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. 1977 Jul 1;74(1):119–135. doi: 10.1083/jcb.74.1.119

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

SK Basu, RGW Anderson, JL Goldstein, MS Brown
PMCID: PMC2109871  PMID: 194905

Abstract

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson R. G., Goldstein J. L., Brown M. S. Localization of low density lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2434–2438. doi: 10.1073/pnas.73.7.2434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Basu S. K., Goldstein J. L., Anderson G. W., Brown M. S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3178–3182. doi: 10.1073/pnas.73.9.3178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown M. S., Dana S. E., Goldstein J. L. Cholesterol ester formation in cultured human fibroblasts. Stimulation by oxygenated sterols. J Biol Chem. 1975 May 25;250(10):4025–4027. [PubMed] [Google Scholar]
  4. Brown M. S., Dana S. E., Goldstein J. L. Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2925–2929. doi: 10.1073/pnas.72.8.2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown M. S., Dana S. E., Goldstein J. L. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Feb 10;249(3):789–796. [PubMed] [Google Scholar]
  6. Brown M. S., Faust J. R., Goldstein J. L. Role of the low density lipoprotein receptor in regulating the content of free and esterified cholesterol in human fibroblasts. J Clin Invest. 1975 Apr;55(4):783–793. doi: 10.1172/JCI107989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown M. S., Goldstein J. L. Familial hypercholesterolemia: A genetic defect in the low-density lipoprotein receptor. N Engl J Med. 1976 Jun 17;294(25):1386–1390. doi: 10.1056/NEJM197606172942509. [DOI] [PubMed] [Google Scholar]
  8. Brown M. S., Goldstein J. L. Familial hypercholesterolemia: defective binding of lipoproteins to cultured fibroblasts associated with impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Proc Natl Acad Sci U S A. 1974 Mar;71(3):788–792. doi: 10.1073/pnas.71.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown M. S., Goldstein J. L. Receptor-mediated control of cholesterol metabolism. Science. 1976 Jan 16;191(4223):150–154. doi: 10.1126/science.174194. [DOI] [PubMed] [Google Scholar]
  10. Brown M. S., Goldstein J. L. Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Cell. 1975 Nov;6(3):307–316. doi: 10.1016/0092-8674(75)90182-8. [DOI] [PubMed] [Google Scholar]
  11. Brown M. S., Goldstein J. L. Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J Biol Chem. 1974 Nov 25;249(22):7306–7314. [PubMed] [Google Scholar]
  12. Brown M. S., Ho Y. K., Goldstein J. L. The low-density lipoprotein pathway in human fibroblasts: relation between cell surface receptor binding and endocytosis of low-density lipoprotein. Ann N Y Acad Sci. 1976;275:244–257. doi: 10.1111/j.1749-6632.1976.tb43358.x. [DOI] [PubMed] [Google Scholar]
  13. Brown M. S., Sobhani M. K., Brunschede G. Y., Goldstein J. L. Restoration of a regulatory response to low density lipoprotein in acid lipase-deficient human fibroblasts. J Biol Chem. 1976 Jun 10;251(11):3277–3286. [PubMed] [Google Scholar]
  14. Danon D., Goldstein L., Marikovsky Y., Skutelsky E. Use of cationized ferritin as a label of negative charges on cell surfaces. J Ultrastruct Res. 1972 Mar;38(5):500–510. doi: 10.1016/0022-5320(72)90087-1. [DOI] [PubMed] [Google Scholar]
  15. FAWCETT D. W. SURFACE SPECIALIZATIONS OF ABSORBING CELLS. J Histochem Cytochem. 1965 Feb;13:75–91. doi: 10.1177/13.2.75. [DOI] [PubMed] [Google Scholar]
  16. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  17. Goldstein J. L., Basu S. K., Brunschede G. Y., Brown M. S. Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans. Cell. 1976 Jan;7(1):85–95. doi: 10.1016/0092-8674(76)90258-0. [DOI] [PubMed] [Google Scholar]
  18. Goldstein J. L., Brown M. S. Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Aug 25;249(16):5153–5162. [PubMed] [Google Scholar]
  19. Goldstein J. L., Brown M. S. The LDL pathway in human fibroblasts: a receptor-mediated mechanism for the regulation of cholesterol metabolism. Curr Top Cell Regul. 1976;11:147–181. doi: 10.1016/b978-0-12-152811-9.50011-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Goldstein J. L., Brunschede G. Y., Brown M. S. Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. J Biol Chem. 1975 Oct 10;250(19):7854–7862. [PubMed] [Google Scholar]
  22. 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]
  23. Goldstein J. L., Dana S. E., Brunschede G. Y., Brown M. S. Genetic heterogeneity in familial hypercholesterolemia: evidence for two different mutations affecting functions of low-density lipoprotein receptor. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1092–1096. doi: 10.1073/pnas.72.3.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goldstein J. L., Dana S. E., Faust J. R., Beaudet A. L., Brown M. S. Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease. J Biol Chem. 1975 Nov 10;250(21):8487–8495. [PubMed] [Google Scholar]
  25. Goldstein J. L., Sobhani M. K., Faust J. R., Brown M. S. Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus. Cell. 1976 Oct;9(2):195–203. doi: 10.1016/0092-8674(76)90110-0. [DOI] [PubMed] [Google Scholar]
  26. Grinnell F., Tobleman M. Q., Hackenbrock C. R. The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells. J Cell Biol. 1975 Sep;66(3):470–479. doi: 10.1083/jcb.66.3.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hoare D. G., Koshland D. E., Jr A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J Biol Chem. 1967 May 25;242(10):2447–2453. [PubMed] [Google Scholar]
  28. Jackson R. L., Morrisett J. D., Gotto A. M., Jr Lipoprotein structure and metabolism. Physiol Rev. 1976 Apr;56(2):259–316. doi: 10.1152/physrev.1976.56.2.259. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Lucky A. W., Mahoney M. J., Barrnett R. J., Rosenberg L. E. Electron microscopy of human skin fibroblasts in situ during growth in culture. Exp Cell Res. 1975 May;92(2):383–393. doi: 10.1016/0014-4827(75)90392-4. [DOI] [PubMed] [Google Scholar]
  31. Margolis S., Langdon R. G. Studies on human serum beta-1-lipoprotein. I. Amino acid composition. J Biol Chem. 1966 Jan 25;241(2):469–476. [PubMed] [Google Scholar]
  32. ROTH T. F., PORTER K. R. YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L. J Cell Biol. 1964 Feb;20:313–332. doi: 10.1083/jcb.20.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmidt F. H., von Dahl K. Zur Methode der enzymatischen Neutralfett-Bestimmung in biologischem Material. Z Klin Chem Klin Biochem. 1968 May;6(3):156–159. [PubMed] [Google Scholar]
  34. Weiss L. The cell periphery. Int Rev Cytol. 1969;26:63–105. doi: 10.1016/s0074-7696(08)61634-4. [DOI] [PubMed] [Google Scholar]

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