo apoA-I are identical to VLDL (lane 7), HDL (lane 8), and VHDL (lane 9) from exo apoA-I in ABCA1−/− hepatocytes. Similarly, for endo apoA-I, the VLDL (lane 4), HDL (lane 5), and VHDL (lane 6) for the control hepatocytes were very similar to the VLDL (lane 10), HDL (lane 11) and VHDL (lane 12) from ABCA1−/− hepatocytes. We know that the quantity of hapoA-I found in VLDL and HDL fractions is significantly reduced in ABCA1−/− hepatocytes (Fig. 3,A and B), but, importantly, the nature of the lipoprotein particles formed is unchanged. From the apoA-I-deficient hepatocytes, the calculated relative distribution of endo apoA-I in the different lipoprotein fractions is shown in Fig.6 A. Interestingly, ∼20% of the total endo apoA-I secreted was found in HDL2/3-sized fractions. As well, a smaller but significant percentage of secreted apoA-I was also found associated with the VLDL pool. This result is in good general agreement with previous results in monkey hepatocytes (4Castle C.K. Pape M.E. Marotti K.R. Melchior G.W. J. Lipid Res. 1991; 32: 439-447Abstract Full Text PDF PubMed Google Scholar) and in HepG2 cells, although the latter do not secrete VLDL and therefore have no apoA-I-containing lipoproteins in this lipoprotein size (27Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. J. Lipid Res. 2002; 43: 36-44Abstract Full Text Full Text PDF PubMed Google Scholar, 35Forte T.M. Nichols A.V. Selmek-Halsey J. Caylor L. Shore V.G. Biochim. Biophys. Acta. 1987; 920: 185-194Crossref PubMed Scopus (6) Google Scholar). The association of [3H]choline phospholipids with hapoA-I in the three-lipoprotein pools was estimated by immunoprecipitation of hapoA-I under native conditions. Equal volumes of the pooled FPLC lipoprotein fractions (identified as VLDL, HDL2/3, and VHDL in Fig. 2) were immunopre" @default.
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