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.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.     

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.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Determination of total intracellular lipid content by flow cytofluorometry

It was demonstrated that perylenoyl-labeled triglyceride, rac-1,2-dioleoyl-3-[9(3-perylenoyl)nonanoyl]glycerol (PLTG), specifically stains the intracellular lipids in formalin-fixed and non-fixed cells in situ and in suspension. The fluorescence of labeled cells was registered on a FACS-II cytofluorometer. It was found that the mean intensity of fluorescence in PLTG-labeled cells of various types, both fixed and non-fixed, correlates with their total lipid content. There was no correlation, however, between the mean intensity of fluorescence and intracellular levels of lipids belonging to different classes: phospholipids, triglycerides, cholesterol or cholesteryl esters.     

Inhibition of acylcoenzyme A:cholesterol acyltransferase activity in CaCo-2 cells results in intracellular triglyceride accumulation

The activity of acylcoenzyme A:cholesterol acyltransferase (ACAT) in CaCo-2 cells was inhibited by the ACAT inhibitor, 58-035. The inhibitory effect of this acylamide was specific for cholesterol esterification catalyzed by ACAT; the rates of triglyceride, phospholipid, and cholesterol synthesis were not inhibited by this agent. Cholesteryl esters were depleted in CaCo-2 cells 24 hr after inhibition of ACAT activity, whereas the unesterified cholesterol content increased by 56% after 96 hr. Moreover, inhibiting ACAT activity with 58-035 resulted in a time-dependent 2.5-fold increase in intracellular triglycerides. This accumulation of triglycerides in CaCo-2 cells was associated with a 37% increase in triglyceride synthesis by 96 hr in the presence of 58-035. Triglyceride-rich lipoprotein secretion (d less than 1.006 g/ml) was not affected by inhibiting ACAT activity for up to 6 hr. However, triglyceride-rich lipoprotein secretion was significantly decreased in CaCo-2 cells that were preincubated with 58-035 for 24 to 96 hr. Lipoproteins of density less than 1.006 g/ml that were isolated from CaCo-2 cells incubated with the ACAT inhibitor were deficient in cholesteryl esters and triglycerides compared to lipoproteins isolated from control cells. The data suggest that triglycerides accumulate in CaCo-2 cells in which ACAT activity has been inhibited by 58-035. This accumulation of triglycerides is associated with a modest increase in triglyceride synthesis and a decrease in triglyceride secretion. Altering intracellular cholesterol pools by regulating ACAT activity in the gut could result in the decrease of triglyceride transport and/or the secretion of triglyceride-rich lipoprotein particles of abnormal composition.     

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively.Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

The effect of interleukin 1 beta on the biosynthesis of cholesterol, phosphatidylcholine, and sphingomyelin in fibroblasts, and on their efflux from cells to lipid-free apolipoprotein A-I.

In this study we have investigated the effect of interleukin 1beta (IL-1beta) on the metabolism of cholesterol and choline-phospholipids in cultured fibroblasts, and also measured efflux of these lipids to lipid-free apo A-I as a function of IL-1beta treatment. Long-term exposure (up to 48 h) of cells to IL-1beta (1 ng.mL-1) markedly increased the rate of cholesterol esterification, as determined by the incorporation of [3H]oleic acid into cholesteryl esters. This treatment also led to a substantially increased mass of cholesteryl esters in the cells. The accumulation of cholesteryl esters in IL-1beta-treated cells could be blocked using compound 58-035 to inhibit the activity of acyl-CoA cholesterol acyl transferase. The activation of cholesterol esterification by IL-1beta was evident within a few hours after initiation of the IL-1beta treatment. Cholesterol biosynthesis was inhibited by 25% by IL-1beta (after 48 h exposure), and this eventually led to a 20% decrease in cell cholesterol mass. Treatment of cells with IL-1beta for 48 h also reduced the synthesis of sphingomyelin and caused a 30% decrease in cell sphingomyelin mass (after 48 h at 1 ng.mL-1 of IL-1beta). IL-1beta did not stimulate an acute (within a few minutes up to an hour) degradation of cell [3H]sphingomyelin. This suggests that IL-1beta did not activate an endogenous sphingomyelinase in these cells, but only affected rates of synthesis. The rate of phosphatidylcholine synthesis was barely affected, but mass was moderately reduced by a 48-h treatment of cells with IL-1beta. Finally, the efflux of cell [3H]cholesterol, [3H]sphingomyelin, and [3H]phosphatidylcholine to lipid-free apolipoprotein A-I was markedly increased from cells treated with IL-1beta for 24 and 48 h. We conclude that long-term exposure of cells to IL-1beta had marked effects on the cellular homeostasis of cholesterol and choline-containing phospholipids.     

The effect of low density lipoproteins, cholesterol, and 25-hydroxycholesterol on apolipoprotein B gene expression in HepG2 cells.

The purpose of the present study was to examine the effects of exogenous cholesterol on the apolipoprotein (Apo) B gene expression in HepG2 cells. Pure cholesterol had no significant effect on either the cellular content of cholesteryl esters or the net accumulation of neutral lipids and ApoB in the culture medium. By contrast, addition of 25-hydroxycholesterol increased the net accumulation of cholesteryl esters in cells and medium by 2-3-fold and decreased that of unesterified cholesterol by 50% in both compartments. A 33% reduction in the cellular content of triglycerides was commensurate with a 40% increase in their accumulation in the medium. A significant 3-fold increase in the net accumulation of ApoB in the medium was predominantly due to enhanced secretion of newly synthesized ApoB as established by pulse-chase studies. The stimulation in ApoB secretion was accompanied by a 55% increase in cellular ApoB mRNA. Under these experimental conditions, the low density lipoprotein receptor activity was decreased by only 12-20%. Addition of progesterone prevented the effects of 25-hydroxycholesterol. The changes in the concentration of neutral lipids and ApoB were reflected in the composition of secreted "low-density" lipoproteins. These particles had increased percentage contents of cholesteryl esters and ApoB and a decreased percentage content of unesterified cholesterol in comparison with lipoproteins produced by control cells. The rate of ApoB production was not correlated with the triglyceride mass in the cells but was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. With the exception of unchanged cellular unesterified cholesterol and ApoB mRNA levels, plasma low density lipoprotein had similar, although less pronounced, effects on the production of neutral lipids and ApoB. These results demonstrate that in HepG2 cells the synthesis and secretion of ApoB and cholesteryl esters are tightly coupled and that 25-hydroxycholesterol increased the concentration of ApoB-containing lipoproteins primarily by stimulating their production rather than reducing their catabolism.     

Differential effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells

The effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells were examined to identify fatty acid specific changes in lipid metabolism that might indicate a basis for the hypolipidemic effect attributed to eicosapentaenoic acid and related n-3 fatty acids. Cellular glycerolipid synthesis, as determined by [3H]glycerol incorporation, increased in a concentration-dependent manner in cells incubated 4 h with either eicosapentaenoic acid or oleic acid at concentrations between 10 and 300 microM. [3H]Glycerol-labeled triglyceride was the principal lipid formed and increased approximately fourfold with the addition of 300 microM oleic acid or eicosapentaenoic acid. Both fatty acids also produced a 20-40% increase in the total cellular triglyceride mass. Although both fatty acids increased triglyceride synthesis to similar extents, eicosapentaenoic acid-treated cells secreted 40% less [3H]glycerol-labeled triglyceride than cells fed oleic acid. Cellular synthesis of [3H]glycerol-labeled phosphatidylethanolamine and phosphatidylcholine was also reduced by 40% and 30%, respectively, in cells given eicosapentaenoic acid versus cells given oleic acid. Similar results were obtained in determinations of radiolabeled oleic acid and eicosapentaenoic acid incorporation. At a fatty acid concentration of 300 microM, incorporation of radiolabeled eicosapentaenoic acid into cellular triglycerides was greater than the incorporation obtained with radiolabeled oleic acid, while the reverse relationship was observed for the formation of phosphatidylcholine from the same fatty acids. Eicosapentaenoic acid is as potent as oleic acid in inducing triglyceride synthesis but eicosapentaenoic acid is a poorer substrate than oleic acid for phospholipid synthesis. The intracellular rise in de novo-synthesized triglyceride in eicosapentaenoic acid-treated cells without corresponding increases in triglyceride secretion suggests that eicosapentaenoic acid is less effective than oleic acid in promoting the transfer of de novo-synthesized triglyceride to nascent very low density lipoproteins.     

Blood glycerol is an important precursor for intramuscular triacylglycerol synthesis.

The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.     

Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Cholesterol esterification in rabbit plasma

1. When [4-(14)C]cholesterol, attached to beta-globulin or dispersed with Tween 20, was incubated with fresh rabbit (New Zealand albino females) plasma, 30-47% esterification was observed. The optimum pH was 6.8. This esterification was accomplished by the transfer of fatty acids from the C-2 position of lecithin (phosphatidylcholine) to cholesterol. 2. There was no evidence that triglycerides or free fatty acids participated directly in this reaction. Lecithins with labelled palmitic acid, oleic acid and linoleic acid in the 2-position yielded 3.2, 4.8 and 6.8% of cholesteryl esters respectively. This pattern reflects that which is normally observed in the cholesteryl esters of rabbit plasma and supports the concept that plasma cholesteryl esters originate from the plasma. 3. Snake venom (containing phospholipase A), sulphoevernan [an alpha-(1-->3,1-->4)-sulphopolyglucan with 12% sulphur], thiol-blocking agents (p-chloromercuribenzoate and N-ethylmaleimide), or an atherogenic diet (stock diet supplemented with 1% cholesterol for 8 weeks) were all effective inhibitors of this cholesterol esterification.     

Smooth microsomes. a trap for cholesteryl ester formed in hepatic microsomes

Acyl-CoA:cholesterol acyltransferase was found predominantly (85%) in RNA-rich microsomes, the rest being in RNA-poor and smooth microsomes. However, the esterified cholesterol concentration of smooth microsomes was 2-fold greater than that of RNA-rich microsomes, suggesting the possibility of an interaction between RNA-rich and smooth microsomes. The distribution of cholesteryl ester between microsome subfractions was examined after incubation of a mixture of RNA-rich and smooth microsomes with [1-14C]palmitoyl-CoA. Based upon specific acyl-CoA:cholesterol acyltransferase activities of the individual fractions, only 31 +/- 3% of the total cholesteryl ester radioactivity should have been found in the smooth component. However, the smooth microsomes contained 54 +/- 3% (p < 0.01) of the radioactive cholesteryl esters. The entrapment of radioactive cholesteryl ester in the smooth microsomes could not be accounted for by passive transfer of cholesteryl ester from RNA-rich microsomes to smooth microsomes. It is proposed that cholesterol in the smooth microsomal membranes may have been esterified by acyl-CoA:cholesterol acyltrasferase located on the surface of RNA-rich microsomes with the resulting cholesteryl ester retained in the smooth microsomes. This hypothesis was strengthened by the observation that acyl-CoA:cholesterol acyl-transferase was located on the cytoplasmic surface of the RNA-rich microsomal vesicle.     

Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts.

This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.     

Hepatic lipid droplets. Isolation, morphology and composition

The floating lipid layer isolated centrifugation of rat liver was examined for composition and ultrastructure. It was chiefly composed of triglycerides and cholesterol esters plus much smaller amounts of free cholesterol, diglycerides, phospholipid and protein. No free fatty acids were detected. The triglyceride and cholesterol ester fractions consisted mostly of esters of linoleic acid, oleic acid and palmitic acid. Electron micrographs of the floating lipid layer revealed numerous spherical osmiophilic droplets having a mean diameter of 0.5-2mum with a very-thin dense outer coat. Similar structures were observed as organelles in electron micrographs of the intact liver cell. The amount of triglyceride in the layer decreased in rats starved for 72h, but pellet triglyceride (homogenate minus the floating lipid layer) was unchanged. These results suggest that the floating lipid layer is the representative in vitro of lipid-rich organelles which probably function as a depot form of hepatic-cell neutral lipid.     

Effects of dietary n-3 fatty acids on the composition of cholesteryl esters and triglycerides in plasma and liver perfusate of the rat

The effects of polyunsaturated fatty acids of the omega-3 family (PUFA n-3), (addition of fish oil), on the molecular composition of cholesteryl esters and triglycerides in plasma and liver perfusate of rats were studied. Rats fed a diet rich in saturated fatty acids (addition of lard) served as controls. Supplemention with PUFA n-3 not only decreases the plasma concentrations of free cholesterol, cholesteryl esters, and triglycerides, it also significantly alters the plasma composition of cholesteryl esters and triglycerides. Analyses of liver perfusate indicate a decrease in triglycerides secretion by in vitro perfused liver and reciprocal changes in relative contents of cholesteryl esters fractions with C(16) and C(20) acyl chains. This finding may be a result of chain-shortening of long-chain fatty acids probably in peroxisomal beta-oxidative system. Alterations in plasma cholesteryl esters and triglycerides composition of the fish oil group could be affected further by additional factors such as increased plasma cholesterol esterification activity and presence of triglyceride species of intestinal origin.