Altered cholesterol ester proportions in embryonic tissues of dystrophic chicken

The concentrations of cholesterol esters in tissues of dystrophic chicken embryos are altered from normal. These changes are accompanied by significant changes in the proportions of the esterified fatty acids (the fatty acid profile). In serum and pectoral muscles there is a shift to a higher proportion of unsaturated fatty acids (in particular 18:1). Thigh muscle esters are little changed and in liver and brain the proportion of unsaturated fatty acids decreases.     

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.     

Fiber-specific cholesterol changes in murine dystrophy

The cholesterol concentration in dystrophic mouse muscle is reported to be increased compared to normal. The muscles studied are, however, composed in most cases of more than one fiber type. As a result, the observed concentration increase may be due to a general increase or may be due to changes in the proportion of individual fiber types which themselves differ in cholesterol concentration. To decide between these possibilities we have measured the cholesterol concentrations (both free cholesterol and cholesterol esters) in normal and dystrophic whole gastrocnemius muscles and compared the values with the concentrations in fast-glycolytic muscle tissue alone. The cholesterol concentrations in both whole and fast-glycolytic sections of dystrophic muscle are increased compared to normal, with the largest increase in the cholesterol ester fraction. Furthermore, the concentration changes in fast-glycolytic fibers are due mainly to cholesterol ester differences in both membrane and sarcoplasm fractions, with differences in the latter being larger. The data show that changes in whole muscle concentrations cannot be ascribed solely to altered fiber type proportions.     

A defect in mobilization of cholesteryl esters in rabbit macrophages

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.     

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.     

Effect of up-regulating individual steps in the reverse cholesterol transport pathway on reverse cholesterol transport in normolipidemic mice

Cholesterol acquired by extrahepatic tissues (from de novo synthesis or lipoproteins) is returned to the liver for excretion in a process called reverse cholesterol transport (RCT). We undertook studies to determine if RCT could be enhanced by up-regulating individual steps in the RCT pathway. Overexpression of 7alpha-hydroxylase, Scavenger receptor B1, lecithin:cholesterol acyltransferase (LCAT), or apoA-I in the liver did not stimulate cholesterol efflux from any extrahepatic tissue. In contrast, infusion of apoA-I.phospholipid complexes (rHDL) that resemble nascent HDL markedly stimulated cholesterol efflux from tissues into plasma. Cholesterol effluxed to rHDL was initially unesterified but by 24 h this cholesterol was largely esterified and had shifted to normal HDL (in mice lacking cholesteryl ester transfer protein) or to apoB containing lipoproteins (in cholesteryl ester transfer protein transgenic mice). Most of the cholesterol effluxed into plasma in response to rHDL came from the liver. However, an even greater proportion of effluxed cholesterol was cleared by the liver resulting in a transient increase in liver cholesterol concentrations. Fecal sterol excretion was not increased by rHDL. Thus, although rHDL stimulated cholesterol efflux from most tissues and increased net cholesterol movement from extrahepatic tissues to the liver, cholesterol flux through the entire RCT pathway was not increased.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Metabolism of doubly-labeled chylomicron cholesteryl esters in the rat

Chylomicrons labeled in vitro with doubly-labeled cholesteryl esters were injected intravenously into fasted rats, and the tissue distribution and chemical form of each isotope were observed for 24 hr. The use of doubly-labeled cholesteryl esters provided information about the metabolism of both the sterol and the fatty acid moieties. Similar results were obtained with doubly-labeled cholesteryl palmitate, oleate, and linoleate. In each instance, most (80-90%) of the chylomicron cholesteryl ester was removed from the plasma by the liver; small amounts were also taken up by all other tissues examined. There was no hydrolysis during uptake. In the liver the newly absorbed cholesteryl esters underwent slow hydrolysis (60% after 1 hr and 85-90% after 3.5 hr); the rate of reesterification of the liberated cholesterol was still slower. After 24 hr only 20-28% of the labeled cholesterol present in the animal was found in the liver. Labeled fatty acid disappeared from the liver, and was redistributed among other tissues, much more rapidly than the labeled cholesterol. Most of the labeled fatty acid apparently underwent oxidation, since only 15-20% of the injected labeled fatty acid was present in the animal after 24 hr. At this time the three fatty acids were differently distributed between and within the tissues. These differences reflected some known differences of fatty acid concentration and lipid composition in the various tissues.     

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.     

Effects of phenylmethylsulphonylfluoride on activities of cholesteryl ester synthesis and hydrolysis in testes of rats, and on serum testosterone and LH levels.

PMSF was injected subcutaneously to male rats once a day for 10 days at a dose of 10 mg/day. There was an increase in the concentrations of free and esterified cholesterol in the testes, a decrease in activities of cholesteryl ester synthesis and hydrolysis but no change in activity of cholesterol side-chain cleavage enzyme. Serum testosterone and LH levels were significantly decreased. The only effect of PMSF on the lipids of the testes was a marked elevation of docosapentaenoic (22:5) acid and the cholesteryl esters.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Effect of the stress of unilateral adrenalectomy on the depletion of individual cholesteryl esters in the rat adrenal

Normal rats were subjected to unilateral adrenalectomy and were killed 3 hr later. The concentration and composition of the cholesteryl esters in adrenals removed at operation and after death were compared. The esterified cholesterol concentration was lower in the adrenals obtained 3 hr after surgery. Cholesteryl arachidonate decreased in concentration significantly more than any other ester, followed in order of magnitude by linoleate and oleate. The cholesteryl ester concentration of adrenals removed from sham-operated rats 3 hr after surgery was also greatly reduced. On the basis of comparison with other work on the hydrolysis of cholesteryl esters by adrenal homogenates, it is concluded that the apparent selectivity in depletion of cholesteryl esters is due to differences in their rates of hydrolysis.     

Abnormal cholesterol metabolism in renal clear cell carcinoma

The clear cell form of renal cell carcinoma is known to derive its histologic appearance from accumulations of glycogen and lipid. We have found that the most consistently stored lipid form is cholesteryl ester. Clear cell cancer tissue contained 8-fold more total cholesterol and 35-fold more esterified cholesterol than found in normal kidney. Cholesteryl ester appeared to be formed intracellularly since it was not membrane-bound and since oleate was the predominant form, as opposed to linoleate in lipoprotein cholesteryl esters. The cholesterol in clear cell tumors did not appear to be a result of excessive synthesis from acetate since HMG-CoA reductase (EC 1.1.1.34) activity was lower in cancer tissue than in normal kidney (2.9 +/- 0.8 vs. 7.2 +/- 1.2 pmol/mg of protein per min). In contrast, intracellular activity of fatty acyl-coenzyme A:cholesterol acyl transferase (ACAT, EC 2.3.1.26) was higher in tumor tissue than in normal kidney (2405 +/- 546 vs. 1326 +/- 301 pmol/mg of protein per 20 min) while cytosolic cholesteryl ester hydrolase activity appeared normal. Cholesteryl ester storage in clear cell renal cancer may be a result of a primary abnormality in ACAT activity or it may be a result of reduced release of free cholesterol (relative to cell content) with a secondary elevation in ACAT activity.     

Cholesterol metabolism in dystrophic mice. II. Altered enzyme activities

In a previous study we found that free cholesterol (FC) and cholesterol ester (CE) concentrations in fast-glycolytic (FG) muscle tissue from dystrophic mice are significantly higher than normal. This increase is not due to an increased capacity for de novo cholesterol biosynthesis. HMG-CoA reductase (HMGR) (the enzyme which catalyzes the rate limiting step) activity is significantly decreased in dystrophic muscle compared to normal. This decrease is paralleled by an increased capacity for both CE production and hydrolysis, i.e., both Acyl-CoA:cholesterol acyltransferase (ACAT) activity and the activities of both lysosomal and sarcoplasmic cholesterol ester hydrolases (CEH) are greatly increased. These enzyme changes in dystrophic FG muscle are similar those observed in normal tissues with elevated levels of cholesterol, which suggests that such changes are not the cause of the altered cholesterol concentrations but are rather the response of the tissue to elevated levels of cholesterol.     

In vivo transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins in man.

The fate of cholesteryl esters in high density lipoprotein (HDL) was studied to determine whether the transfer of esterified cholesterol from HDL to other plasma lipoproteins occurred to a significant extent in man. HDL cholesteryl ester, labelled in vitro with [3H] cholesterol, was injected into human subjects. Labelling of cholesteryl esters in very low density (VLDL) occurred rapidly and by 3 h, the esterified cholesterol in VLDL reached peak specific radioactivity. The removal rate of cholesteryl esters from HDL appeared to be exponential and of the order of 0.2/h; calculation of the apparent flux was about 150 mg/h which approximates reported values for total cholesterol esterification in human plasma in vivo. The rapid rate of labelling of VLDL from HDL suggests that the transfer of HDL cholesteryl esters to VLDL may represent a significant pathway for the disposal of HDL cholesterol.     

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.     

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.     

Intracellular processing of exogenously derived non-lipoprotein [3H)cholesterol in normal and mutant human skin fibroblasts deficient in acid sterol ester hydrolase

By studying the incorporation and esterification of non-lipoprotein, free [³H]cholesterol in normal and acid sterol ester hydrolase-deficient human fibroblasts, it was examined whether the esterification reaction of the lysosomal acid sterol ester hydrolase contributed to the formation of cellular [³H|cholesteryl esters. Both the normal and the acid sterol ester hydrolase-deficient cells incorporated exogenous, vesicle-derived free [³H]cholesterol linearly as a function of time. Also, the rate of [³H]cholesteryl ester formation was almost the same in normal and mutant fibroblasts, indicating that the apparent esterification activity of the acid sterol ester hydrolase in normal fibroblasts did not contribute to the formation of [³H]cholesteryl esters in intact cells. To examine whether the incorporated [³H]cholesterol was transported into the endoplasmic reticulum and esterified by the acyl-CoA: cholesterol acyltransferase, the rate of [³H]cholesteryl ester formation was measured in the presence or absence of the acyl-CoA: cholesterol acyltransferase-inhibitor 58-035 (Sandoz Inc.). Results showed that the formation of [³H]cholesteryl esters was reduced markedly when cells were co-incubated with the acyltransferase inhibitor. Maximal inhibition (i.e., 75%) was obtained at an inhibitor concentration of 1 μg/ml. Since the inhibitor 58-035 is very specific for acyl-CoA: cholesterol acyltransferase, this finding clearly shows that exogenous, exchangeable [³H]cholesterol can reach and mix with the intracellular substrate pool of the enzyme.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.     

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.