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• W2139061897 abstract "The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells.We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL. The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL. High density lipoprotein is produced by the reaction of helical apolipoprotein and ABCA1 (1Yokoyama 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). This is considered a main source of plasma HDL, because familial HDL deficiency (Tangier disease) has been identified as the defect of HDL assembly by this reaction (2Francis G.A. Knopp R.H. Oram J.F. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease.J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (373) Google Scholar) caused by the mutation of ABCA1 (3Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. Dam M. van 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. Kasteline J.J.P. Genest J. Hayden M.R. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar, 4Bodzioch 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. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1349) Google Scholar, 5Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1269) Google Scholar). The ABCA1/apolipoprotein reaction is also an important pathway of cellular cholesterol release for its conversion to bile acids in the liver, along with an alternative nonspecific diffusion pathway accelerated by cholesterol esterification with LCAT on HDL particles (1Yokoyama 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 main site for HDL production is generally thought to be the liver and intestine, where the cells synthesize helical apolipoprotein, mainly apolipoprotein A-I (apoA-I), and produce HDL, presumably upon the interaction of this apolipoprotein with its own ABCA1 by removing cellular lipid (6Wellington C.L. Brunham L.R. Zhou S. Singaraja R.R. Visscher H. Gelfer A. Ross C. James E. Liu G. Huber M.T. Yang Y.Z. Parks R.J. Groen A. Fruchart-Najib J. Hayden M.R. Alterations of plasma lipids in mice via adenoviral-mediated hepatic overexpression of human ABCA1.J. Lipid Res. 2003; 44: 1470-1480Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Francone O.L. Subbaiah P.V. Tol A. van Royer L. Haghpassand M. Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1.Biochemistry. 2003; 42: 8569-8578Crossref PubMed Scopus (37) Google Scholar, 8Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways.J. Biol. Chem. 2003; 278: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, it is unclear in which step of the apoA-I production and secretion this reaction takes place for the assembly of HDL. HDL particles have never been clearly identified in the secretory pathway of any HDL-producing cell, including hepatocytes. Probucol is an inhibitor of apoA-I-mediated cellular cholesterol release and HDL assembly (9Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Crossref PubMed Scopus (71) Google Scholar, 10Tsujita M. Tomimoto S. Okumura-Noji K. Okazaki M. Yokoyama S. Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice.Biochim. Biophys. Acta. 2000; 1485: 199-213Crossref PubMed Scopus (41) Google Scholar) and has been identified as an inactivator of ABCA1 (11Wu C-A. Tsujita M. Hayashi M. Yokoyama S. Probucol inactivates ABCA1 in plasma membrane for its function to mediate apolipoprotein binding and HDL assembly and for its proteolytic degradation.J. Biol. Chem. 2004; 279: 30168-30174Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We used this compound in LCAT-deficient mice in attempting to suppress the two major cholesterol-release pathways of somatic cells described above (12Tomimoto S. Tsujita M. Okazaki M. Usui S. Tada T. Fukutomi T. Ito S. Itoh M. Yokoyama S. Effect of probucol in lecithin-cholesterol acyltransferase-deficient mice: inhibition of 2 independent cellular cholesterol-releasing pathways in vivo.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 394-400Crossref PubMed Scopus (30) Google Scholar). To our surprise, no systemic cholesterol accumulation was observed, indicating that cholesterol may leave cells by a nonspecific pathway and that many extracellular acceptors can act as cholesterol transporters, such as albumin and blood cells. However, cholesterol content increased only in the liver when probucol inhibited the ABCA1 pathway in the cholesterol-fed LCAT-deficient mice. Thus, the liver seems to be a major organ from which cholesterol release requires the ABCA1 pathway, and these results indicate that the liver is a major source of plasma HDL (13Yokoyama S. Tajima S. Yamamoto A. The process of dissolving apolipoprotein A-I in an aqueous buffer.J. Biochem. (Tokyo). 1982; 91: 1267-1272Crossref PubMed Scopus (73) Google Scholar), consistent with other reports using genetically engineered animals (6Wellington C.L. Brunham L.R. Zhou S. Singaraja R.R. Visscher H. Gelfer A. Ross C. James E. Liu G. Huber M.T. Yang Y.Z. Parks R.J. Groen A. Fruchart-Najib J. Hayden M.R. Alterations of plasma lipids in mice via adenoviral-mediated hepatic overexpression of human ABCA1.J. Lipid Res. 2003; 44: 1470-1480Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Francone O.L. Subbaiah P.V. Tol A. van Royer L. Haghpassand M. Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1.Biochemistry. 2003; 42: 8569-8578Crossref PubMed Scopus (37) Google Scholar, 8Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways.J. Biol. Chem. 2003; 278: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It is thus important to characterize how HDL is assembled in hepatocytes with helical apolipoproteins synthesized by hepatocytes interacting with their own ABCA1. We used the human hepatoma cell line HepG2 and mouse primary culture hepatocytes as model systems. To identify a role of ABCA1 in HDL production by hepatocytes, we used probucol to inactivate ABCA1 as well as ABCA1-deficient mice. A monoclonal antibody specific against lipid-free apoA-I was used as a tool to trap lipid-free apoA-I to examine whether apoA-I is secreted from the cells as a free form before it interacts with ABCA1 of the cells to generate HDL. ApoA-I and apoA-II were purified from human HDL fraction using delipidation and anion-exchange column chromatography in 6 M urea as previously described (13Yokoyama S. Tajima S. Yamamoto A. The process of dissolving apolipoprotein A-I in an aqueous buffer.J. Biochem. (Tokyo). 1982; 91: 1267-1272Crossref PubMed Scopus (73) Google Scholar, 14Yokoyama S. Tajima S. Yamamoto A. Association of apolipoprotein A-II with egg phosphatidylcholine unilamellar vesicles.J. Biochem. (Tokyo). 1984; 96: 871-880Crossref PubMed Scopus (9) Google Scholar). Apolipoproteins were dissolved in 50 mM sodium phosphate buffer, pH 7.4, containing 6 M guanidine-HCl and thoroughly dialyzed against 10 mM sodium phosphate buffer, pH 7.4, 0.15 M NaCl (PBS). For the specific binding study, apoA-I was labeled with 125I as previously described (9Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Crossref PubMed Scopus (71) Google Scholar) except for the use of Iodo-Beads to activate 125I. The iodine-labeled apoA-I was concentrated by a Ultrafree-15 centrifugal filter device (Millipore Corp.). One milliliter of 125I-apoA-I solution was dissolved in an equal volume of 6 M guanidine-HCl in 50 mM sodium phosphate buffer, pH 7.4, and dialyzed against PBS to remove unbound 125I and guanidine-HCl. Probucol was kindly provided by Daiichi Pharmaceutical Co. LDL was isolated by sequential ultracentrifugation, and control and probucol-containing LDL were prepared by the method previously described (9Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Crossref PubMed Scopus (71) Google Scholar). A monoclonal antibody (IgG) against lipid-free apoA-I, 725-1E2, was among the antibodies provided by Daiichi Pure Chemicals (Tokyo, Japan) and characterized in our laboratory as described previously (15Okuhira K. Tsujita M. Yamauchi Y. Abe-Dohmae S. Kato K. Handa T. Yokoyama S. Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.J. Lipid Res. 2004; 45: 645-652Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Mouse IgG was purchased from Chemicon International and used as a nonspecific control for 725-1E2. HepG2 cells (American Type Culture Collection; ATCC HB8065) were maintained in minimum essential medium Eagle α modification (MEM-α) supplemented with 10% fetal calf serum (FCS) and antibiotics (5 U/ml penicillin and 5 μg/ml streptomycin). For the individual experiments, cells were subcultured onto 35 or 60 mm plates at the density of 0.7 to 1 × 106 cells/ml and maintained in MEM-α with 10% FCS and antibiotics (5 U/ml penicillin and 5 μg/ml streptomycin) by changing the medium every 2 days. On the fifth day, when the cells were 80–90% confluent, the cells were washed extensively with MEM-α and incubated with MEM-α containing 0.02% BSA and antibiotics with and without exogenous human apoA-I and human apoA-II for 16 h. The conditioned medium was collected for further lipid analysis. To load probucol, cells at 65–75% confluence were incubated with LDL or probucol-containing LDL (50 μg/ml as protein) in MEM-α for 24 h before the lipid-release assay. C57Bl/6 mice (9 weeks old) were purchased from the local experimental animal supplier. The mice were fed with normal mouse chow or chow containing 0.5% (w/w) probucol ad libitum. ABCA1-deficient mice were bred from ABCA1 heterozygote mice (DBA/1-Abca1tm1Jdm/J) purchased from Jackson's Animal Laboratories (Stony Brook, NY). The genotypes of all offspring were determined by PCR analysis of tail DNA. The oligonucleotide primer sets 5′-TGGGAACTCCTGCTAAAAT-3′, 5′-CCATGTGGTGTGTAGACA-3′ and 5′-TTTCTCATAGGGTTGGTCA-3′, 5′-TGCAATCCATCTTGTTCAAT-3′ were used to determine the wild-type and mutant alleles, respectively. PCR was performed according to the genotyping method provided by the animal supplier except for an annealing temperature of 58°C. The mouse primary hepatocytes were harvested and cultured according to the method by Noga et al. (16Noga A.A. Stead L.M. Zhao Y. Brosnan M.E. Brosnan J.T. Vance D.E. Plasma homocysteine is regulated by phospholipid methylation.J. Biol. Chem. 2003; 278: 5952-5955Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Mice were anesthetized and the liver was perfused with Hanks' salt solution without calcium containing 0.5 mM EGTA and Hanks' salt solution with calcium and magnesium containing 75 U/ml collagenase (type IV) at 37°C. The hepatocytes were isolated by low-gravity centrifugation and placed onto sterilized collagen-coated 60 mm culture dishes (1.8 × 106 cells/dish). After 2 h, when cells were attached as a monolayer, the unbound cells were washed with DMEM (high glucose) containing 0.02% BSA. The cells were incubated with and without apoA-I for 16 h, and the conditioned medium was collected after the incubation. All of the experiments were completed within 24 h after harvesting the cells. The experimental procedure had been approved by the Animal Welfare Committee of Nagoya City University Graduate School of Medical Sciences according to institutional guidelines. Lipoprotein in the conditioned medium of the hepatocytes was analyzed by HPLC using a gel-permeation column(s) (Lipopropak XL; 7.8 mm × 300 mm; Tosoh) with 0.05 M Tris-buffered acetate, pH 8.0, containing 0.3 M sodium acetate, 0.05% sodium azide, and 0.005% Brij-35 at a flow rate of 0.7 ml/min and an online enzymatic lipid-detection system (10Tsujita M. Tomimoto S. Okumura-Noji K. Okazaki M. Yokoyama S. Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice.Biochim. Biophys. Acta. 2000; 1485: 199-213Crossref PubMed Scopus (41) Google Scholar, 17Okazaki M. Usui S. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. American Association of Clinical Chemistry Press, Washington, DC2000: 647-669Google Scholar, 18Wu C-A. Tsujita M. Okumura-Noji K. Usui S. Kakuuchi H. Okazaki M. Yokoyama S. Cholesteryl ester transfer protein expressed in lecithin cholesterol acyltransferase-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1347-1353Crossref PubMed Scopus (5) Google Scholar, 19Usui S. Hara Y. Hosaki S. Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar). The conditioned medium was centrifuged at 10,000 rpm for 5 min to remove cell debris, and a 200 μl aliquot was applied for HPLC analysis. The method was thoroughly validated against the reference methods of ultracentrifugation and of Superose gel-permeation chromatography, including the criteria of subfraction analysis of HDL (17Okazaki M. Usui S. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. American Association of Clinical Chemistry Press, Washington, DC2000: 647-669Google Scholar, 19Usui S. Hara Y. Hosaki S. Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar). In some experiments, the VLDL/LDL fraction (d < 1.063 g/ml) and the HDL fraction (1.063 < d < 1.21 g/ml) were isolated from the conditioned medium by sequential ultracentrifugation at 1.063 g/ml and 1.21 g/ml in a Himac CS120GX (Hitachi) at 99,000 rpm for 4 h. Lipid was extracted from the total conditioned medium and the VLDL/LDL and HDL fractions with organic solvent, and cholesterol and choline-phospholipid were determined with colorimetric enzyme assay kits (Kyowa Medics Co., Ltd., for cholesterol and Wako Pure Chemical Industries, Ltd., for phospholipid) (20Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (101) Google Scholar). Electrophoretic analysis was also performed for the medium using a Beckman Paragon System on an agarose gel. Apolipoproteins in the HepG2 conditioned medium were analyzed by immunoblotting using rabbit antiserum raised against human apoA-I and goat anti-human apoB IgG (affinity purified; Academy Bio-Medical Co., Inc.). The distribution of apoA-I and apoB in the HPLC-fractionated samples was analyzed. The eluent was fractionated every 30 s. After adding 4 μg of BSA to each sample, protein was precipitated with 15% (w/v) trichloroacetic acid for 30 min on ice and recovered by centrifugation at 15,000 rpm for 10 min. The precipitated protein was washed with 1 ml of acetone and dissolved in 10 μl of sample buffer for SDS-polyacrylamide electrophoresis, incubated at 100°C for 5 min, and 5 μl of 9 M urea was added. The electrophoresis was performed on a polyacrylamide gel with a gradient of 4–20%. The conditioned media and fractionated media by ultracentrifugation were directly analyzed by immunoblotting against apoA-I. Anti-human albumin IgG fraction of rabbit antiserum was purchased from Sigma-Aldrich Inc. and used as a secondary antibody. Blotted bands were visualized by the chemiluminescence method (ECL Western Blotting Detection System; Amersham Pharmacia Biotech). The clearance rate of ABCA1 was analyzed by immunoblotting of the protein in the bulk cellular membrane fraction (21Arakawa R. Yokoyama S. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation.J. Biol. Chem. 2002; 277: 22426-22429Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). HepG2 cells were loaded with control and probucol-containing LDL for 2 h and incubated in the presence of 71 μM cycloheximide to inhibit the synthesis of new protein. The cells were harvested and applied for ABCA1 immunoblotting using a specific antibody raised against a C-terminal peptide of human ABCA1 (21Arakawa R. Yokoyama S. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation.J. Biol. Chem. 2002; 277: 22426-22429Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Cellular lipid was analyzed as described previously (20Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (101) Google Scholar). The sample was also used for probucol analysis using an HPLC system (22Satonin D.K. Coutant J.E. Comparison of gas chromatography and high-performance liquid chromatography for the analysis of probucol in plasma.J. Chromatogr. 1986; 380: 401-406Crossref PubMed Scopus (33) Google Scholar). Protein was measured with the bicinchoninic acid method using BSA as a standard. Apolipoprotein-induced cellular lipid release from HepG2 cells is shown in Fig. 1. There was a significant spontaneous release of cholesterol and choline-phospholipid by HepG2 cells (indicated at zero concentration of apolipoproteins). Both apoA-I and apoA-II, when exogenously added to the culture medium, induced further release of the lipids in a dose-dependent manner. The medium was analyzed for lipoprotein particles using gel-permeation HPLC (Fig. 2). Two major peaks were identified corresponding to LDL and HDL sizes by human plasma reference, whereas no significant peak was detected at the position of VLDL (Fig. 2A). The peaks were indeed verified to correspond to density classification of lipoprotein as fractions d < 1.063 and d > 1.063 (Fig. 2C, E). The conditioned medium incubated with 10 μg/ml human apoA-I showed an increase of the HDL peak (Fig. 2B, D, F). Thus, increase of the release of cholesterol and phospholipid by apolipoprotein was shown to be attributable to the additional production of the HDL-size particles. Generation of lipoprotein was also examined for primary cultured mouse hepatocytes (Fig. 3). The cells were incubated with and without exogenous 10 μg/ml human apoA-I for 24 h, and the conditioned medium was fractionated by ultracentrifugation. Cholesterol content in the HDL fraction (d > 1.063) was increased by 2-fold, and the VLDL-LDL fraction (d < 1.063) was insignificantly decreased. These results indicated that the system is functional both in HepG2 cells and in mouse hepatocytes for generation of the HDL-size lipoprotein by helical apolipoprotein.Fig. 2Lipoprotein analysis of the culture medium of HepG2 cells by HPLC. Cells were incubated with MEM-α containing 0.02% BSA with (B, D, F) or without (A, C, E) 10 μg/ml human apoA-I for 16 h. The conditioned medium of HepG2 cells (100 μl) was analyzed by the HPLC lipoprotein analysis system using two tandem gel-permeation columns (Lipopropak XL; 7.8 mm × 300 mm; Tosoh). The elution profile was monitored by an online assay system for total cholesterol (solid lines) and triacylglycerol (dotted lines). A and B: Whole conditioned medium. C and D: d > 1.063 g/ml fraction. E and F: d < 1.063 g/ml fraction. The eluting positions of human plasma VLDL, LDL, and HDL are indicated by arrows in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Analysis of lipoprotein generated by mouse primary culture hepatocytes. Primary culture hepatocytes of C57Bl/6 mice were harvested using the Hanks'-EDTA and collagenase two-medium method described in Materials and Methods. The cells were incubated in DMEM (high glucose)/BSA medium with or without human apoA-I for 16 h. The conditioned medium was separated by ultracentrifugation at a density 1.063 g/ml. The data represent means ± SEM of three determinations. Difference in HDL between control and apoA-I (+) is significant by P < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the contribution of ABCA1 to this system, the effect of an inactivator of ABCA1, probucol, was analyzed in those cells. Probucol was loaded to HepG2 cells, and the culture medium was analyzed by HPLC after 16 h of incubation with and without 10 μg/ml apoA-I. The data for cellular lipid and probucol are listed in Table 1. There was no difference in lipid composition between the control and probucol-loaded cells. The latter cells contained probucol at ∼1 μg/mg cell proteins, which was equivalent to our previous data with mouse peritoneal macrophages to which probucol was delivered by acetylated LDL (9Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Crossref PubMed Scopus (71) Google Scholar). LDL- and HDL-size particles were identified in the medium, and the HDL fraction was increased by apoA-I (Fig. 4A, B). Probucol markedly decreased the HDL peak with both spontaneous production or exogenously added apoA-I (Fig. 4C, D). This finding was confirmed by electrophoretic analysis of the medium on an agarose gel (data not shown). The medium of mouse primary hepatocytes was also examined. The hepatocytes isolated from ABCA1-deficient mice produced no HDL, and apoA-I did not induce the production of HDL either (Fig. 4E, F). The effect of probucol was examined in hepatocytes prepared from C57Bl/6 mice by feeding control chow or 0.5% probucol-containing chow. The HDL peak decreased by probucol feeding (Fig. 4G, H). Figure 5shows the effect of probucol on the apoA-I-induced lipid release from HepG2 cells. The results indicated again that probucol inhibits the lipid release for both the baseline and an additional part induced by apoA-I. The cell-specific (reversible) binding of apoA-I was examined using 125I-labeled apoA-I for HepG2 cells. Probucol inhibited the binding of apoA-I (Fig. 5C).TABLE 1Chemical compositions of cellular lipids in HepG2 cellsSampleTotal CholesterolTriglyceridePhospholipidProbucolμg/mg proteinControl LDLexo-apoA-I(−)22.02 ± 0.9310.09 ± 2.90201 ± 5.850.00exo-apoA-I(+)21.25 ± 0.3513.91 ± 5.62213 ± 6.610.00Probucol LDLexo-apoA-I(−)19.84 ± 0.6315.12 ± 1.72169 ± 10.451.01 ± 0.21exo-apoA-I(+)21.17 ± 0.8419.08 ± 2.48185 ± 8.601.17 ± 0.27exo-apoA-I, exogenous apolipoprotein A-I. Cellular lipid was analyzed by enzymatic methods, and probucol was measured using an HPLC method. Open table in a new tab Fig. 5Effect of probucol loading on apoA-I-mediated cellular lipid release from HepG2 cells and exchangeable binding apoA-I to HepG2 cells. A and B: Lipid release. HepG2 cells were incubated with either control LDL or probucol-containing LDL for 24 h. Cellular cholesterol and phospholipid release were measured after incubation for 16 h with various amounts of apoA-I. HDL-cholesterol (A) and HDL-phospholipid (B) were determined by an HPLC system. Open squares, control LDL-loaded cells; open circles, probucol-LDL loaded cells. C: ApoA-I binding. The indicated amounts of 125I-apoA-I-incubated HepG2 cells were preloaded with control LDL or probucol-containing LDL for 2 h at 4°C. The cells were washed two times with ice-cold PBS, and the bound 125I-apoA-I was displaced two times by 50 μg/ml nonlabeled apoA-I for 9 h. The difference in the total radioactivity after the first washing and after the second displacement was calculated as displaceable or exchangeable binding of apoA-I to the cells. Squares, binding to the cells loaded with control LDL; circles, binding to the cells loaded with probucol-containing LDL. Error bars indicate ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) exo-apoA-I, exogenous apolipoprotein A-I. Cellular lipid was analyzed by enzymatic methods, and probucol was measured using an HPLC method. ApoB and apoA-I were analyzed in the HPLC fractions of the HepG2-conditioned medium (Fig. 6). ApoB was detected in the LDL fractions, and the probucol treatment did not alter this distribution. ApoA-I in the HDL fractions was markedly decreased by the probucol treatment. ApoA-I was also present in the free protein fractions, and this is consistent with the findings from the analysis of human plasma by HPLC in the diluted condition equivalent to the culture medium (×250) (data not shown). Although this HPLC method was thoroughly validated for lipoprotein lipid elution profile by its excellent correlation with the other gel-permeation method with a Superose column, apoA-I may dissociate from HDL particles during the analysis, presumably as a result of the use of 0.005% Brij-35 and extreme dilution of lipoprotein (17Okazaki M. Usui S. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. American Association of Clinical Chemistry Press, Washington, DC2000: 647-669Google Scholar, 19Usui S. Hara Y. Hosaki S. Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar). Because of this limitation of the HPLC method, the medium was analyzed by ultracentrifugation to identify the status of apoA-I in the medium. When ABCA1 was inactivated by probucol in HepG2 cells, overall secretion of apoA-I was not influenced, as demonstrated in Fig. 7A. However, although apoA-I was localized in the HDL fraction (1.063 < d <1.21 g/ml) in the control conditioned medium, it was found exclusively in the d > 1.21 g/ml fraction in the medium of probucol-loaded cells (Fig. 7B). Thus, apoA-I is secreted by the cells mostly in its free form when ABCA1 is inactivated. Stabilization and increase of ABCA1 by additional apoA-I was not observed in this cell line, presumably because it is already affected by endogenously secreted apolipoproteins (Fig. 7C). However, ABCA1 was resistant against degradation in the probucol-loaded HepG2 cells (Fig. 7C, D) in the same manner as we demonstrated in fibroblasts (11Wu C-A. Tsujita M. Hayashi M. Yokoyama S. Probucol inactivates ABCA1 in plasma membrane for its function to mediate apolipoprotein binding and HDL assembly and for its proteolytic degradation.J. Biol. Chem. 2004; 279: 30168-30174Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Finally, we attempted to inactivate free apoA-I in the medium by trapping it using a monoclonal antibody to lipid-free apoA-I, 725-1E2 (15Okuhira K. Tsujita M. Yamauchi Y. Abe-Dohmae S. Kato K. Handa T. Yokoyama S. Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.J. Lipid Res. 2004; 45: 645-652Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As shown in Fig. 8, production of the HDL-size particles by both endogenous and exogenous apoA-I was effectively suppressed when this antibody was present in the medium during the incubation of HepG2 cells (Fig. 8Ag, Ah), whereas control IgG did not influence the profile (Fig. 8Ac, Ad). On the other hand, this antibody did not change lipoprotein profile when incubated with the HepG2-conditioned medium after removal of the cells (Fig. 8Ba, Bb). These results are quantitatively illustrated in Fig. 9A. Alternatively, the medium was analyzed by ultracentrifugation after the same experiments, and these results are shown in Fig. 9B. Similar to the results of the HPLC analysis, HDL production was selectively suppressed by the lipid-free apoA-I monoclonal antibody 725-1E2.Fig. 8Inhibition of HDL production by HepG2 cells by a monoclonal antibody against lipid-free apoA-I. A: HepG2 cells were incubated with 0.02% BSA-containing MEM-α medium in the presence of 1 mg/ml monoclonal antibody 725-1E2 specific to lipid-free apoA-I and 10 μg/ml apoA-I for 16 h, and the medium was analyzed for its lipoprotein profile using HPLC with one gel-permeation column (Lipopropak XL; 7.8 mm × 300 mm; Tosoh). A-a: The medium of the cells by plain incubation. A-b: The medium of the cells incubated in the presence of apoA-I. A-c: The medium after incubation in the presence of control mouse IgG. A-d: The medium incubated in the presence of apoA-I and control IgG. A-e: The medium of the probucol (Pb)-preloaded cells. A-f: The medium of the cell preloaded with probucol after incubation in the presence of apoA-I. A-g: The medium of the cells incubated in the presence of the free apoA-I-specific antibody 725-1E2. A-h: The medium of the cells incubated with the antibody 725-1E2 in the presence of apoA-I. B: The conditioned medium of HepG2 cells was incubated for 16 h in the absence (B-a) and presence (B-b) of the antibody, and the lipoprotein profile was analyzed by HPLC. Solid lines, cholesterol; dotted lines, choline-phospholipid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 9Effect of free apoA-I-specific monoclonal antibody on HDL production by HepG2 cells. A: Cholesterol and phospholipid in HDL in the medium of HepG2 cells incubated in the presence of the antibody 725-1E2 and apoA-I, based on HPLC analysis. The data represent means ± SEM of triplicate assays. B: Cholesterol and phospholipid in HDL in the medium of HepG2 cells incubated in the presence of the antibody 725-1E2 and apoA-I, based on ultracentrifugal analysis. Data represent means ± SEM of triplicate assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results of this work are summarized as follows. 1) HepG2 cells and mouse primary cultured hepatocytes produce HDL with endogenously synthesized apolipoprotein as well as with exogenously added apolipoprotein. 2) Production of HDL by both pathways is inhibited by the inactivation of ABCA1, but secretion of endogenous apoA-I was not decreased as much. ApoA-I is secreted as a free form when ABCA1 is inactivated. 3) A monoclonal antibody against lipid-free apoA-I, 725-1E2, suppresses the production of HDL by HepG2 cells but has no effect on lipoprotein when directly incubated with the HepG2-conditioned medium. Based on these observations, we conclude that the majority of HDL produced by hepatocytes are generated by the interaction of apoA-I that is secreted in a free or lipid-poor form with ABCA1 of the hepatocytes in an autocrine-like manner. Many reports have characterized the HDL-like particles secreted in the culture medium by HepG2 cells and other liver cell lines (23Thrift R.N. Forte T.M. Cahoon B.E. Shore V.G. Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions.J. Lipid Res. 1986; 27: 236-250Abstract Full Text PDF PubMed Google Scholar, 24McCall M.R. Forte T.M. Shore V.G. Heterogeneity of nascent high density lipoproteins secreted by the hepatoma-derived cell line, Hep G2.J. Lipid Res. 1988; 29: 1127-1137Abstract Full Text PDF PubMed Google Scholar, 25Forte T.M. McCall M.R. Knowles B.B. Shore V.G. Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2.J. Lipid Res. 1989; 30: 817-829Abstract Full Text PDF PubMed Google Scholar). We essentially confirmed these findings with HepG2 cells and, in addition, demonstrated the increase of production of such particles by exogenously added apoA-I and apoA-II. Thus, the HepG2 cells have the same pathway to generate HDL as many other cells upon interaction with helical apolipoproteins, presumably dependent on ABCA1 (26Sahoo D. Trischuk T.C. Chan T. Drover V.A. Ho S. Chimini G. Agellon L.B. Agnihotri R. Francis G.A. Lehner R. ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from primary rat and mouse hepatocytes.J. Lipid Res. 2004; 45: 1122-1131Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Probucol has been shown to inhibit the apolipoprotein-cell interaction and accordingly to suppress the generation of HDL (9Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Crossref PubMed Scopus (71) Google Scholar, 10Tsujita M. Tomimoto S. Okumura-Noji K. Okazaki M. Yokoyama S. Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice.Biochim. Biophys. Acta. 2000; 1485: 199-213Crossref PubMed Scopus (41) Google Scholar). We recently demonstrated that this compound inactivates ABCA1 in the plasma membrane with respect to its activity as well as its calpain-mediated degradation (11Wu C-A. Tsujita M. Hayashi M. Yokoyama S. Probucol inactivates ABCA1 in plasma membrane for its function to mediate apolipoprotein binding and HDL assembly and for its proteolytic degradation.J. Biol. Chem. 2004; 279: 30168-30174Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Probucol inhibited the production of HDL, whether by endogenous or exogenous apolipoprotein, in hepatocytes. These findings suggested that endogenous and exogenous apolipoprotein, mainly apoA-I, generate HDL by a common mechanism, most likely by the ABCA1 pathway. Under this condition, the amount of the secreted apoA-I did not change, and it was secreted mostly in its free form (Fig. 7A, B). Therefore, the interaction of apoA-I with ABCA1 does not influence the rate of its secretion, but most apoA-I remains in a free form in the medium when it is unable to assemble HDL. A monoclonal antibody raised against apoA-I, 725-1E2, has been characterized to be selective for lipid-free apoA-I (15Okuhira K. Tsujita M. Yamauchi Y. Abe-Dohmae S. Kato K. Handa T. Yokoyama S. Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.J. Lipid Res. 2004; 45: 645-652Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). By using this antibody, we attempted to alter the production of HDL by HepG2 cells. In the presence of this antibody in the culture medium of HepG2 cells, the production of HDL markedly decreased. This finding strongly suggested that most apoA-I is secreted in its free form and trapped by the antibody, resulting in a decrease of HDL production. This view was supported by the finding that the incubation of the antibody with the HepG2-conditioned medium did not cause a change of the lipoprotein profile. Therefore, the effect of the antibody was not likely caused by the reaction with HDL after its generation by the cells. It was reported that a certain portion of apoA-I (20%) was intracellularly lipidated in HepG2 cells and in primary cultured hepatocytes (8Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways.J. Biol. Chem. 2003; 278: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 27Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.J. Lipid Res. 2002; 43: 36-44Abstract Full Text Full Text PDF PubMed Google Scholar), and some of the lipidation may not be mediated by ABCA1 (8Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways.J. Biol. Chem. 2003; 278: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, the present results indicated that the majority of HDL particles are produced by a common mechanism for endogenous and exogenous apolipoproteins, both of which can be inhibited by probucol. Also, most of the HDL production was inhibited by lipid-free apoA-I-specific antibody, indicating an autocrine mechanism: apoA-I is secreted as a free form and then interacts with hepatocytes to generate HDL. These findings by no means exclude the possibility of intracellular lipidation of apoA-I and the presence of the ABCA1-independent pathway as a minor source of HDL production. ApoA-I is secreted as pro-apoA-I and converted to a mature form by a metalloprotease in blood plasma (28Edelstein C. Gordon J.I. Toscas K. Sims H.F. Strauss A.W. Scanu A.M. In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.J. Biol. Chem. 1983; 258: 11430-11433Abstract Full Text PDF PubMed Google Scholar). Both pro-apoA-I and mature apoA-I were identically capable of generating HDL by reacting with ABCA1 (15Okuhira K. Tsujita M. Yamauchi Y. Abe-Dohmae S. Kato K. Handa T. Yokoyama S. Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.J. Lipid Res. 2004; 45: 645-652Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), so perhaps there is no need to consider apoA-I maturation for this autocrine mechanism. The authors thank Michi Hayashi for her preparation of apolipoproteins. This work was supported in part by grants-in-aid from the Ministry of Science, Education, Culture, and Sports and the Ministry of Health, Welfare, and Labor. It was also partly sponsored by a research grant from the Pharmaceuticals and Medical Device Agency." @default.
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• W2139061897 title "On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway" @default.
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