Tissue culture in the study of atherosclerosis.
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Biomedical subjects
Publications and source records attributed to G H Rothblat.
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The Fu5AH rat hepatoma cell line accumulates cholesteryl ester (CE) upon incubation in medium supplemented with hyperlipemic serum or hyperlipemic serum lipoproteins. This cell line was used to investigate the origin of the fatty acids esterified to cholesterol in intracellular accumulations of CE. The intracellular CE-fatty acid distribution was found to be markedly different from that of the lipoprotein which stimulated the accumulation. Free fatty acids added to the culture medium were found esterified to cholesterol in the cells, demonstrating that cellular esterification contributes to the accumulation of CE. Using a subline of Fu5AH cells containing radioactivity labeled intracellular fatty acids, it was found that about one-third of the fatty acid moiety of CE accumulated by the cells during a 24 hr incubation with hyperlipemic serum was derived from endogenous fatty acids. The drug chloroquine was found to inhibit cellular cholesterol esterification, so that only 4% of CE-fatty acids were derived from endogenous fatty acids. Evidence is presented suggesting a major role for cellular esterification in CE accumulation by Fu5AH cells.
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Previous investigations had demonstrated that Fu5AH rat hepatoma cells accumulated large quantities of esterified cholesterol when grown in hyperlipemic rabbit serum. The present investigation has determined the sources of the cellular esterified cholesterol when the cells were grown in hyperlipemic serum. Cellular esterification of endogenous and exogenous free cholesterol contributed 10% and 30%, respectively. The remaining 60% of the accumulated cellular esterfied cholesterol was derived from exogenous (serum) cholesteryl esters. Evidence for the hydrolysis of a portion of the incorporated esterified cholesterol is presented. A stimulation of free cholesterol incorporation and cellular esterification is elicited by hyperlipemic serum and serum lipoproteins when compared to normolipemic serum present at equivalent exogenous cholesterol concentrations. The effect of hyperlipemic serum is reduced by Tween-80 and Triton WR-1339. Comparative data on esterified cholesterol accumulation, free cholesterol incorporation, and cellular cholesterol esterification in Fu5-5 rat hepatoma cells, L-cells, and rabbit aortic medial cells are presented.
A rapid procedure for the preparation of delipidized serum protein is described. The delipidized protein can be used for the maintenance and growth of tissue culture cells in a lipid-free environment. The extraction procedure greatly reduces all serum lipid classes and the delipidized protein supports the growth of a variety of cells in culture.
The influence of lipoprotein composition on free cholesterol (RC) esterification and accumulation of esterified cholesterol (EC) was studied in cells of Fu5AH rat hepatoma exposed to culture media supplemented with (FC/P) ranging from 0.6 to 2.8. In the presence of normal human serum, FC-lecithin dispersions witb a FC/P greater than one stimulated both the esterification of FC and the accumulation of cellular EC. Conversely, dispersions with FC/P values of approximately one had no effect on either FC esterification or the accumulation of EC when compared to cells grown in the absence of lipid dispersions. No stimulation of cellular response was obtained when FC-rich dispersions were added to cells in the absence of serum; however, stimulation was observed when cells were exposed to isolated human serum lipoproteins (very low density, low density, and high density). With each lipoprotein, FC-lecithin dispersions with FC/P values less than one inhibited cholesteryl ester accumulation, FC-rich dispersions stimulated, and dispersions with FC/P values of approximately one had an effect equivalent to that of isolated lipproteins alone. These studies extablish that cellular FC esterification and EC content are not influenced solely by the FC concentration of medium, but rather respond to the ratio of FC to phospholipidl These studies also suggest that serum lipoproteins participate in the transfer of FC carried by FC-rich lecithin dispersions to cells.
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Hybrids between different human cells (which synthesize cholesterol) and mouse cells (whose end-product of sterol synthesis is desmosterol) were analyzed for the ability to convert desmosterol to cholesterol. Conversion of [(14)C]desmosterol to cholesterol and incorporation of [(14)C]acetate into the end-product sterol were studied in the parental and hybrid cells. Concordant segregation of the conversion of desmosterol to cholesterol and the human chromosome F-20 was observed.
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The incorporation of host cell sterol into vesicular stomatitis virus can be effectively studied in an L cell system. The end product of de novo sterol synthesis in the L cell is desmosterol, and as the concentration of cholesterol in the medium is increased the cells incorporate the exogenous cholesterol and the synthesis of desmosterol decreases. L cells which contained desmosterol as their sole sterol produced virus whose sterol content was similarly composed of only desmosterol. Virus grown in L cells which had a constantly changing sterol ratio also contained a mixture of cholesterol and desmosterol, but the virus was found to be more enriched in cholesterol than in the L cells in which it was grown. Viral stability, growth, and plaquing efficiency were tested and found not to be affected by the alteration of its sterol composition, i.e., by partially or completely replacing cholesterol with desmosterol.
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A number of steroids have been tested in an L-cell tissue culture system to determine their effects on cellular sterol biosynthesis and cellular growth. Cholesterol, desmosterol, lathosterol, 7-dehydrocholesterol, and cholestanone reduce de novo synthesis and produce only limited toxicity at high concentrations of exogenous sterol. Considerable cellular toxicity is observed when cells are grown in the presence of coprostanol and Delta(4)-cholestenone. No marked effect on either cell growth or sterol biosynthesis is produced by cholestanol, beta-sitosterol, stigmasterol, campesterol, ergosterol, cholesteryl oleate, or cholestane.
The following data have been obtained from comparative studies on the metabolism of cholesterol, cholestanol, and beta-sitosterol by L-cell mouse fibroblasts. (1) When the sterols are added to the growth medium under similar conditions, cellular incorporation of cholesterol > cholestanol > beta-sitosterol; (2) only limited cellular esterification of these compounds occurs; (3) no metabolic products arising from the sterols could be detected; (4) influx of all sterols is dependent upon the concentration; and (5) exogenous cholesterol reduces mevalonate incorporation into cellular sterol to a lesser extent than acetate or glucose. The metabolism of these sterols is discussed in relation to their ability to influence de novo sterol biosynthesis.
The principal sterol synthesized by L-cell mouse fibroblasts is desmosterol. Cholesterol was not detected in these cells when they were grown in a sterol-free culture medium. These findings indicate that, in cells, cholesterol can be replaced by desmosterol. Sterol analyses of six other tissue culture cell lines revealed cholesterol synthesis.
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