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PubMed1995

A comparison of the esterification of steroids by rat lecithin:cholesterol acyltransferase and acyl coenzyme A:cholesterol acyltransferase.

Although fatty acid esters of several steroids have been found in both blood and tissues, their biosynthetic origins are uncertain. For example, the fatty acid esters of delta 5-3 beta-hydroxysteroids pregnenolone and dehydroepiandrosterone (DHEA) are synthesized in tissues by an acyl coenzyme A:acyltransferase. These esters are not secreted, and the circulating esters are formed in blood by lecithin:cholesterol acyltransferase (LCAT). Fatty acid esters of corticosterone (B) and estradiol (E2) are also present in both blood and tissues, but unlike the delta 5-3 beta-hydroxysteroids, their structures are so different from cholesterol that it would not necessarily follow that they are esterified by the same enzyme. We have examined the esterification of the steroids DHEA, B, and E2 in blood and tissue, in comparison to the esterification of cholesterol, using as a model plasma and hepatic microsomes from the rat. All of the steroids were esterified in plasma, but at very different rates: cholesterol > DHEA >> E2 = B. The LCAT inhibitor, 5.5'-dithiobis-(2-nitrobenzoic acid), inhibited the esterification of all of the substrates. DHEA inhibited the esterification of cholesterol, albeit only at high concentration. The fatty acid compositions of the cholesterol and DHEA esters were analyzed, and they were found to be identical, with arachidonate the predominant ester, greater than 60%. In hepatic microsomes, the rate of esterification was different than plasma: cholesterol > E2 > or = DHEA >> B. Although B was esterified in both plasma and hepatic microsomes, the rate was exceedingly slow in both. The acyl coenzyme A:cholesterol acyltransferase inhibitor, N'-(2,4-difluorophenyl)-N-[[4-(2,2-dimethylpropyl)phenyl]- methyl]-N-heptylurea, blocked the esterification of cholesterol almost completely, but surprisingly, it had no effect on the esterification of the other steroids. The fatty acid esters of cholesterol, E2, and DHEA synthesized in the hepatic microsomes were analyzed. The composition of the cholesterol esters from the microsomes was very different than the esters of DHEA and E2. These results show that all of the steroids tested are esterified by LCAT, and consequently that blood LCAT is the probable source of the circulating steroidal esters. Most interesting are the studies of microsomal esterification. It has been presumed that similar to blood, the esterification of steroids in tissues is carried out by the same enzyme that esterifies cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Role of multidrug resistance P-glycoproteins in cholesterol esterification.

Cholesterol esterification, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), plays a central role in cellular cholesterol homeostasis and in physiologic processes that lead to coronary heart disease. Although ACAT resides in the endoplasmic reticulum (ER), the cholesterol substrate for esterification originates in the plasma membrane and must be transported to the ER for esterification. Progesterone inhibits esterification, possibly by blocking the transport of cholesterol to the ER. Recent studies suggest that progesterone acts by inhibiting the activity of one or more of the multidrug-resistant (MDR) P-glycoproteins. In the current manuscript, we demonstrate that progesterone's ability to inhibit esterification is not mediated through the progesterone receptor. We evaluate a series of steroid hormones and find a strong correlation between a steroid hormone's hydrophobicity and its ability to inhibit both cholesterol esterification and MDR-catalyzed drug efflux. We also find that cholesterol esterification is inhibited by nonsteroidal MDR inhibitors, and that this inhibition specifically affects the esterification of cholesterol derived from the plasma membrane. MDR inhibitors also inhibit cholesterol esterification in a wide range of cultured human cell lines. These observations suggest that MDR activity normally functions in a general process of intracellular cholesterol transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1981

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

The relationship between plasma lecithin:cholesterol acyltransferase mass and enzyme activity and between mass and plasma cholesterol esterification rate was determined in 25 adult volunteers without overt disease (14 normolipidemic and 11 hyperlipidemic). Furthermore, the relationship of lecithin:cholesterol acyltransferase mass and cholesterol esterification rate to lipids, apoproteins, age, and ideal body weight was assessed. Lecithin:cholesterol acyltransferase mass determined by radioimmunoassay was highly correlated with enzyme activity assayed using a heated plasma substrate (r = 0.636) and with the molar cholesterol esterification rate determined either by radioassay (r = 0.809) or by measurement of the decrease of unesterified cholesterol (r = 0.621). Lecithin:cholesterol acyltransferase mass was also positively correlated with total cholesterol (r = 0.608), unesterified cholesterol (r = 0.562), age (r = 0.544), and percent ideal body weight (r = 0.619), but was not significantly correlated with log triglyceride, high density lipoprotein cholesterol, or apolipoproteins A-I, A-II, or D. Plasma cholesterol esterification rate by both methods was highly positively correlated with total cholesterol, unesterified cholesterol, log triglyceride, and age, but was inversely correlated with high density lipoprotein cholesterol. Upon partial correlation analysis with lecithin:cholesterol acyltransferase mass kept constant the cholesterol esterification rate remained significantly positively related to total cholesterol, unesterified cholesterol, and log triglyceride and inversely related to high density lipoprotein cholesterol. Two subjects had normal lecithin:cholesterol acyltransferase but approximately half normal molar cholesterol esterification rate. Measurement of lecithin:cholesterol acyltransferase mass and activity along with plasma cholesterol esterification rate will permit differentiation of abnormalities of enzyme from qualitative or quantitative substrate or cofactor abnormalities. Also, the finding that the regression line between LCAT mass and the plasma esterification rate by direct determination of unesterified cholesterol passes through the origin suggests that all immunodetectable LCAT in plasma is active in normal subjects.-Albers, J. J., C-H. Chen, and J. L. Adolphson. Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Adult↗
PubMed1985

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.

Acyltransferases↗
PubMed1995

Esterification of oxysterols by human plasma lecithin-cholesterol acyltransferase.

In the present study, lecithin-cholesterol acyltransferase (LCAT) catalyzed esterification of oxysterols was investigated by using discoidal bilayer particles (DBP) containing various oxysterols, phosphatidylcholines, and apolipoprotein A-I. The esterified oxysterols were analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. LCAT esterified all oxysterols tested that are known to be present in human plasma. The esterification yields in almost all cases were relatively high, often as high as the yield of cholesterol esterification. When DBP preparations containing 27-hydroxycholesterol and various phosphatidylcholines were used for the LCAT reaction, both monoesters and diesters were produced. The mass spectrometry analysis showed that the monoester was produced by the esterification of the 3 beta-hydroxyl group and not the 27-hydroxyl group. The diesters were apparently produced by the esterification of the 27-hydroxyl group only after the esterification of the 3 beta-hydroxyl group. Phosphatidylcholine containing a saturated acyl group at sn-1 position and an unsaturated acyl group at sn-2 position gave generally high esterification yield. The esterification of various oxysterols was compared by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols produced 3 beta-oleoyl monoesters. Unlike 27-hydroxycholesterol, 25-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or cholestanetriol did not produce diesters. Various factors influencing the formation of the monoesters and diesters from 27-hydroxycholesterol were investigated. When dioleoyl-phosphatidylcholine was used as the acyl donor, prolonged dialysis of DBP preparations and increase in the ratio of the enzyme concentration to substrate particle concentration increased the diester formation. Significant amounts of diesters were also produced by using 1-palmitoyl-2-oleoyl-phosphatidylcholine and other phosphatidylcholines as the acyl donors. By analyzing the conditions of monoester and diester formation, a scheme for the LCAT reaction pathway was proposed.

Apolipoprotein A-I↗
PubMed1999

Regiospecific esterification of estrogens by lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT), the enzyme that esterifies cholesterol in blood, also esterifies other steroids at the 3beta-hydroxyl. These steroids, like cholesterol, are delta5-3beta-hydroxysteroids, such as pregnenolone and dehydroepiandrosterone. One unusual LCAT substrate is the estrogen, estradiol, which is esterified at the 17beta-hydroxyl. The esterification of estradiol by LCAT has been reported to produce a powerful antioxidant that protects low density lipoprotein (LDL) from oxidation. We investigated the substrate specificity of LCAT, comparing the esterification of four different steroids (estradiol, estriol, testosterone, and 5-androstene-3beta, 17beta-diol) by human LCAT in blood and by acyl-coenzyme A:acyltransferase in tissue (placenta and fat). Estradiol was esterified only at the D ring 17beta-hydroxyl group in both blood and tissue. In contrast, although testosterone has a D ring structure identical to that of estradiol, and it was esterified at the 17beta-hydroxyl by acyl-coenzyme A:acyltransferase in tissue, it was not esterified by LCAT. When 5-androstenediol was the substrate in the tissues, both the 3beta- and 17beta-esters were synthesized, but the major product was the 17beta-ester. Conversely, although 5-androstenediol was an excellent substrate for LCAT, only the 3beta-hydroxyl was esterified. No 17beta-ester was formed. The comparison of the esterification of estriol by acyl-coenzyme A:acyltransferase and LCAT was also surprising. In the tissues, estriol is esterified at both D ring hydroxyls, and both are esterified about equally. Although estriol is an extremely polar estrogen, it is esterified by LCAT, albeit at a very slow rate. Although again both D ring hydroxyls were esterified, the LCAT esterification site was mainly at the 17beta-hydroxyl. Esterification of estriol at the 17beta-hydroxyl in preference to the 16alpha-hydroxyl is especially striking, because the 17beta-hydroxyl group is sterically shielded by the C-18 methyl group, making esterification at this position energetically much more difficult. Furthermore, these studies demonstrate that esterification of the 17beta-hydroxyl group by LCAT is unique to estrogens. It suggests that this unusual regiospecific esterification of C-17 of the estrogens underlies a distinct stereochemical requirement for the powerful antioxidant action that has reported for the estradiol esters formed by LCAT.

Acyl Coenzyme A↗
PubMed1991

Regulation of retinol uptake and esterification in MCF-7 and HepG2 cells by exogenous fatty acids.

The influence of extracellular fatty acids on the uptake and esterification of [3H]retinol bound to human retinol-binding protein (RBP), to RBP-transthyretin (TTR), or in dispersed form by the human hepatoma, HepG2, and human mammary epithelial carcinoma, MCF-7, cell lines was studied. The esterification of [3H]retinol was significantly increased in cells incubated with myristic, palmitic, stearic oleic, or linoleic acid-albumin complexes and was observed for all forms of [3H]retinol. Enhancement of [3H]retinol uptake was also observed in cells incubated with these fatty acids, but this increase was relatively small for the dispersed form as compared to that observed for [3H]retinol bound to RBP or RBP-TTR. Comparing equal concentrations of the [3H]retinol donors, cell uptake and esterification was greatest from the dispersed form and least from that bound to RBP-TTR. After preincubation of cells with oleate, uptake and esterification of [3H]retinol was increased but not to the extent observed when oleate and [3H]retinol donor were co-incubated. Incubation of cells with oleate resulted in rapid and correlated increases in the rates of [3H]retinol uptake and esterification which persisted until the steady state for [3H]retinol uptake was achieved. Beyond this time, net esterification of [3H]retinol continued in the presence of oleate. This kinetic pattern was observed for all [3H]retinol donors. These effects on [3H]retinol uptake and esterification were dose-dependent as the oleate to albumin ratio was varied from 0.5 to 3.0 and were observed across a physiological concentration range of RBP-3H-retinol. The data indicate that: 1) the fatty acid status of cells is a determinant of retinol uptake and esterification; and 2) the form of retinol presentation to cells is not qualitatively important for these processes.

Animals↗
PubMed1992

Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis.

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.

Cell Line↗
PubMed1994

The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages.

Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-coenzyme A: cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the ability of these cells to store lipoprotein-cholesterol and in the eventual development of atheroma foam cells. Herein, we provide evidence that the actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. When the actin cytoskeleton of cultured mouse peritoneal macrophages was disrupted by treatment with cytochalasin D or Clostridial C2 toxin, the ability of beta very low density lipoprotein (beta-VLDL) to stimulate cholesterol esterification was decreased 3-6-fold, even under conditions in which beta-VLDL protein degradation, cholesteryl ester hydrolysis, or net cholesterol delivery to the cells was matched. Esterification of cellular phospholipids and triglycerides was not affected by this treatment. Cytochalasin D treatment of macrophages also inhibited the ability of acetyl-low density lipoprotein, another foam cell-forming lipoprotein, to stimulate cholesterol esterification, but stimulation of cholesterol esterification by 25-hydroxycholesterol was not inhibited by cytochalasin D. Cytochalasin D was found to inhibit neither the exit of beta-VLDL-derived cholesterol from lysosomes nor the ability of beta-VLDL to down-regulate endogenous cholesterol synthesis. From these data we conclude that an intact actin cytoskeleton is necessary for efficient stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. Although the exact function of actin in the cholesterol esterification pathway remains to be determined, our data rule out a general role for actin in intracellular cholesterol trafficking or maintenance of ACAT enzyme activity. Rather, we speculate that actin filaments play a role in specific cellular entry processes of atherogenic lipoproteins and/or in establishing transport or contact between the plasma membrane cholesterol substrate pool and the ACAT enzyme in macrophages.

Actins↗
PubMed2004

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases.

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte-derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]-oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA-induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal-regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.

Cholesterol Esters↗
PubMed2004

In vitro and in vivo trans-esterification of 1-[2(R)-(2-amino-2-methylpropionylamino)-3-(1H-indol-3-yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester and the effects of ethanol on its pharmacokinetics in rats.

PURPOSE: To investigate the in vitro trans-esterification of 1-[2(R)-(2-amino-2-methylpropionylamino)-3-(1H-indol-3-yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester (compound A) and to determine the effects of ethanol on its in vivo pharmacokinetics in male Sprague-Dawley rats. METHODS: The effects of deuterated [d5]ethanol on the hydrolysis and trans-esterification of compound A in rat plasma and rat liver microsomes in the presence or absence of bis(p-nitrophenyl) phosphate (BNPP), a carboxylesterase inhibitor, were investigated. Following an oral pretreatment with deuterated ethanol in conjunction with an intravenous dose of compound A to rats, the pharmacokinetics of compound A and deuterated compound A were evaluated. RESULTS: It was observed that the amount of deuterated compound A generated increased with increasing amounts of deuterated ethanol in incubates, whereas the amount of hydrolyzed product (compound B) decreased. BNPP inhibited both the hydrolysis and the trans-esterification of compound A. Furthermore, the pharmacokinetics of compound A in rats receiving ethanol was altered, such that the plasma clearance decreased by 1.5-fold and the elimination rate constant decreased by 2-fold. Deuterated compound A was determined, confirming that trans-esterification proceeded in vivo; approximately one third of the intravenous dose of compound A underwent trans-esterification. CONCLUSIONS: In the presence of ethanol, compound A underwent trans-esterification catalyzed by carboxylesterases. Ethanol pretreatment resulted in a decrease in the in vivo clearance of compound A mainly due to trans-esterification with ethanol.

Administration, Oral↗
PubMed1997

Effect of lacidipine on cholesterol esterification: in vivo and in vitro studies.

1. Cholesterol esterification and accumulation in the arterial wall is a hallmark of atherogenesis. Several preclinical studies suggest that calcium antagonists may exert antiatherosclerotic activity by directly affecting atherogenesis in the arterial wall. We investigated the effect of the second generation dihydropyridine calcium antagonist lacidipine on cholesterol metabolism in vivo in the aortic arch of cholesterol fed rabbits, and in vitro in mouse cultured peritoneal macrophages. 2. Treatment of cholesterol-fed rabbits with 1, 3 and 10 mg kg-1 day-1 of lacidipine for two months reduced, in a dose-dependent manner, cholesterol esterification in the aortic arch: 24 +/- 6, 30 +/- 12, and 41 +/- 8% inhibition, respectively (P < 0.001 vs HC control). Concomitantly, drug treatment reduced total cholesterol content of the vessel wall. Lacidipine 3 and 10 mg kg-1 day-1 reduced cholesterolaemia (approximately 20%); no effect was observed at the lowest dose used (1 mg kg-1 day-1). These results suggest that the action of lacidipine on cholesterol esterification in the arterial wall involves, at least in part, a direct effect on cellular cholesterol metabolism. Inhibition of cholesterol esterification in the arterial wall was observed also in a reference group of animals treated with the specific ACAT inhibitor CI-976. 3. To evaluate the action of lacidipine on intracellular cholesterol metabolism we performed in vitro experiments with murine macrophages, the main cell type that accumulates cholesterol in the arterial wall. Lacidipine almost completely inhibited cholesterol esterification in cholesterol loaded macrophages in culture. The effect was observed independently of esterification stimuli and in cell free homogenates. The drug modified intracellular cholesterol distribution, doubling the free- to esterified sterol ratio, but did not influence the cellular rate of cholesteryl ester hydrolysis in the cell. All together these results indicate an inhibitory effect of lacidipine on cholesterol esterification catalyzed by the enzyme ACAT in murine macrophages. 4. We concluded that lacidipine influences cellular cholesterol metabolism. This effect may contribute to the potential antiatherosclerotic activity of this drug.

Animals↗
PubMed1979

Development of fatty acid esterification mechanisms in rat small intestine.

Fatty acid esterification was measured in fetal jejunal and ileal isografts implanted under the kidney capsules of adult host rats and compared to the age-controlled intestine grown in situ. Studies were conducted on the 21st, 35th, 49th, and 63rd postconceptional days, corresponding to prenatal, suckling, weaning, and weaned rats. Substantial fatty acid esterification activity was found in prenatal jejunum but not in ileum. A proximal-distal gradient of fatty acid esterification was observed in all groups grown in situ, but not in isografts. The monoglyceride pathway (MG-P) accounted for about one-third of total fatty acid esterification (TFAE) in jejunum grown in situ and remained constant through the study. In the ileum, MG-P was the major esterification pathway during the first 4 postnatal weeks, but decreased progressively after weaning to become insignificant in adult rats. TFAE fell in the jejunal isografts, whereas it increased in the ileum. MG-P remained as the major pathway in the implanted jejunum and ileum. Our studies suggest that luminal contents are probably the most important modulator for the development and maintenance of intestinal fatty acid esterification, and "fetal programming" manifested by changes in fatty acid esterification mechanisms in the isografts is less important.

Animals↗
PubMed1999

Retinol esterification activity contributes to retinol transport in stellate cells.

The mechanisms of retinol transport and accumulation in hepatic stellate cells (HSC) remain to be elucidated. Our previous studies suggested that retinol esterification activity, particularly lecithin:retinol acyltransferase (LRAT) activity, in liver retinoid metabolism is important to elucidate the relationship between retinol uptake by HSC and the esterification of retinol. In the present study, using a human HSC-like cell line, LI90, we demonstrated that retinol esterification activity of LI90 cells is similar to that of primary cultures of rat HSC and higher than that of a human hepatoma cell line. Further, since progesterone or diphospho-lauroyl-phosphatidylcholine increased retinol esterification activity of LI90 cells, it is likely that LRAT contributes to retinol esterification in LI90. We examined retinol esterification in LI90 cells and clearance of retinol from culture medium. The percentages of both retinol and esterified retinol in LI90 cells increased in a manner dependent on retinol concentration in medium, whereas that of retinol in medium decreased. The percentages of esterified and unesterified retinol in LI90 cells and of retinol in medium were linearly dependent on the logarithm of the initial concentration of retinol in the medium. These results suggest that retinol esterification activity contributes to retinol uptake by HSC and maintenance of non-toxic retinol levels in plasma.

Actins↗
PubMed1989

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

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

Acetylation↗
PubMed1988

Eicosapentaenoic acid inhibits cholesterol esterification in cultured parenchymal cells and isolated microsomes from rat liver.

The effects of eicosapentaenoic acid on synthesis and secretion of cholesterol and cholesterol ester by cultured rat hepatocytes were studied. In the presence of eicosapentaenoic acid cellular cholesterol esterification was decreased by 50-75% compared to oleic acid as measured by radioactive precursors and mass. Secretion of cholesterol ester was reduced by 50-60% in the presence of eicosapentaenoic acid as evaluated by radiolabeled fatty acids, mevalonolactone, and mass measurement. Oleic, palmitic, and stearic acid increased, whereas eicosapentaenoic and docosahexaenoic acid decreased synthesis and secretion of cholesterol ester as compared to a fatty acid-free control. Cellular and secreted free cholesterol were unaffected by eicosapentaenoic acid in comparison with oleic acid. The reduced cholesterol esterification was observed within 1 h and lasted for at least 20 h. Eicosapentaenoic acid caused lower cholesterol esterification than oleic acid in the concentration range 0.2-1.0 mM fatty acid and reduced the stimulatory effect of oleic acid on cholesterol ester formation. Cholesterol esterification and release of cholesterol ester were markedly increased by 25-hydroxycholesterol in the presence of eicosapentaenoic acid as well as oleic acid. Experiments with liver microsomes revealed that radioactive eicosapentaenoic acid and eicosapentaenoyl-CoA were poorer substrates (7-30%) for cholesterol esterification than oleic acid and oleoyl-CoA. Reduced formation of cholesterol ester was also observed when eicosapentaenoyl-CoA was given together with labeled oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linolenoyl-CoA, and arachidonoyl-CoA had no inhibitory effect. In conclusion, eicosapentaenoic acid reduced cellular cholesterol esterification by inhibiting the activity of acyl-CoA:cholesterol acyltransferase. The lowered cholesterol esterification caused by eicosapentaenoic acid secondly decreased secretion of very low density lipoprotein cholesterol ester.

Animals↗
PubMed1988

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein.

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.

Animals↗
PubMed1988

Hepatic perfusate very low density lipoproteins obtained from fat-fed nonhuman primates stimulate cholesterol esterification in macrophages.

The livers of both baboons and rhesus monkeys fed a high fat, high cholesterol diet secreted very low density lipoproteins (VLDL) that were enriched in cholesteryl ester and apoe as compared to VLDL secreted by the livers of chow-fed animals. Stimulation of macrophage cholesterol esterification by the experimental VLDL was compared to that produced by the standard beta-VLDL obtained from the plasma of a rhesus monkey fed 25% coconut oil plus 2% cholesterol. This standard beta-VLDL stimulated 7- to 10-fold more esterification than did the bovine albumin control. Hepatic VLDL from fat-fed animals stimulated esterification in J774 macrophages 50 to 150% as well as did the standard beta-VLDL, even though hepatic VLDL did not display beta electrophoretic mobility on agarose gel electrophoresis. Plasma VLDL from lard-fed baboons did not exhibit beta electrophoretic mobility but did stimulate esterification in macrophages. Baboons were divided into high and low responders based on the change in plasma cholesterol levels in response to a high fat, high cholesterol diet. Both plasma and hepatic VLDL from high responders stimulated cholesterol esterification, whereas hepatic VLDL obtained from low responders or chow-fed baboons did not stimulate cholesterol esterification in macrophages. There was a strong positive correlation (r = 0.866) between the number of apoE molecules per VLDL particle in VLDL obtained from chow-fed, lard-fed, or coconut oil-fed primates and the rate of cholesterol esterification in macrophages. Our results show that hepatic perfusate VLDL obtained from fat- and cholesterol-fed primates have compositional and functional properties usually ascribed to circulating beta-VLDL, without displaying beta mobility, and indicate that the liver may be an important source of atherogenic lipoproteins.

Animals↗