Reverse cholesterol transport.
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Animals↗
Biomedical subjects
G H Rothblat
Publications and source records attributed to G H Rothblat.
Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions.
A possible mechanism for the formation of smooth muscle foam cells in the atherosclerotic lesion was explored. Cultured macrophages (J774 cell line) were induced to form cytoplasmic cholesteryl ester inclusions by exposure to acetylated low density lipoprotein in the presence of cholesterol-rich phospholipid dispersions. The macrophages were disrupted by brief sonication, and the inclusions were isolated by flotation. When these inclusions were placed in direct contact with cultured smooth muscle cells, cellular uptake of the inclusions in a time- and dose-dependent manner was observed. Light and electron microscopy indicated the presence of lipid inclusions throughout the cytoplasm of the cells. Uptake of inclusion lipid by the smooth muscle cells was inhibited by several metabolic inhibitors, indicating that the process is dependent on metabolic activity. A modest but significant hydrolysis of the cholesteryl ester was observed, showing that the stored cholesteryl esters are metabolically available.
Arteriosclerosis↗
Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture.
Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid, and cholesteryl ester by cells has been compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated, and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances the incorporation of HDL-esterified cholesterol. However, the amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C nuclear magnetic resonance spectroscopy (90.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Metabolism of high density lipoproteins reconstituted with [3H]cholesteryl ester and [14C]cholesterol in the rat, with special reference to the ovary.
In order to study the metabolism of high density lipoprotein (HDL)-carried sterol in the rat, human HDL was reconstituted with [14C]cholesterol and [3H]cholesteryl ester. After iv injection into immature PMSG-human CG primed rats pretreated with 4-aminopyrazolopyrimidine and aminoglutethimide, there was time-dependent accumulation of 3H and 14C in various organs which reached a maximum by 15-90 min. On a milligram wet weight basis, uptake of 3H and 14C was greatest in the adrenals, next in ovaries, followed by the liver, with little uptake by kidneys and spleen. On an organ basis, accumulation was greatest by the liver. At 15-45 min post injection, 60% of the 3H in the ovary was in free sterol, indicating hydrolysis of the accumulated cholesteryl esters, whereas 95% of the 3H in serum remained in sterol esters associated with HDL. Coadministration of excess unlabeled HDL, but not human low density lipoprotein, reduced accumulation of radioactivity by the ovaries and adrenals by 60%, indicating a specific and saturable uptake process. Granulosa cells cultured in lipoprotein-deficient medium with reconstituted HDL formed 3H- and 14C-labeled 20 alpha-hydroxypregn-4-en-3-one. Over a 24-h period, utilization of both [14C]cholesterol and [3H]cholesteryl ester was linear, but rates of utilization of the two sterol moieties were not parallel. There was preferential uptake and utilization of free sterol. A dose-response study demonstrated a Michaelis-Menten constant (Km) of 40-60 micrograms sterol/ml for both free and esterified cholesterol. Lysosomotropic agents (chloroquine and NH4Cl) had no effect on utilization of either free or esterified cholesterol for steroidogenesis but reduced degradation of 125I-labeled low density lipoprotein apoprotein. These findings lend further support to the concept of a distinct HDL pathway in steroidogenic cells of the rat, which involves 1) preferential uptake and utilization of free cholesterol from HDL and 2) does not require lysosomal activity.
20-alpha-Dihydroprogesterone↗
Lipid composition and physical state effects on cellular cholesteryl ester clearance.
The influence of lipid composition and physical state on the rate of cholesteryl ester clearance from cytoplasmic inclusions has been investigated. Our findings demonstrate that the increased rate of clearance correlates with an increased cellular triglyceride content and a more fluid cholesteryl ester physical state. Cultured rat hepatoma cells were induced to accumulate esterified cholesterol in a smectic liquid-crystalline state by exposure to free cholesterol-rich phospholipid dispersions. Addition of cis-unsaturated fatty acids to this loading medium (either oleate, linoleate, linolenate, or eicosadienoate) resulted in a substantial increase in cellular triglyceride content (greater than 7 times non-fatty acid-treated), cellular cholesteryl esters in a liquid state, and a rate of cholesteryl ester clearance twice that of control (approximately 34% versus 17% in 12 h). In studies with oleic acid, storage of cellular cholesteryl esters in a liquid state was found to be dependent on the presence of triglycerides, and the rate at which these cells hydrolyzed cholesteryl esters was proportional to triglyceride levels. Cells exposed to either linoleic or linolenic acid hydrolyzed cholesteryl esters at the faster rate, but in contrast to findings with oleate and eicosadienoate, the storage of cholesteryl esters in a liquid state may also be a consequence of the modified fatty acyl composition of the cholesteryl esters themselves. Addition of a saturated fatty acid (palmitate) or a fatty acid with a trans-double bond (elaidate) to the cholesterol loading media had little effect on cellular triglyceride content, cholesteryl ester physical state, or the rate of cholesteryl ester clearance.
Animals↗
Role of the plasma membrane in the mechanism of cholesterol efflux from cells.
In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg/ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t 1/2) for at least one-third of the cell cholesterol of 3.2 +/- 0.6 and 14.3 +/- 1.5 h, respectively. Plasma membrane vesicles (0.5-5.0 micron diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t 1/2 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 +/- 0.5 and 11.2 +/- 0.7 h, respectively. These t 1/2 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rates indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 +/- 0.1 and 2.9 +/- 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t 1/2 values for cholesterol efflux from these cell lines.
Animals↗
Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035.
Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]pro panamide) has been found to inhibit the accumulation of cholesteryl esters in both rat hepatoma (Fu5AH) cells and arterial smooth muscle cells in culture. To explore the specificity of 58-035, we have studied the esterification of cholesterol, retinol, and glycerides by the Fu5AH cell and by isolated membranes. Exposure of Fu5AH to cholesterol/phospholipid dispersions and 58-035 (greater than 100 ng/ml) for 24 h resulted in greater than 95% inhibition of cholesterol esterification while cellular free cholesterol increased slightly. Inhibition was also rapid; incorporation of [3H]oleate into cholesteryl [3H]oleate equaled only 12% of control value after 30 min with 58-035 at 5 micrograms/ml. In contrast, there was no decrease in [3H]oleate incorporation into phospholipids or diglycerides, nor was the esterification of [3H]retinol inhibited by 58-035. In microsomal fractions, acyl-CoA:cholesterol acyltransferase could be inhibited completely by 58-035, while activities of acyl-CoA: retinol acyltransferase and triglyceride synthesis proceeded at 75-100% of control values. These observations that 58-035 is highly selective allow the inference that acyl-CoA:cholesterol acyltransferase is a separate microsomal enzyme whose activity can be modulated independently from acyl-CoA:retinol acyltransferase and other cellular acyltransferases.
Acyltransferases↗
The conformation of apolipoprotein E isoforms in phospholipid complexes and their interaction with human Hep G2 cells.
The human hepatoma cell line, Hep G2, has been used to compare the metabolism by isolated liver cells of purified isoforms of human apolipoprotein E (apo E). Complexes of [125I]apo E-3/3, 2/2, 3/2 and 4/3 with dimyristoyl phosphatidylcholine (DMPC) were prepared by a detergent-dialysis method: discoidal, bilayer complexes with a stoichiometry of 125 +/- 15 mol DMPC/mol apo E resulted. The predominant phenotype apo E-3/3, and the phenotype apo E-2/2 characteristic of patients with Type III hyperlipoproteinemia, interact similarly with DMPC and adopt the same conformation with 60-70% alpha-helix, as monitored by circular dichroism spectroscopy. The uptake and degradation at 37 degrees C, and binding at 4 degrees C by Hep G2 cells, of [125I]apo E-3/3/DMPC and [125I]apo E-2/2/DMPC complexes were compared. Apo E-3/3 was degraded more rapidly than apo E-2/2 suggesting that the diminished catabolism of the latter phenotype by intact livers is due to lack of recognition by the hepatocytes. The observed degradation of apo E was 3-4 times greater than that which could be attributed to fluid phase endocytosis and low-affinity adsorptive endocytosis. The degradation of [125I]apo A-I by Hep G2 cells can be accounted for by the above endocytotic mechanisms. The distinction between apo E-3/3 and apo E-2/2 isoforms is attributed to the presence of a cell-surface receptor on Hep G2 cells which binds apo E-3/3 with a higher affinity than apo E-2/2.
Apolipoproteins↗
Cellular cholesteryl ester clearance. Relationship to the physical state of cholesteryl ester inclusions.
The hypothesis that clearance of cellular cholesteryl ester deposits may be a function of the physical state of the stored lipid has been investigated. Cultured rat hepatoma cells were induced to store cholesteryl ester in either anisotropic inclusions by exposure to free cholesterol-rich phospholipid dispersions or isotropic inclusions by exposure to identical dispersions supplemented with oleic acid. Differential scanning calorimetry demonstrated an order/disorder transition at 43 degrees C for cholesteryl esters stored in anisotropic inclusions; the enthalpy of this transition was consistent with a smectic liquid crystalline to liquid transition. Lipids in cells with isotropic inclusions displayed no order/disorder transitions over the range 20-80 degrees C, indicating that the lipids are in a liquid state. The presence of oleic acid did not influence the mass of cholesteryl ester stored but increased the amount of stored triglyceride. Fatty acyl compositions of the cholesteryl esters were different under the two loading conditions; in particular, there was 38% cholesteryl oleate in anisotropic inclusions and 65% cholesteryl oleate in isotropic inclusions. Kinetics of cholesteryl ester clearance from cells with either anisotropic or isotropic inclusions were studied during a 12-h exposure to acceptors of free cholesterol. In both cases, cholesteryl ester clearance is essentially linear over 12 h and is directly proportional to the initial content of cholesteryl ester. However, the fraction of initial content of cholesteryl ester cleared in 12 h is 0.17 +/- 0.05 for cells with anisotropic inclusions and 0.34 +/- 0.09 for cells with isotropic inclusions. Our data demonstrate that the more rapid clearance of cholesteryl ester by cells with isotropic inclusions can be correlated with the physical state of the cholesteryl ester.
Animals↗
Hepatic lipase stimulates the uptake of high density lipoprotein cholesterol by hepatoma cells.
The objective of this study was to determine whether high density lipoproteins (HDL) that have been treated with hepatic lipase have an enhanced ability to deliver cholesterol to cells. Human HDL was incubated with rat hepatic lipase, reisolated, and subjected to compositional analysis. Approximately 28% of the HDL phosphatidylcholine was hydrolyzed by the hepatic lipase but no change was detected in the cholesterol or apoprotein content of the HDL compared to HDL incubated with heat-inactivated hepatic lipase. Cultured rat hepatoma cells exposed to hepatic lipase-modified HDL showed an increased uptake of HDL free cholesterol relative to cells exposed to control HDL. This increased delivery of HDL free cholesterol was demonstrated by both isotopic and mass determinations and it contributed to a 1.6-fold increase in total cellular cholesterol content relative to cells treated with control HDL. The free cholesterol delivered by the HDL is functionally available to the cell as evidenced by the conversion of radiolabeled free cholesterol to cholesteryl ester. The stimulation of free cholesterol delivery was dose-dependent up to a level of 100 micrograms of HDL free cholesterol/ml of extracellular medium, and was directly related to the extent of phosphatidylcholine hydrolysis. The enhanced cellular accumulation of HDL free cholesterol observed with hepatic lipase appears to be due to the phospholipase activity of this enzyme, since similar results were obtained with HDL that had been modified by snake venom phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mechanism of cholesterol efflux from cells. Effects of acceptor structure and concentration.
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Animals↗
Mobilization of cholesterol from cholesterol ester-enriched tissue culture cells by phospholipid dispersions.
The accumulation of cholesterol esters in foam cells of the arterial intima is an important characteristic of fatty streak lesions of atherosclerosis. We wished to know if cholesterol ester accumulations in cells could be mobilized by altering their external milieu. Thus, phospholipid dispersions were used to remove cholesterol from a cholesterol ester-enriched cell line. Rat hepatoma cells, Fu5AH, were loaded with cholesterol esters by incubation in medium supplemented with hyperlipemic rabbit serum. After removing the loading medium, we incubated the cells in serum-free medium containing egg phosphatidylcholine dispersions. Unesterified cellular cholesterol level decreased in the first 4 h and then remained at a constant level. The cholesterol esters decreased after a lag time of about 2 h and the triacylglycerol level increased after 3 h. The decrease in cellular cholesterol ester depended on the amount of phospholipid in the medium. Cellular cholesterol ester decreased with increasing concentration of medium phospholipid to 2 mumols/ml and then plateaued. The removed cellular sterols appeared in the medium as free cholesterol. Since there was no measurable cholesterol esterase activity in the medium, the cholesterol ester in the cells was hydrolyzed before it appeared in the medium. The fatty acyl composition of the cellular cholesterol esters remained unchanged after significant reduction, suggesting that the hydrolysis of cholesterol esters was not specific for the acyl chain. Sphingomyelin and dimyristoyl phosphatidylcholine dispersions, though cytotoxic, were also effective in reducing cellular cholesterol esters. These experiments demonstrate that cholesterol ester accumulations in these cells can be reduced when phospholipid dispersions are used as cholesterol acceptors in the extracellular medium.
Animals↗
The effect of lidocaine on cholesterol influx, esterification, and accumulation in cultured cells.
Lidocaine, a local anesthetic, inhibited cholesterol esterification in various cultured cells derived from tissues of the mouse, rat, hamster, monkey, and man. Esterification of exogenous [1-14C]oleate, fatty acid synthesized in situ from [1-14C]acetate, and exogenous [7-3H]cholesterol was reduced 20-50% with 1.5 mM lidocaine in the culture medium. In Fu5AH rat hepatoma cells, incubated for 24 h with hyperlipemic serum lipoproteins and increasing levels of lidocaine up to 1.5 mM, unesterified (free) cholesterol mass of the cells increased about 25% whereas the cholesteryl ester mass fell about 40%. The net result was a reduction in total cellular sterol. When the cells were incubated with lidocaine and hyperlipemic serum lipoproteins labeled with [7-3H(N)]cholesterol of known specific activity, incorporation of exogenous free cholesterol into cellular free cholesterol was constant, whereas there was a dose-dependent reduction in the amount of exogenous cholesterol appearing in cholesteryl esters. Additionally, a comparison of specific activities of cellular free cholesterol and cholesteryl esters at intervals over a 24-h period suggests that lidocaine also inhibits lysosomal cholesterol ester hydrolase (EC 3.1.1.13) in the Fu5AH cell line.
Acetates↗
Lipoprotein apolipoprotein synthesis by human hepatoma cells in culture.
Lipoprotein synthesis was demonstrated by double diffusion with low density lipoprotein antibody, and by 3H-labeled amino acid incorporation into proteins of the d less than 1.063 g/ml centrifugally isolated lipoprotein fraction. Radioactive label was incorporated predominantly into apolipoprotein B (60%), apolipoprotein A-I (20%) and apolipoprotein C (12%), as determined by Sepharose column chromatography and polyacrylamide gel electrophoresis. Incorporation of radioactive label into apolipoprotein B was inhibited by the presence of albumin in the medium, and was restored to control levels with the addition of 1 mM oleic acid, indicating that cell synthesis of apolipoproteins could be modified by culture conditions. The human hepatoma cell line, Hep G2, provides a potential in vitro model for the study of regulation of human hepatic lipoprotein and apolipoprotein synthesis.
Amino Acids↗
Effects of metabolic inhibitors on the synthesis and release of lipoprotein lipase in cultured cells derived from the stromal-vascular fraction of rat adipose tissue.
Cultured cells derived from the stromal-vascular fraction of rat epididymal adipose tissue have been used to study the release of lipoprotein lipase in response to heparin. In the presence of heparin, lipoprotein lipase appeared in the medium biphasically, with an initial rapid rise followed by a slower linear release. The initial increase in activity in the medium was paralleled by a decrease in cell-associated activity. Over the course of the slower phase of release, cell-associated lipoprotein lipase levels remained constant. Treatment of cultures with cycloheximide or 2-deoxyglucose eliminated the second phase of release as well as cell-associated lipoprotein lipase activity. Treatment of cultures with Colcemid decreased the second phase of release but increased cell-associated lipoprotein lipase activity. These data suggest that exposure to heparin results in a rapid release of pre-formed enzyme and that continued release is dependent on both protein synthesis and transport of the enzyme to the cell surface.
Adipose Tissue↗
Stimulation of esterified cholesterol accumulation in tissue culture cells exposed to high density lipoproteins enriched in free cholesterol.
Human high density lipoprotein enriched in free cholesterol was obtained by exposing the lipoprotein to lipid dispersions having a free cholesterol/lecithin molar ratio greater than two. The metabolism of cholesterol was studied in tissue culture cells exposed to normal and cholesterol-enriched lipoproteins. Incubation of Fu5-AH rat hepatoma cells in medium containing cholesterol-enriched lipoprotein resulted in the accumulation of cellular cholesterol whereas normal high density lipoprotein produced no change in cellular content. The accumulated sterol was recovered primarily as esterified cholesterol and was derived almost entirely from lipoprotein free cholesterol. The esterification of incorporated free cholesterol and the cellular cholesterol content were directly related to the molar ratio of free cholesterol to phospholipid in the lipoprotein and to the concentration of lipoprotein in the culture medium. Isotopic experiments utilizing lipoprotein labeled with 125I or [4-14C]cholesteryl oleate demonstrated that a large fraction of the cholesterol incorporated from lipoprotein enriched in free cholesterol occurred by mechanisms that did not result in lipoprotein internalization and degradation. The response of other tissue culture cells to cholesterol/phospholipid dispersions is presented. The data indicate that the lipid composition of a lipoprotein can regulate free cholesterol uptake and esterification as well as cellular cholesterol content.
Animals↗
Lipid requirement for cell cycling. The effect of selective inhibition of lipid synthesis.
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Biotin↗
Release of lipoprotein lipase from rat adipose tissue cells grown in culture.
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Adipose Tissue↗