Composition and synthesis of fatty acids in atherosclerotic aortas of the pigeon

The composition, synthesis, and esterification of fatty acids were studied in aortas of White Carneau and Show Racer pigeons after perfusion of the aortas with a medium containing acetate-1-(14)C. For both breeds of pigeons the principal change in aortic fatty acids, in response to an atherogenic diet, was a marked increase in the percentage of oleic acid in the cholesteryl ester fraction. In atherosclerotic aortas incorporation of acetate-1-(14)C into the phospholipid and glyceride fractions increased 2-fold, while a much greater increase (up to 10-fold) was seen in incorporation into cholesteryl esters. In those birds receiving the atherogenic diet, palmitic acid accounted for approximately 50% of the fatty acid radioactivity, compared with approximately 25% from control aortas. Calculation of fatty acid synthesis showed the major newly synthesized fatty acids to be stearic acid in the phospholipid fraction; stearic, palmitic, and oleic acids in the glycerides; and oleic acid in the cholesteryl esters. The pattern of fatty acid synthesis was closely similar to the actual fatty acid composition of the aorta. In atherosclerotic aortas an increased synthesis of all fatty acids was seen, but the greatest increase was seen in the synthesis of oleic acid and its esterification to cholesterol.     

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.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Moderate intake of myristic acid in sn-2 position has beneficial lipidic effects and enhances DHA of cholesteryl esters in an interventional study

Among the saturated fatty acids (SFA), myristic acid is known to be one of the most atherogenic when consumed at high levels. Our purpose was to compare the effects of two moderate intakes of myristic acid on plasma lipids in an interventional study. Twenty-five male monks without dyslipidemia were given two isocaloric diets for 5 weeks each. In diet 1, 30% of the calories came from fat (8% SFA, 0.6% myristic acid) and provided 200 mg cholesterol/day. Calories of diet 2 were 34% fat (11% SFA, 1.2% myristic acid) with the same levels of oleate, linoleate, alpha-linolenate and cholesterol. A baseline diet was provided before each diet. In comparison with baseline, diets 1 and 2 induced a decrease in total cholesterol, LDL-cholesterol and triglycerides (P<.001); HDL-cholesterol was not modified and the apo A-I/apo B ratio increased (P<.001). Plasma triglycerides were lower after diet 2 than after diet 1 whereas HDL-cholesterol was higher (P<.05). In phospholipids, myristic acid, oleic acid, linoleic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased after diet 2 vs. baseline (P<.01) and diet 1 (P<.05). Both diets were associated with an increase in alpha-linolenate of cholesteryl esters (P<.05), but only diet 2 was associated with an increase in DHA of cholesteryl esters (P<.05). In diet 2, myristic acid intake was positively correlated with myristic acid of phospholipids, and alpha-linolenic acid intake was correlated with alpha-linolenic acid of cholesteryl esters. Moderate intake (1.2% of total calories) of myristic acid has beneficial lipidic effects and enhances DHA of cholesteryl esters.     

Short-term effect of dietary cholesterol on tissue n-6 fatty acids in fat-deficient rats

Weanling female rats raised on a fat-free diet for 8 weeks were then given the same diet supplemented with 0, 0.25, 0.5, or 1% by weight of cholesterol in addition to 10% of safflower oil for 3 days. Fatty acid compositions of cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in liver and plasma were examined. Cholesterol feeding increased plasma and liver cholesterol contents and also affected the patterns of n-6 polyunsaturated fatty acids. There were no consistent changes in either plasma and liver TG which contained little 20:3n-6 and 20:4n-6. The levels of 20:3n-6 increased in plasma and liver PL, while proportions of 20:4n-6 decreased in liver and plasma CE. However, the absolute amount of 20:4n-6 in cholesteryl esters increased because of a threefold rise in cholesteryl ester levels. The changes might be attributable to an increased utilization of 20:4n-6 for cholesterol transport and/or an inhibition of delta 5-desaturation of n-6 fatty acids by cholesterol feeding.     

Influence of diet on the composition of plasma cholesterol esters in man

The effect on the plasma cholesterol esters of diets rich in either carbohydrate, chocolate, or safflower oil was studied sequentially in two men. The changes in the cholesterol esters of the major plasma lipoproteins were studied by measuring (a) the distribution of fatty acids in the esters and (b) the distribution of radioactivity among the esters after the administration of cholesterol-4-(14)C labeled lipoproteins. Similar changes were found in the cholesterol esters of the two major lipoproteins; these changes became apparent within 24 hr after changing diets. Monounsaturated esters predominated with carbohydrate-rich diets. When the chocolate-rich diet was substituted, the proportion of saturated and monounsaturated esters fell and that of cholesteryl linoleate rose. This indicated the utilization of preexisting linoleate in preference to the more saturated fatty acids which abounded in the diet. The substitution of safflower oil led to further increments of cholesteryl linoleate. The possible reasons underlying the preferential incorporation of cholesteryl linoleate in man are discussed.     

Secretion of cholesteryl-ester-rich lipoproteins by the perfused livers of rabbits fed a wheat-starch-casein diet

Rabbits fed a cholesterol-free semi-synthetic wheat-starch-casein diet had a high plasma cholesterol concentration; most of the cholesterol was associated with low-density lipoproteins (LDL). Chemical analyses of plasma lipoproteins revealed that very-low-density lipoproteins (VLDL), intermediate lipoproteins and LDL from casein-fed rabbits contained more cholesteryl ester than that of lipoproteins isolated from chow-fed animals. The fatty acid composition of cholesteryl esters of plasma lipoproteins showed that there were higher contents of oleic acid than linoleic acids in lipoproteins from casein-fed rabbits. Lipoproteins isolated from liver perfusates of casein-fed rabbits had higher cholesteryl oleate content than lipoproteins from chow-fed rabbit liver perfusates. There was a marked increase in secretion of apolipoproteins from perfused livers of casein-fed rabbits. We conclude that the high levels of plasma cholesterol in casein-fed rabbits are of hepatic origin and that one of the hypercholesterolemic actions of dietary casein in rabbits is the induction of hepatic synthesis and secretion of cholesteryl-ester-rich lipoproteins.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

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.     

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

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

Effects of different types of polyunsaturated fatty acids on cholesterol esterification in human fibroblasts.

We have enriched human fibroblasts with oleic acid, with linoleic acid and with eicosapentaenoic acid. The accumulation of cholesteryl esters in the cells and the rate of esterification of cholesterol by microsomal acyl-CoA:cholesterol acyltransferase (ACAT) were measured in these cells. Cholesteryl ester levels were lower in cells enriched with eicosapentaenoic acid compared with cells enriched with oleate or linoleate. We also observed significantly lower ACAT activities in the microsomes from fibroblasts enriched with the n-3 polyunsaturated fatty acids relative to cells enriched with oleic acid or linoleic acid. We suggest that the presence of n-3 polyunsaturated fatty acids might suppress cholesteryl ester accumulation and inhibit atherogenesis.     

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.     

Selective incorporation of n-3 and n-6 fatty acids in essential fatty acid deficient rats in response to short-term oil feeding

Essential fatty acid deficient male Sprague Dawley rats were fed for 7 days a fat-free semi-synthetic diet supplemented with 10% by weight of different oil supplements. The oil supplement was a mixture of olive, safflower and linseed oils prepared at different proportions so the dietary n-9/n-6/n-3 ratios were approximate 2/1/1, 1/2/1, 1/1/2, and 1/1/1. The fatty acid compositions of plasma and liver lipids were then examined. Our results show polyunsaturated n-6 and n-3 fatty acids were selectively incorporated into plasma and liver phospholipids, and also into plasma cholesteryl esters. A preferential incorporation of n-6 over n-3 fatty acids into plasma cholesteryl esters and phospholipids was also observed.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography

An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

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.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.