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. 1993 Sep 15;294(Pt 3):771–778. doi: 10.1042/bj2940771

Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters.

W Sattler 1, R Stocker 1
PMCID: PMC1134528  PMID: 8379932

Abstract

We have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL3 on lipoprotein particle metabolism and 'selective uptake' of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL3. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL3, [3H]cholesteryl linoleate ([3H]Ch18:2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL3 by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL3 was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL3 was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 microM chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.

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Selected References

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  1. Aden D. P., Fogel A., Plotkin S., Damjanov I., Knowles B. B. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature. 1979 Dec 6;282(5739):615–616. doi: 10.1038/282615a0. [DOI] [PubMed] [Google Scholar]
  2. Barter P. J., Hopkins G. J., Ha Y. C. The role of lipid transfer proteins in plasma lipoprotein metabolism. Am Heart J. 1987 Feb;113(2 Pt 2):538–542. doi: 10.1016/0002-8703(87)90628-4. [DOI] [PubMed] [Google Scholar]
  3. Bond H. M., Morrone G., Venuta S., Howell K. E. Characterization and purification of proteins which bind high-density lipoprotein. A putative cell-surface receptor. Biochem J. 1991 Nov 1;279(Pt 3):633–641. doi: 10.1042/bj2790633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowry V. W., Ingold K. U., Stocker R. Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant. Biochem J. 1992 Dec 1;288(Pt 2):341–344. doi: 10.1042/bj2880341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowry V. W., Stanley K. K., Stocker R. High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10316–10320. doi: 10.1073/pnas.89.21.10316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dashti N., Wolfbauer G., Alaupovic P. Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2. Biochim Biophys Acta. 1985 Jan 9;833(1):100–110. doi: 10.1016/0005-2760(85)90257-7. [DOI] [PubMed] [Google Scholar]
  7. DeLamatre J. G., Carter R. M., Hornick C. A. Evidence for extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): a model for delivery of high-density lipoprotein cholesterol. J Cell Physiol. 1991 Jan;146(1):18–24. doi: 10.1002/jcp.1041460104. [DOI] [PubMed] [Google Scholar]
  8. Frei B., Stocker R., Ames B. N. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9748–9752. doi: 10.1073/pnas.85.24.9748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glass C., Pittman R. C., Civen M., Steinberg D. Uptake of high-density lipoprotein-associated apoprotein A-I and cholesterol esters by 16 tissues of the rat in vivo and by adrenal cells and hepatocytes in vitro. J Biol Chem. 1985 Jan 25;260(2):744–750. [PubMed] [Google Scholar]
  10. Glass C., Pittman R. C., Weinstein D. B., Steinberg D. Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5435–5439. doi: 10.1073/pnas.80.17.5435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldberg D. I., Beltz W. F., Pittman R. C. Evaluation of pathways for the cellular uptake of high density lipoprotein cholesterol esters in rabbits. J Clin Invest. 1991 Jan;87(1):331–346. doi: 10.1172/JCI114991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein J. L., Basu S. K., Brown M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. doi: 10.1016/0076-6879(83)98152-1. [DOI] [PubMed] [Google Scholar]
  13. Green S. R., Pittman R. C. Comparative acyl specificities for transfer and selective uptake of high density lipoprotein cholesteryl esters. J Lipid Res. 1991 Mar;32(3):457–467. [PubMed] [Google Scholar]
  14. Hough J. L., Zilversmit D. B. Comparison of various methods for in vitro cholesteryl ester labeling of lipoproteins from hypercholesterolemic rabbits. Biochim Biophys Acta. 1984 Mar 7;792(3):338–347. doi: 10.1016/0005-2760(84)90202-9. [DOI] [PubMed] [Google Scholar]
  15. Jessup W., Mander E. L., Dean R. T. The intracellular storage and turnover of apolipoprotein B of oxidized LDL in macrophages. Biochim Biophys Acta. 1992 Jun 22;1126(2):167–177. doi: 10.1016/0005-2760(92)90287-6. [DOI] [PubMed] [Google Scholar]
  16. Kan C. C., Yan J., Bittman R. Rates of spontaneous exchange of synthetic radiolabeled sterols between lipid vesicles. Biochemistry. 1992 Feb 18;31(6):1866–1874. doi: 10.1021/bi00121a040. [DOI] [PubMed] [Google Scholar]
  17. Kelley J. L., Kruski A. W. Density gradient ultracentrifugation of serum lipoproteins in a swinging bucket rotor. Methods Enzymol. 1986;128:170–181. doi: 10.1016/0076-6879(86)28067-2. [DOI] [PubMed] [Google Scholar]
  18. Kilsdonk E. P., Van Gent T., Van Tol A. Characterization of human high-density lipoprotein subclasses LP A-I and LP A-I/A-II and binding to HepG2 cells. Biochim Biophys Acta. 1990 Aug 6;1045(3):205–212. doi: 10.1016/0005-2760(90)90121-d. [DOI] [PubMed] [Google Scholar]
  19. Knecht T. P., Pittman R. C. A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins. Biochim Biophys Acta. 1989 Apr 26;1002(3):365–375. doi: 10.1016/0005-2760(89)90351-2. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Leblond L., Marcel Y. L. The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells. J Biol Chem. 1991 Apr 5;266(10):6058–6067. [PubMed] [Google Scholar]
  22. Leitersdorf E., Israeli A., Stein O., Eisenberg S., Stein Y. The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells. Biochim Biophys Acta. 1986 Oct 3;878(3):320–329. doi: 10.1016/0005-2760(86)90239-0. [DOI] [PubMed] [Google Scholar]
  23. Li Q. T., Sawyer W. H. Effect of unesterified cholesterol on the compartmentation of a fluorescent cholesteryl ester in a lipoprotein-like lipid microemulsion. J Lipid Res. 1992 Apr;33(4):503–512. [PubMed] [Google Scholar]
  24. Mackinnon M., Savage J., Wishart R., Barter P. Metabolism of high density lipoproteins by the perfused rabbit liver. J Biol Chem. 1986 Feb 25;261(6):2548–2552. [PubMed] [Google Scholar]
  25. Mahlberg F. H., Rothblat G. H. Cellular cholesterol efflux. Role of cell membrane kinetic pools and interaction with apolipoproteins AI, AII, and Cs. J Biol Chem. 1992 Mar 5;267(7):4541–4550. [PubMed] [Google Scholar]
  26. Maiorino M., Chu F. F., Ursini F., Davies K. J., Doroshow J. H., Esworthy R. S. Phospholipid hydroperoxide glutathione peroxidase is the 18-kDa selenoprotein expressed in human tumor cell lines. J Biol Chem. 1991 Apr 25;266(12):7728–7732. [PubMed] [Google Scholar]
  27. Miller K. W., Small D. M. Surface-to-core and interparticle equilibrium distributions of triglyceride-rich lipoprotein lipids. J Biol Chem. 1983 Nov 25;258(22):13772–13784. [PubMed] [Google Scholar]
  28. Nagano Y., Arai H., Kita T. High density lipoprotein loses its effect to stimulate efflux of cholesterol from foam cells after oxidative modification. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6457–6461. doi: 10.1073/pnas.88.15.6457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Noy N., Leonard M., Zakim D. The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin. Biophys Chem. 1992 Feb;42(2):177–188. doi: 10.1016/0301-4622(92)85007-q. [DOI] [PubMed] [Google Scholar]
  30. Pfeuffer M. A., Richard B. M., Pittman R. C. Probucol increases the selective uptake of HDL cholesterol esters by Hep G2 human hepatoma cells. Arterioscler Thromb. 1992 Jul;12(7):870–878. doi: 10.1161/01.atv.12.7.870. [DOI] [PubMed] [Google Scholar]
  31. Phillips M. C., Johnson W. J., Rothblat G. H. Mechanisms and consequences of cellular cholesterol exchange and transfer. Biochim Biophys Acta. 1987 Jun 24;906(2):223–276. doi: 10.1016/0304-4157(87)90013-x. [DOI] [PubMed] [Google Scholar]
  32. Pieters M. N., Schouten D., Bakkeren H. F., Esbach B., Brouwer A., Knook D. L., van Berkel T. J. Selective uptake of cholesteryl esters from apolipoprotein-E-free high-density lipoproteins by rat parenchymal cells in vivo is efficiently coupled to bile acid synthesis. Biochem J. 1991 Dec 1;280(Pt 2):359–365. doi: 10.1042/bj2800359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pittman R. C., Glass C. K., Atkinson D., Small D. M. Synthetic high density lipoprotein particles. Application to studies of the apoprotein specificity for selective uptake of cholesterol esters. J Biol Chem. 1987 Feb 25;262(6):2435–2442. [PubMed] [Google Scholar]
  34. Pittman R. C., Knecht T. P., Rosenbaum M. S., Taylor C. A., Jr A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters. J Biol Chem. 1987 Feb 25;262(6):2443–2450. [PubMed] [Google Scholar]
  35. Rinninger F., Pittman R. C. Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells. J Biol Chem. 1989 Apr 15;264(11):6111–6118. [PubMed] [Google Scholar]
  36. Rinninger F., Pittman R. C. Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells. J Lipid Res. 1988 Sep;29(9):1179–1194. [PubMed] [Google Scholar]
  37. Rothblat G. H., Mahlberg F. H., Johnson W. J., Phillips M. C. Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol efflux. J Lipid Res. 1992 Aug;33(8):1091–1097. [PubMed] [Google Scholar]
  38. Shepherd J., Gotto A. M., Jr, Taunton O. D., Caslake M. J., Farish E. The in vitro interaction of human apolipoprotein A-I and high density lipoproteins. Biochim Biophys Acta. 1977 Dec 21;489(3):486–501. doi: 10.1016/0005-2760(77)90169-2. [DOI] [PubMed] [Google Scholar]
  39. Sinn H. J., Schrenk H. H., Friedrich E. A., Via D. P., Dresel H. A. Radioiodination of proteins and lipoproteins using N-bromosuccinimide as oxidizing agent. Anal Biochem. 1988 Apr;170(1):186–192. doi: 10.1016/0003-2697(88)90107-8. [DOI] [PubMed] [Google Scholar]
  40. Smaby J. M., Brockman H. L. Acyl unsaturation and cholesteryl ester miscibility in surfaces. Formation of lecithin-cholesteryl ester complexes. J Lipid Res. 1987 Sep;28(9):1078–1087. [PubMed] [Google Scholar]
  41. Sparrow C. P., Pittman R. C. Cholesterol esters selectively taken up from high-density lipoproteins are hydrolyzed extralysosomally. Biochim Biophys Acta. 1990 Apr 2;1043(2):203–210. doi: 10.1016/0005-2760(90)90297-b. [DOI] [PubMed] [Google Scholar]
  42. Stein Y., Dabach Y., Hollander G., Halperin G., Stein O. Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether. Biochim Biophys Acta. 1983 Jun 16;752(1):98–105. doi: 10.1016/0005-2760(83)90237-0. [DOI] [PubMed] [Google Scholar]
  43. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C., Witztum J. L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989 Apr 6;320(14):915–924. doi: 10.1056/NEJM198904063201407. [DOI] [PubMed] [Google Scholar]
  44. Stocker R., Bowry V. W., Frei B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does alpha-tocopherol. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1646–1650. doi: 10.1073/pnas.88.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tabas I., Tall A. R. Mechanism of the association of HDL3 with endothelial cells, smooth muscle cells, and fibroblasts. Evidence against the role of specific ligand and receptor proteins. J Biol Chem. 1984 Nov 25;259(22):13897–13905. [PubMed] [Google Scholar]
  46. Tosaki A., Blasig I. E., Pali T., Ebert B. Heart protection and radical trapping by DMPO during reperfusion in isolated working rat hearts. Free Radic Biol Med. 1990;8(4):363–372. doi: 10.1016/0891-5849(90)90102-o. [DOI] [PubMed] [Google Scholar]
  47. Yamamoto Y., Brodsky M. H., Baker J. C., Ames B. N. Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography. Anal Biochem. 1987 Jan;160(1):7–13. doi: 10.1016/0003-2697(87)90606-3. [DOI] [PubMed] [Google Scholar]
  48. Yamamoto Y., Niki E. Presence of cholesteryl ester hydroperoxide in human blood plasma. Biochem Biophys Res Commun. 1989 Dec 29;165(3):988–993. doi: 10.1016/0006-291x(89)92700-9. [DOI] [PubMed] [Google Scholar]

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