Internalized Plasma Membrane Cholesterol Passes through an Endosome Compartment That Is Distinct from the Acid Vesicle–Lysosome Compartment

Cholesterol from the plasma membrane of MA-10 Leydig tumor cells is internalized into the cell and either esterified or used as substrate for steroid hormone synthesis. In the present studies we show that chloroquine and sphinganine cause LDL cholesterol and cholesteryl esters to accumulate in the cells. A lysosome fraction contained the excess cholesterol and cholesteryl esters. Both inhibitors blocked the conversion of plasma membrane cholesterol into intracellular cholesteryl esters and caused dose-dependent inhibition of dibutyryl-cAMP-stimulated progesterone synthesis. Radiolabeled cholesterol applied to the plasma membrane of MA-10 cells accumulated in the lysosome fraction of chloroquine and sphinganine-treated cells. Evidence that these inhibitors did not require the Golgi was provided by experiments using brefeldin A. Experiments utilizing a fluorescent cholesterol analogue and a lysosomal marker indicated that cholesterol entered the cells in structures that were different than the acidic vesicle–lysosome compartment. Consistent with this observation was the observation that the peak fluorescence fractions of cells subjected to density gradient centrifugation was of lower density than the lysosome fraction.     

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.     

Quantification of LDL cholesteryl ester contribution to biliary steroids in the rat

The proportion of LDL cholesteryl ester converted to biliary steroids was quantified in the rat. The pre-existing pool of bile was allowed to drain for 10-12 h through a bile duct cannula. A single intravenous pulse injection of LDL labelled with [3H]cholesterol linoleyl ester was made, followed by a constant infusion of the same material in order to maintain constant specific radioactivity in plasma. A new steady state was achieved within 6 h and bile samples were then collected hourly until 12 h. Although substantial amounts (53-61 micrograms/h) of cholesteryl ester were released into the liver during LDL catabolism, only a very small fraction (0.8-1.90 micrograms/h) was found in biliary steroids. The proportion of LDL cholesteryl esters contributing to biliary steroids was only 1-2%. These results perhaps explain why perturbations to accelerate bile acid excretion have no effect on plasma LDL cholesterol concentration in the rat.     

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

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.     

Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts

The kinetics of low density lipoprotein (LDL) cholesterol transport to the plasma membrane of Chinese hamster ovary (CHO) cells was studied. LDL was reconstituted with [3H]cholesteryl linoleate and added to CHO cells in a pulse-chase experiment. The internalization and lysosomal cleavage of reconstituted LDL (rLDL) [3H]cholesteryl linoleate to free [3H]cholesterol occurred with a half-time of 37 min after a 30-min lag. The rate of transport of released [3H]cholesterol to the plasma membrane was measured by brief (20-30 sec) cholesterol oxidase treatment of intact, adherent cells: the half-time of transport was 42 min. The similarity in the rate of free cholesterol release from rLDL and transport of this cholesterol to the plasma membrane suggests very rapid transport of rLDL cholesterol from the lysosome to the plasma membrane. Cells were shown to be intact throughout the cholesterol oxidase treatment by the absence of cell-derived lactate dehydrogenase (LDH) activity or K+ in the assay buffer.     

Short-term metabolism of cholesteryl ester from low-density lipoprotein in primary monolayers of bovine adrenal cortical cells

The uptake and metabolism of [14C]cholesteryl ester in bovine LDL to cortisol and to cholesteryl ester was studied in monolayer cultures of bovine adrenal cortical cells over short time periods of up to 8 h. The experiments were designed to determine the intracellular pathway followed by the cholesterol derived from the LDL cholesteryl ester and how this is modified in the short term by the tropic hormone ACTH. The cells were cultured in the presence of mevinolin to remove the contribution of endogenous synthesis of cholesterol for supply of substrate for steroidogenesis. The specific activity of the cortisol secreted by the cells was measured under a variety of conditions. Control incubations showed a relatively steady specific activity in the cortisol secreted over an 8 h period. In the presence of ACTH the specific activity of the cortisol was significantly reduced for the first 2 h of the experiment. This is consistent with dilution of the [14C]cholesterol from the LDL with non-radioactive free cholesterol released from the intracellular stores of cholesteryl ester in the presence of ACTH. The inhibitor of acyl-CoA:cholesterol acyltransferase, Sandoz compound 58-035, increased the specific activity of the secreted cortisol in the absence of ACTH, indicating that much of the incoming cholesterol would normally be esterified but was here diverted to steroidogenesis. In the presence of ACTH this increase was observed only during the first 2 h of the experiment, after which inhibition of acyl-CoA:cholesterol acyltransferase had no effect on the specific activity of the cortisol. The adrenal cells were further fractionated into mitochondrial, lysosomal and microsomal plus cytosol fractions and the appearance of free and esterified cholesterol from the labelled LDL measured in these fractions over a period of up to 8 h. ACTH stimulated the uptake of LDL-cholesteryl ester into the cells and tended to increase the relative amounts of free cholesterol in the cells, consistent with its role in promoting supply of cholesterol for steroidogenesis. These experiments allow the roles of endogenous cholesteryl ester and lipoprotein-derived cholesteryl ester in the bovine adrenal cortical cells to be observed over a short time scale. They show that the cells make a substantial change in the internal flux of cholesterol in a short time after stimulation with ACTH and in these cultures the full expression of the presence of ACTH takes up to 2 h.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Intravascular metabolism of lipoprotein cholesteryl esters in African green monkeys: differential fate of doubly labeled cholesteryl oleate

High density lipoproteins (HDL), doubly labeled with [3H]cholesteryl oleate and cholesteryl [14C]oleate, were reinjected to study HDL cholesteryl ester metabolism in African green monkeys. The transfer of labeled HDL cholesteryl ester to low density lipoprotein (LDL) was rapid and equilibration of the [3H]cholesteryl oleate and cholesteryl [14C]oleate specific activities in LDL and HDL occurred within 90 min after reinjection. The apparent rates of disappearance from the circulation of the two moieties of the cholesteryl ester were different. In the same four animals, the residence time for the turnover of plasma [3H]cholesterol averaged 6.1 days while the residence time for the removal of cholesteryl [14C]oleate from plasma was approximately 2.1 days. These results suggest that for some lipoprotein cholesteryl esters removed from plasma, the cholesterol moiety subsequently reappeared in plasma. The difference between the rate of decay of the 14C-labeled fatty acid moiety, which represents all of the cholesteryl ester removed from plasma (0.48 pools/day) and the decay of the 3H-labeled cholesterol moiety, which represents the sum of cholesteryl ester removal and cholesterol reappearance (0.16 pools/day), is the fraction of the cholesteryl ester pool recycled per day (0.32 pools/day or 22.5 mg/kg per day). In other words, approximately 68% of the cholesterol moiety that was removed from plasma as cholesteryl oleate reappeared in the plasma cholesterol pool. These studies support the concept that an efficient reutilization cycle for plasma cholesterol occurs, i.e., the cholesteryl ester molecule can exit and the cholesterol moiety can re-enter plasma without effective equilibration of the cholesterol moiety with extravascular cholesterol pools.     

Cholesteryl ester enrichment of plasma low-density lipoproteins in hamsters fed cereal-based diets containing cholesterol

Male Syrian hamsters were fed 0.02, 0.03, or 0.05% cholesterol to test the hypothesis that moderate cholesterol intake increases the cholesteryl ester content of the plasma low-density lipoproteins (LDL). Dietary cholesterol levels of 0.02%-0.05% were chosen to reflect typical human intakes of cholesterol. Hamsters were fed ad libitum a cereal-based diet (modified NIH-07 open formula) for 15 weeks. Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased plasma LDL and high-density lipoprotein cholesterol concentration, increased liver cholesterol concentration, and increased total aorta cholesterol content. The cholesteryl ester content of plasma LDL was determined as the molar ratio of cholesteryl ester to apolipoprotein B and to surface lipid (i.e., phospholipid + free cholesterol). Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased cholesteryl ester content of LDL particles. Furthermore, cholesteryl ester content of LDL was directly associated with increased total aorta cholesterol, whereas a linear relationship between plasma LDL cholesterol concentration and aorta cholesterol was not observed. Thus, the data suggest that LDL cholesteryl ester content may be an important atherogenic feature of plasma LDL.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

Cyclic AMP mediated modification of cholesterol traffic in Leydig tumor cells

The level of nonesterified cholesterol within MA-10 Leydig Tumor cells is regulated acutely by trophic hormones (Freeman, D. A., and Ascoli, M. (1982) J. Biol. Chem. 257, 14231-14238). In the present studies, we localize the site of this steroidogenic cholesterol to the plasma membrane and characterize the means by which this membrane becomes cholesterol-depleted. It is possible to detect the translocation of both newly synthesized cholesterol and cholesterol derived from lipoproteins from the cell interior to the plasma membrane. Stimulated MA-10 cells that are actively producing steroid hormones divert cholesterol from the normal intracellular or plasma membrane acceptor sites into the steroid biosynthetic pathway. Another important effect of steroidogenic stimulation is to cause internalization of plasma membrane cholesterol. Changes in cholesterol traffic in stimulated cells can be blocked by preventing the utilization of cholesterol for steroidogenesis. This later finding indicates that the changes in cholesterol transport induced by trophic hormones are consequences rather than primary causes of steroidogenic stimulation.     

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters.     

Influence of adrenocorticotropin on transport of a cholesteryl linoleate-low density lipoprotein complex into adrenal tumor cells.

The action of adrenocorticotropin (ACTH) on the specific (receptor-mediated) uptake of cholesteryl linoleate . low density lipoprotein complexes was examined in Y-1 mouse adrenal tumor cells. High affinity binding (KA 4.1 X 10(8) M) was observed with ACTH; lower affinity was seen in the absence of ACTH. The effect of ACTH was observed within 10 min at physiological concentrations of low density lipoprotein (100 microgram/ml). Binding was followed by uptake (internalization) of the ester . lipoprotein complex which was transported to lysosomes. The site of action of ACTH was localized to the uptake process (internalization) since no effect of ACTH was observed on binding to the cell membrane nor on movement of internalized complex to lysosomes. ACTH increases the transport of cholesterol derived from cholesterol ester to the mitochondria. This cholesterol is converted to 20 alpha-hydroxypregn-4-en-3-one and this conversion is accelerated by ACTH. Dibutyryl cyclic AMP (but not butyrate) also stimulates uptake of cholesteryl linoleate . low density lipoprotein. The process stimulated by ACTH and dibutyryl cyclic AMP is specific for low density (as opposed to high density) lipoprotein and for ACTH as distinct from other peptide hormones. The possible physiological importance of this response is considered.     

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

Cholesteryl ester turnover in human plasma lipoproteins during cholestyramine and clofibrate therapy

The effects of cholestyramine and of clofibrate on the turnover rates of individual cholesteryl esters in whole human plasma and in each of the three classes of plasma lipoproteins have been studied. Four hyperlipidemic patients (two under treatment with each of the two drugs) were injected intravenously with cholesterol-(14)C, and serial plasma samples were collected after 3-4 hr, 8 hr, 24 hr, and 4-5 days. The plasma samples were separated into three classes of lipoproteins by ultracentrifugation. The cholesteryl esters and free cholesterol were isolated from each sample, and the specific radioactivity of the free and esterified cholesterol was determined. The specific radioactivity of each individual cholesteryl ester was then determined for each sample, by separately measuring the distribution of cholesterol mass and of radioactivity among four different cholesteryl ester groups, namely the saturated, mono-, di-, and tetra-unsaturated esters. In all subjects the plasma cholesteryl esters were metabolically heterogeneous, and could be divided into three pools corresponding to the three classes of plasma lipoproteins. High density lipoprotein (d > 1.063) cholesteryl esters showed the greatest fractional turnover rate, and low density lipoprotein (d 1.019-1.063) cholesteryl esters showed the smallest fractional turnover rate. In each subject the cholesteryl ester composition of the three classes of plasma lipoprotein was almost identical. Within each lipoprotein, and in whole plasma, all the different individual cholesteryl esters were found to turn over at the same fractional rate. In all respects these results were similar to those previously obtained with normal subjects. The results suggest that neither drug has a strongly selective effect on the turnover of one particular cholesteryl ester, or on the turnover or composition of the cholesteryl esters in one particular plasma lipoprotein.     

Growth-related modulation of human skin fibroblast cholesterol distribution and metabolism

The relationship between cell growth state and the metabolism and distribution of cellular cholesterol was studied in human skin fibroblasts. Cells made quiescent by serum starvation maintained a smaller fraction of total free cholesterol in a pool susceptible to oxidation by added cholesterol oxidase compared to growing cells. The growth-related differences in the distribution of free cholesterol were magnified in cells which were preincubated in low-density lipoprotein. These latter cells hydrolyzed cholesteryl ester which had accumulated in the presence of LDL, resulting in an increased level of cellular free cholesterol after growth activation. By preincubating cells in [3H]cholesterol linoleate-labeled LDL, it could be demonstrated that activation of cell growth facilitated the appearance of LDL-derived cholesterol in a pool accessible to cholesterol oxidase. These studies suggest that onset of growth in fibroblasts leads to a redistribution of free cholesterol from intracellular to plasma membrane compartments. Furthermore, activation of cell growth in cholesterol loaded cells leads to the net conversion of cholesteryl ester to free cholesterol and most of the latter is in the plasma membrane.     

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Mechanisms and consequences of cellular cholesterol exchange and transfer

It is apparent from consideration of the reactions involved in cellular cholesterol homeostasis that passive transfer of unesterified cholesterol molecules plays a role in cholesterol transport in vivo. Studies in model systems have established that free cholesterol molecules can transfer between membranes by diffusion through the intervening aqueous layer. Desorption of free cholesterol molecules from the donor lipid-water interface is rate-limiting for the overall transfer process and the rate of this step is influenced by interactions of free cholesterol molecules with neighboring phospholipid molecules. The influence of phospholipid unsaturation and sphingomyelin content on the rate of free cholesterol exchange are known in pure phospholipid bilayers and similar effects probably occur in cell membranes. The rate of free cholesterol clearance from cells is determined by the structure of the plasma membrane. It follows that the physical state of free cholesterol in the plasma membrane is important for the kinetics of cholesterol clearance and cell cholesterol homeostasis, as well as the structure of the plasma membrane. Bidirectional flux of free cholesterol between cells and lipoproteins occurs and rate constants characteristic of influx and efflux can be measured. The direction of any net transfer of free cholesterol is determined by the relative free cholesterol/phospholipid molar ratios of the donor and acceptor particles. Cholesterol diffuses down its gradient of chemical potential generally partitioning to the phospholipid-rich particle. Such a surface transfer process can lead to delivery of cholesterol to cells. This mechanism operates independently of any lipoprotein internalization by receptor-mediated endocytosis. The influence of enzymes such as lecithin-cholesterol acyltransferase and hepatic lipase on the direction of net transfer of free cholesterol between lipoproteins and cells can be understood in terms of their effects on the pool sizes and the rate constants for influx and efflux. Excess accumulation of free cholesterol in cells stimulates the rate of cholesteryl ester formation and induces deposition of cholesteryl ester inclusions in the cytoplasm similar to the situation in the 'foam' cells of atherosclerotic plaque. Clearance of cellular cholesteryl ester requires initial hydrolysis to free cholesterol followed by efflux of this free cholesterol. The rate of clearance of cholesteryl ester from cytoplasmic droplets is influenced by the physical state of the cholesteryl ester; liquid-crystalline cholesteryl ester is removed more slowly than cholesteryl ester in a liquid state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of the atherogenic dyslipoproteinemia induced by dietary cholesterol in rhesus monekys (Macaca mulatta).

Hypercholesterolemia was induced in adult male rhesus monkeys with a high-fat diet containing an elevated cholesterol level (0.5%). Plasma lipoproteins were chromatographically separated into four size populations (regions) that were subdivided by density until fractions with single electrophoretic mobilities were obtained. The region III lipoproteins (LDL) contained 80% of plasma cholesterol and were present in the highest concentration of all fractions. Their molecular weight was increased over that of controls so that each particle averaged 1.8 times the number of cholesteryl ester molecules as did control LDL. Region II lipoproteins, a heterogeneous group, were present in next highest concentration. Most were cholesteryl ester-rich, beta-migrating lipoproteins that overlapped the VLDL and LDL density ranges; apoB was the predominant apoprotein. One region II subfraction had pre beta 2 migration and the density range. 1.050 less than d less than 1.10. Another subfraction, cholesteryl ester-rich VLDL including only about 1% of plasma cholesterol, had pre beta 1 migration and apoB and apoC as the predominant apoproteins with no apoprotein E. Region I lipoproteins were larger sized, slow beta-migrating cholesteryl ester-rich VLDL that included 5% of plasma cholesterol. ApoB and apoE were the predominant apoproteins. Region IV lipoproteins (HDL) contained 4% of the plasma cholesterol; their concentration was decreased to about 1/3 of the control level. Atherogenic features of the diet-induced dyslipoproteinemia included the increased plasma concentrations and cholesteryl ester contents of the region I, II, and III lipoproteins in addition to the decreased HDL concentration.