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. 1987 Nov 1;247(3):739–746. doi: 10.1042/bj2470739

Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

L M Havekes 1, E C de Wit 1, H M Princen 1
PMCID: PMC1148474  PMID: 3426559

Abstract

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bilheimer D. W., Goldstein J. L., Grundy S. M., Starzl T. E., Brown M. S. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia. N Engl J Med. 1984 Dec 27;311(26):1658–1664. doi: 10.1056/NEJM198412273112603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown M. S., Goldstein J. L. Lipoprotein receptors in the liver. Control signals for plasma cholesterol traffic. J Clin Invest. 1983 Sep;72(3):743–747. doi: 10.1172/JCI111044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown M. S., Ho Y. K., Goldstein J. L. The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters. J Biol Chem. 1980 Oct 10;255(19):9344–9352. [PubMed] [Google Scholar]
  5. Cohen L. H., Griffioen M., Havekes L., Schouten D., van Hinsbergh V., Kempen H. J. Effects of compactin, mevalonate and low-density lipoprotein on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2. Biochem J. 1984 Aug 15;222(1):35–39. doi: 10.1042/bj2220035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dashti N., Wolfbauer G., Koren E., Knowles B., Alaupovic P. Catabolism of human low density lipoproteins by human hepatoma cell line HepG2. Biochim Biophys Acta. 1984 Jul 26;794(3):373–384. doi: 10.1016/0005-2760(84)90003-1. [DOI] [PubMed] [Google Scholar]
  7. Edge S. B., Hoeg J. M., Triche T., Schneider P. D., Brewer H. B., Jr Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor. J Biol Chem. 1986 Mar 15;261(8):3800–3806. [PubMed] [Google Scholar]
  8. Ellsworth J. L., Erickson S. K., Cooper A. D. Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid. J Lipid Res. 1986 Aug;27(8):858–874. [PubMed] [Google Scholar]
  9. Erickson S. K., Fielding P. E. Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2. J Lipid Res. 1986 Aug;27(8):875–883. [PubMed] [Google Scholar]
  10. Everson G. T., Polokoff M. A. HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation. J Biol Chem. 1986 Feb 15;261(5):2197–2201. [PubMed] [Google Scholar]
  11. Fainaru M., Havel R. J., Imaizumi K. Apoprotein content of plasma lipoproteins of the rat separated by gel chromatography or ultracentrifugation. Biochem Med. 1977 Jun;17(3):347–355. doi: 10.1016/0006-2944(77)90040-0. [DOI] [PubMed] [Google Scholar]
  12. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  13. Goldstein J. L., Dana S. E., Brown M. S. Esterification of low density lipoprotein cholesterol in human fibroblasts and its absence in homozygous familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4288–4292. doi: 10.1073/pnas.71.11.4288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harders-Spengel K., Wood C. B., Thompson G. R., Myant N. B., Soutar A. K. Difference in saturable binding of low density lipoprotein to liver membranes from normocholesterolemic subjects and patients with heterozygous familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6355–6359. doi: 10.1073/pnas.79.20.6355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Havekes L. M., Schouten D., de Wit E. C., Cohen L. H., Griffioen M., van Hinsbergh V. W., Princen H. M. Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions. Biochim Biophys Acta. 1986 Feb 12;875(2):236–246. doi: 10.1016/0005-2760(86)90173-6. [DOI] [PubMed] [Google Scholar]
  16. Havekes L. M., Verboom H., de Wit E., Yap S. H., Princen H. M. Regulation of low density lipoprotein receptor activity in primary cultures of human hepatocytes by serum lipoproteins. Hepatology. 1986 Nov-Dec;6(6):1356–1360. doi: 10.1002/hep.1840060623. [DOI] [PubMed] [Google Scholar]
  17. Havekes L., van Hinsbergh V., Kempen H. J., Emeis J. The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2. Biochem J. 1983 Sep 15;214(3):951–958. doi: 10.1042/bj2140951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kosykh V. A., Preobrazhensky S. N., Ivanov V. O., Tsibulsky V. P., Repin V. S., Smirnov V. N. High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture. FEBS Lett. 1985 Apr 8;183(1):17–20. doi: 10.1016/0014-5793(85)80944-3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lange Y., Ramos B. V. Analysis of the distribution of cholesterol in the intact cell. J Biol Chem. 1983 Dec 25;258(24):15130–15134. [PubMed] [Google Scholar]
  21. Leichtner A. M., Krieger M., Schwartz A. L. Regulation of low density lipoprotein receptor function in a human hepatoma cell line. Hepatology. 1984 Sep-Oct;4(5):897–901. doi: 10.1002/hep.1840040518. [DOI] [PubMed] [Google Scholar]
  22. Oram J. F. Effects of high density lipoprotein subfractions on cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells. Arteriosclerosis. 1983 Sep-Oct;3(5):420–432. doi: 10.1161/01.atv.3.5.420. [DOI] [PubMed] [Google Scholar]
  23. Patsch W., Schonfeld G., Gotto A. M., Jr, Patsch J. R. Characterization of human high density lipoproteins by zonal ultracentrifugation. J Biol Chem. 1980 Apr 10;255(7):3178–3185. [PubMed] [Google Scholar]
  24. Ross A. C., Go K. J., Heider J. G., Rothblat G. H. Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035. J Biol Chem. 1984 Jan 25;259(2):815–819. [PubMed] [Google Scholar]
  25. Schmitz G., Assmann G., Bowyer D. E. A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography. I. Sample preparation, chromatography, and densitometry. J Chromatogr. 1984 Apr 13;307(1):65–79. doi: 10.1016/s0378-4347(00)84073-6. [DOI] [PubMed] [Google Scholar]
  26. Schmitz G., Niemann R., Brennhausen B., Krause R., Assmann G. Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase. EMBO J. 1985 Nov;4(11):2773–2779. doi: 10.1002/j.1460-2075.1985.tb04003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Slater H. R., Smith E. B., Robertson F. W. The effect of delipidated high density lipoprotein on human leukocyte sterol synthesis. Atherosclerosis. 1980 Jan;35(1):41–49. doi: 10.1016/0021-9150(80)90026-x. [DOI] [PubMed] [Google Scholar]
  28. Steinberg D. Lipoproteins and atherosclerosis. A look back and a look ahead. Arteriosclerosis. 1983 Jul-Aug;3(4):283–301. doi: 10.1161/01.atv.3.4.283. [DOI] [PubMed] [Google Scholar]
  29. Suckling K. E., Stange E. F. Role of acyl-CoA: cholesterol acyltransferase in cellular cholesterol metabolism. J Lipid Res. 1985 Jun;26(6):647–671. [PubMed] [Google Scholar]
  30. Tabas I., Weiland D. A., Tall A. R. Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation. J Biol Chem. 1986 Mar 5;261(7):3147–3155. [PubMed] [Google Scholar]
  31. Thrift 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 Mar;27(3):236–250. [PubMed] [Google Scholar]
  32. Wu G. Y., Wu C. H., Rifici V. A., Stockert R. J. Activity and regulation of low density lipoprotein receptors in a human hepatoblastoma cell line. Hepatology. 1984 Nov-Dec;4(6):1190–1194. doi: 10.1002/hep.1840040615. [DOI] [PubMed] [Google Scholar]

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