Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Induction of macrophage growth by lipids

Lipoproteins from ascitic tumors in mice and lipids extracted from these lipoproteins induced growth of murine peritoneal macrophages in vitro. The lipid components with activity were examined by use of lipid vesicles or liposomes. Liposomes prepared from egg-yolk PC alone did not induce macrophage growth, but those prepared from mixtures of egg-yolk PC and cholesterol or cholesteryl esters other than cholesteryl oleate, or triglycerides other than triolein, enhanced 3H-TdR incorporation into macrophages. The free fatty acids examined had no effect on 3H-TdR incorporation. These results suggest that growth of macrophages is induced by ordinary lipids present in lipoproteins or cell membranes that the macrophages scavenge in the body.     

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.     

Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by relieving product inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.     

The effect of calcium antagonists on cholesterol metabolism in human aortal intima cells and macrophage lines]

Effects of two Ca-antagonists, verapamil and nifedipine, on the total cellular cholesterol content and accumulation, as well as on the synthesis and hydrolysis of cholesteryl esters in human aortic intimal smooth muscle cells and P388D1 cell line have been studied. Verapamil and nifedipine used at 10(-6) M and higher concentrations decreased the total cellular cholesterol content (by 25-40%) in intimal cells isolated from atherosclerotic lesions without any effect on the cholesterol content in normal intimal cells or P388D1 cells. At 2 x 10(-5) M verapamil and nifedipine prevented the accumulation of cholesterol induced by atherogenic blood serum or atherogenic low density lipoproteins in both types of cells. At 10(-5) M and higher concentrations verapamil and nifedipine inhibited (2-3-fold) cholesteryl ester synthesis in intimal cells and, used at 10(-6) M and higher doses, in P388D1 cells as well. Verapamil and nifedipine (2 x 10(-5) M) enhanced the hydrolysis of cholesteryl esters in both types of cells. The Ca-channel agonist Bay K8644 had no effect on cholesteryl ester synthesis, nor did it suppress its inhibition by Ca-antagonist. The beta-receptor blocker propranolol induced the accumulation of cholesterol in intimal cells and inhibited the synthesis and hydrolysis of cholesterol esters in these cells. The data obtained suggest that the antiatherosclerotic action of Ca-blockers is determined by their ability to reduce the cellular cholesterol content which is suggested to be the result of enhanced hydrolysis of cellular cholesteryl esters.     

Preferential enrichment of large-sized very low density lipoprotein populations with transferred cholesteryl esters

The effect of lipid transfer proteins on the exchange and transfer of cholesteryl esters from rat plasma HDL2 to human very low (VLDL) and low density (LDL) lipoprotein populations was studied. The use of a combination of radiochemical and chemical methods allowed separate assessment of [3H]cholesteryl ester exchange and of cholesteryl ester transfer. VLDL-I was the preferred acceptor for transferred cholesteryl esters, followed by VLDL-II and VLDL-III. LDL did not acquire cholesteryl esters. The contribution of exchange of [3H]cholesteryl esters to total transfer was highest for LDL and decreased in reverse order along the VLDL density range. Inactivation of lecithin: cholesterol acyltransferase (LCAT) and heating the HDL2 for 60 min at 56 degrees C accelerated transfer and exchange of [3H]cholesteryl esters. Addition of lipid transfer proteins increased cholesterol esterification in all systems. The data demonstrate that large-sized, triglyceride-rich VLDL particles are preferred acceptors for transferred cholesteryl esters. It is suggested that enrichment of very low density lipoproteins with cholesteryl esters reflects the triglyceride content of the particles.     

Turnover of cholesteryl esters of plasma lipoproteins in the rat

Turnover of individual classes of cholesteryl esters (classified on the basis of the degree of unsaturation of the fatty acid moiety) in rat plasma lipoproteins and liver was studied after the administration of mevalonic acid-5-(3)H and mevalonic acid-2-(14)C. The relative turnover rate was greatest in the d < 1.019 lipoproteins, with monoenes > saturated = dienes > tetraenes. In the d > 1.063 lipoproteins, all cholesteryl esters had slower turnover rates, but tetraenes = pentaenes > dienes > monoenes = saturated. Comparisons of specific activities of individual cholesteryl ester classes of liver subcellular fractions and lipoproteins suggest that the d < 1.019 lipoprotein cholesteryl esters are synthesized from newly synthesized cholesterol in the liver and are rapidly released into this lipoprotein. Tetraenoic cholesteryl esters, however, may originate from esterification of free cholesterol in plasma. Tetraenoic esters are formed from cholesterol in plasma during incubation or ultracentrifugation unless a thiol-reacting or alkylating agent is added. Failure to add such a reagent to plasma results in erroneous specific activities. In the adrenal, relative rates of synthesis of cholesteryl esters are monoenes = dienes > tetraenes > trienes = pentaenes > saturated. It is concluded that cholesteryl ester turnover in the rat, as opposed to man, is determined not only by the particular lipoprotein class but also by the fatty acid moiety of the ester.     

Effect of unesterified cholesterol on the compartmentation of a fluorescent cholesteryl ester in a lipoprotein-like lipid microemulsion.

The access of enzymes and lipid transfer proteins to neutral lipids located predominantly in the core compartment of lipoproteins may be determined to some degree by the solubility of the neutral lipids in the surface monolayer of phospholipid. This report concerns the hypothesis that unesterfied cholesterol can affect the partition of a cholesteryl ester between the surface monolayer of a lipid emulsion and the internal core compartment, thus controlling the degree to which the cholesteryl ester is presented at the emulsion surface. For microemulsions composed of dimyristoyl phosphatidylcholine and cholesteryl oleate, the addition of unesterified cholesterol results in an increase in the particle size from about 170 nm diameter to 210 nm diameter at 13.5 mol% unesterified cholesterol. Fluorescent quenching methods were devised to determine the apparent partition of a fluorescent cholesteryl ester (cholesteryl anthracene-9-carboxylate) between surface and core compartments. The addition of unesterified cholesterol resulted in the movement of the fluorescent cholesteryl ester from the surface monolayer to the core compartment. The apparent partition coefficient, defined as the ratio of the concentration of probe in the monolayer to that in the core, decreased from 1.03 in the absence of unesterfied cholesterol to 0.54 at 28 mol% unesterified cholesterol in the emulsion. In this process, the fluorescent cholesteryl ester becomes less accessible to a quencher (5-doxyl stearate) located in the surface monolayer. The decrease in the surface curvature resulting from incorporation of unesterified cholesterol into the particle does not influence this quenching process. We conclude that the presence of unesterified cholesterol in the emulsion causes the fluorescent cholesteryl ester to become less soluble in the surface monolayer.     

Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol

The distribution of apolipoprotein A-I, apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein in fasting normal human plasma was determined by two-dimensional electrophoresis followed by immunoblotting. The synthesis and transfer of labeled cholesteryl esters generated in plasma briefly incubated with [3H]cholesterol-labeled fibroblasts was followed in terms of the lipoprotein species containing these antigens. Following the early appearance of labeled free cholesterol in two pre beta-migrating apolipoprotein A-I species (Castro, G. R., and Fielding, C. J. (1988) Biochemistry 27, 25-29), labeled esters were first detected, after a 2-min delay, in a third pre beta-migrating species which also contained apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein. Pulse-chase experiments determined that label generated in this fraction was the precursor of at least a major part of labeled cholesteryl esters in the bulk of alpha-migrating high density lipoprotein. Over the maximum time course of these experiments (15 min, 37 degrees C), less than 10% of labeled cholesteryl esters were recovered in low or very low density lipoproteins separated by electrophoresis, immunoaffinity, or heparin-agarose chromatography. These data suggest channeling of cell-derived cholesterol and cholesteryl esters derived from it through a preferred pathway involving several minor pre beta-migrating lipoproteins to alpha-migrating high density lipoprotein.     

Microemulsions of cholesteryl oleate and dimyristoylphosphatidylcholine: a model for cholesteryl ester rich very low density lipoproteins

This study describes the preparation, purification, and characterization of a cholesteryl oleate/dimyristoylphosphatidylcholine microemulsion as a model for the interaction of lipid domains in cholesteryl ester rich very low density lipoproteins. These lipids were chosen specifically because their thermal transitions were distinct from each other, and their differences in chemical structure permitted the motion(s) of each lipid component to be monitored independently by 13C nuclear magnetic resonance (NMR). The model particles were formed by cosonication of cholesteryl oleate and dimyristoylphosphatidylcholine in a 4:1 molar ratio for 45 min at 55-60 degrees C (above both lipid phase transition temperatures). The crude microemulsion was fractionated by low-speed centrifugation and Sepharose CL-2B chromatography. Microemulsion particles which eluted from the column at a volume similar to that of cholesteryl ester rich very low density lipoproteins had high cholesteryl ester:phospholipid ratios (2.5:1----6:1). Electron micrographs of negatively stained particles showed them to be large spheres devoid of multilamellar or unilamellar vesicle structures. Particle size calculated from a simple compositional model correlated well with sizes determined by electron microscopy (500-1000 A) for various column fractions. Differential scanning calorimetry studies of the microemulsion revealed two thermal transitions for the model particles, at 31.0 and 46.6 degrees C, which were tentatively assigned to the surface phospholipid and core cholesteryl ester domains, respectively. These assignments were confirmed by 13C NMR which demonstrated that, at temperatures near the lower thermotropic transition, only resonances derived from carbon atoms of dimyristoylphosphatidylcholine (DMPC) were observable. As the temperature was raised to 38.6 degrees C, resonances from the olefinic carbons in the cholesteryl ester acyl chain appeared in the spectrum. At 46.6 degrees C, the center of the higher temperature endotherm, resonances from both the steroid ring and remaining acyl chain carbons of cholesteryl oleate became observable in the spectrum. Further increases in temperature did not result in the appearance of new resonances; however, those that were present narrowed and increased in intensity. The elevation in transition temperature for DMPC in these particles (31 degrees C) as compared to that for DMPC in small unilamellar (18 degrees C) and large multilamellar (23 degrees C) vesicles suggested a stabilization of the phospholipid monolayer, possibly by interaction with the nonpolar core lipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.     

Differential effects of cis and trans fatty acid isomers, oleic and elaidic acids, on the cholesteryl ester transfer protein activity.

In a previous publication (Lagrost, L. and Barter, P.J. (1991) Biochim. Biophys. Acta 1085, 209-216), saturated and cis unsaturated non-esterified fatty acids have been shown to modulate the rate at which cholesteryl esters are transferred from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the presence of the human cholesteryl ester transfer protein (CETP). In the present report, the effects of cis (oleic acid) and trans (elaidic acid) monounsaturated isomers on the CETP-mediated transfer of cholesteryl esters between HDL and LDL were compared. Mixtures of human LDL and HDL3, containing or not radiolabelled cholesteryl esters, were incubated at 37 degrees C with CETP in the presence or in the absence of either stearic (18:0), oleic (18:1 cis) or elaidic (18:1 trans) acids. It was observed that oleic acid and elaidic acid had different effects on the CETP-mediated redistribution of radiolabelled cholesteryl esters as well as on the net mass transfer of cholesterol from HDL3 to LDL. In particular, at high non-esterified fatty acid/lipoprotein ratio, the transfer of cholesteryl esters was significantly inhibited by the cis isomer and increased by the trans isomer.     

Triglyceride and cholesteryl ester hydrolysis in a cell culture model of smooth muscle foam cells

Cultured rabbit aortic smooth muscle cells were converted to foam cells by exposure to sonicated lipid droplets of defined composition using an inverted culture technique. Uptake of the lipid droplets by the cells was shown to be dependent on the time of exposure to the droplets and on the mass of droplets presented to the cells. A comparison of the hydrolysis of triolein and cholesteryl oleate by cells that had been exposed to isotropic lipid droplets containing equimolar amounts of the two lipids revealed that the rate of hydrolysis of triglyceride was 3 to 4 times faster than that for cholesteryl ester. The hydrolysis of cholesteryl oleate from cells loaded with the isotropic droplets was approximately 1.5 times as fast as that from cells loaded with anisotropic droplets containing only cholesteryl oleate. A comparison of the hydrolysis of cholesteryl ester in the presence and absence of Sandoz compound 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, by cells loaded with isotropic droplets showed that about 30% of the free cholesterol liberated by hydrolysis was reesterified regardless of the mass of free cholesterol. We conclude that cultured smooth muscle cells have a greater capacity to hydrolyze triglyceride than cholesteryl ester, and that the rate of hydrolysis of cholesteryl ester appears to be related to the physical state of the droplet in which the cholesteryl ester is stored. In addition, it appears that the smooth muscle cells have a cholesteryl ester cycle that is inefficient in the reesterification of excess free cholesterol.     

Inhibitors of neutral cholesteryl ester hydrolase

p-Nitrophenyl N-butyl, N-octyl, and N-dodecyl carbamates and a newly synthesized diethyl phosphate compound were studied as potential inhibitors of the cholesteryl ester hydrolases of Fu5AH rat hepatoma cells. Whole homogenates of Fu5AH cells were used as an enzyme source for the assay of cholesteryl ester hydrolase activity. All four compounds led to marked inhibition (70-80%) of neutral cholesteryl ester hydrolase activity (assayed at pH 7) at concentrations where the activity of acid cholesteryl ester hydrolase (assayed at pH 4) was unaffected. Cholesteryl ester hydrolysis was also evaluated in intact cultured cells induced to accumulate cholesteryl esters in cytoplasmic lipid droplets by exposure to cholesterol-rich phospholipid dispersions. Hydrolysis was then assessed during subsequent incubations in the presence of an inhibitor of cholesterol esterification. All compounds caused significant inhibition of cholesterol ester hydrolysis with the diethyl phosphate being the most effective. At a concentration that caused greater than 90% inhibition of the hydrolysis of cytoplasmic cholesteryl esters, the compound had only a minimal effect on lysosomal hydrolysis of cholesteryl esters. These results suggest that diethyl phosphates and N-alkylcarbamates may be of value in future studies on the substrate specificities, regulation, and physiological role(s) of cholesteryl ester hydrolases.     

Phospholipid/cholesteryl ester microemulsions containing unesterified cholesterol: model systems for low density lipoproteins

As models for the effects of unesterified cholesterol (UC) on the lipid organization of low density lipoprotein (LDL), microemulsions containing either egg yolk phosphatidylcholine (EYPC) or dimyristoyl phosphatidylcholine (DMPC) as the surface component, cholesteryl oleate (CO) as the core component, and varying amounts of unesterified cholesterol were prepared by sonication. Gel filtration chromatography showed coelution of each of the lipid components, demonstrating the formation of well-defined microemulsion populations. Unesterified cholesterol incorporation into the microemulsions was proportional to the composition of the original mixture at low unesterified cholesterol compositions, but reached saturation at compositions of approximately 15 and 10 mol% unesterified cholesterol for EYPC/CO and DMPC/CO microemulsions, respectively. The Stokes' radius of the microemulsions was constant and similar to native LDL for initial compositions less than 15 mol% unesterified cholesterol, but increased at compositions above 15 mol%. In both EYPC/CO/UC and DMPC/CO/UC microemulsions, no significant changes were observed for the calorimetric or Van't Hoff enthalpy for the thermal transition of the core cholesteryl ester; however, increases in the transition temperature as a function of increasing unesterified cholesterol composition suggests that unesterified cholesterol has a stabilizing effect on the core transition. In DMPC/CO/UC microemulsions, the effect of unesterified cholesterol on the surface-located DMPC could be clearly observed as a broadening of the thermal transition of the acyl chains. These results demonstrate that unesterified cholesterol is located primarily in the surface of these protein-free lipid model systems for LDL.     

Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3

Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [(3)H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters approximately 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway.     

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.     

Concerted actions of cholesteryl ester transfer protein and phospholipid transfer protein in type 2 diabetes: effects of apolipoproteins

PURPOSE OF REVIEW: Type 2 diabetes frequently coincides with dyslipidemia, characterized by elevated plasma triglycerides, low high-density lipoprotein cholesterol levels and the presence of small dense low-density lipoprotein particles. Plasma lipid transfer proteins play an essential role in lipoprotein metabolism. It is thus vital to understand their pathophysiology and determine which factors influence their functioning in type 2 diabetes. RECENT FINDINGS: Cholesteryl ester transfer protein-mediated transfer is increased in diabetic patients and contributes to low plasma high-density lipoprotein cholesterol levels. Apolipoproteins A-I, A-II and E are components of the donor lipoprotein particles that participate in the transfer of cholesteryl esters from high-density lipoprotein to apolipoprotein B-containing lipoproteins. Current evidence for functional roles of apolipoproteins C-I, F and A-IV as modulators of cholesteryl ester transfer is discussed. Phospholipid transfer protein activity is increased in diabetic patients and may contribute to hepatic very low-density lipoprotein synthesis and secretion and vitamin E transfer. Apolipoprotein E could stimulate the phospholipid transfer protein-mediated transfer of surface fragments of triglyceride-rich lipoproteins to high-density lipoprotein, and promote high-density lipoprotein remodelling. SUMMARY: Both phospholipid and cholesteryl ester transfer proteins are important in very low and high-density lipoprotein metabolism and display concerted actions in patients with type 2 diabetes.     

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.     

Effect of free cholesterol on incorporation of triolein in phospholipid bilayers

Triacylglycerols are the major substrates for lipolytic enzymes that act at the surface of emulsion-like particles such as triglyceride-rich lipoproteins, chylomicrons, and intracellular lipid droplets. This study examines the effect of cholesterol on the solubility of a triacylglycerol, triolein, in phospholipid surfaces. Solubilities of [carbonyl-13C]triolein in phospholipid bilayer vesicles containing between 0 and 50 mol % free cholesterol, prepared by cosonication, were measured by 13C NMR. The carbonyl resonances from bilayer-incorporated triglyceride were shifted downfield in the 13C NMR spectra from those corresponding to excess, nonincorporated material. This enabled solubilities to be determined directly from carbonyl peak intensities at most cholesterol concentrations. The bilayer solubility of triolein was inversely proportional to the cholesterol/phospholipid mole ratio. In pure phospholipid vesicles the triolein solubility was 2.2 mol %. The triglyceride incorporation decreased to 1.1 mol % at a cholesterol/phospholipid mole ratio of 0.5, and at a mole ratio of 1.0 for the bilayer lipids, the triolein solubility was reduced to just 0.15 mol %. The effects of free cholesterol were more pronounced and progressive than observed previously on the bilayer solubility of cholesteryl oleate (Spooner, P. J. R., Hamilton, J. A., Gantz, D. L., & Small, D. M. (1986) Biochim. Biophys. Acta 860, 345-353]. As with cholesteryl oleate, we suggest that cholesterol also displaces solubilized triglyceride to deeper regions of the bilayer.