Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.     

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.     

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.     

Specific stimulation of steroid 5 alpha-reductase solubilized from rat liver microsomes by endogenous phosphatidylserine

Dilauroylphosphatidylcholine caused a marked increase in progesterone 5 alpha-reductase activity solubilized from rat liver microsomes, whereas naturally occurring phosphatidylcholines from biological sources as well as dioleoylphosphatidylcholine had not effect on the activity. Therefore, the stimulatory effect of phospholipids normally found in rat liver microsomes was examined. The lipid extracts were prepared from the fraction which was freed from 5 alpha-reductase activity by DEAE-cellulose chromatography, and found to exhibit a strong stimulatory effect. The lipid extracts were then separated into phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine by chromatography on silicic acid column and preparative thin-layer plate. Among these endogenous phospholipids, only phosphatidylserine stimulated the 5 alpha-reductase, suggesting that the lipid requirement is specific for phosphatidylserine in steroid 5 alpha-reductase from liver microsomes.     

Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol.

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.     

beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.     

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.     

In vitro regulation of bovine adrenal cortical acyl-CoA: cholesterol acyltransferase and comparison with the rat liver enzyme

Acyl-CoA:cholesterol acyltransferase activity in bovine adrenal cortical microsomes was increased by preincubation of microsomes in vitro in the presence of MgCl2. The acyltransferase activity in the microsomes could be inhibited by further incubation in the presence of ATP/MgCl2. These effects appear to complement the known ATP-dependent activation of adrenal cytosolic cholesterol ester hydrolase, which is consistent with the role of the hydrolase in supplying cholesterol for steroidogenesis. These effects are, however, opposite to those recently demonstrated for the rat liver and intestine. Acyl-CoA:cholesterol acyltransferase activity in rat liver can be increased by the addition of cholesterol, as substrate, or by 25-hydroxycholesterol. Such activation was not observed in adrenal microsomal preparations, further suggesting that the mechanisms of regulation of cholesterol esterification differs between these tissues.     

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.     

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.     

Changes in both acyl-CoA:cholesterol acyltransferase activity and microsomal lipid composition in rat liver induced by distal-small-bowel resection.

The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of hepatic microsomal membrane were investigated 6 weeks after both 50 and 75% distal-small-bowel resection (SBR). A significant decrease in hepatic cholesteryl ester levels was observed after SBR, with a significant increase in the cholesteryl ester content of the livers of 75% SBR compared with the 50% SBR. Hepatic total acylglycerols, free cholesterol and phospholipid levels were not modified after the surgical operation. Microsomal free cholesterol was increased after both 50 and 75% SBR. However, a decrease in both microsomal ACAT activity and cholesteryl ester levels were found in microsomes (microsomal fractions) of resected rats, both changes being higher after 75 than after 50% resection. The total phospholipid content of the microsomes did not change after the surgical operation. The microsomal phospholipid fatty acid composition indicated higher changes after 75 than after 50% SBR. These results demonstrated that, in resected animals: (1) the activity of the enzyme responsible for catalysing cholesterol esterification (ACAT) is decreased, and (2) hepatic microsomal free cholesterol does not appear to influence the activity of ACAT.     

Compared with Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase,ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol

The capacity of acyl-CoA:cholesterol O-acyltransferase (ACAT) 2 to differentiate cholesterol from the plant sterol, sitosterol, was compared with that of the sterol esterifying enzymes, ACAT1 and lecithin:cholesterol acyltransferase (LCAT). Cholesterol-loaded microsomes from transfected cells containing either ACAT1 or ACAT2 exhibited significantly more ACAT activity than their sitosterol-loaded counterparts. In sitosterol-loaded microsomes, both ACAT1 and ACAT2 were able to esterify sitosterol albeit with lower efficiencies than cholesterol. The mass ratios of cholesterol ester to sitosterol ester formed by ACAT1 and ACAT2 were 1.6 and 7.2, respectively. Compared with ACAT1, ACAT2 selectively esterified cholesterol even when sitosterol was loaded into the microsomes. To further characterize the difference in sterol specificity, ACAT1 and ACAT2 were compared in intact cells loaded with either cholesterol or sitosterol. Despite a lower level of ACAT activity, the ACAT1-expressing cells esterified 4-fold more sitosterol than the ACAT2 cells. The data showed that compared with ACAT1, ACAT2 displayed significantly greater selectively for cholesterol compared with sitosterol. The plasma cholesterol esterification enzyme lecithin:cholesterol acyltransferase was also compared. With recombinant high density lipoprotein particles, the esterification rate of cholesterol by LCAT was only 15% greater than for sitosterol. Thus, LCAT was able to efficiently esterify both cholesterol and sitosterol. In contrast, ACAT2 demonstrated a strong preference for cholesterol rather than sitosterol. This sterol selectivity by ACAT2 may reflect a role in the sorting of dietary sterols during their absorption by the intestine in vivo.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol-induced microsomal changes modulate desaturase activities

The effect of 1% dietary cholesterol and 0.5% cholate on the rat liver microsomal composition and fatty acid desaturase activities was studied over various periods of time. The cholesterol content of liver microsomes increased as well as that of phosphatidylcholine. Cholesterol/phosphatidylcholine and phosphatidylcholine/phosphatidylethanolamine ratios were also elevated. Phosphatidylinositol decreased, but it recovered its original values at the end of the experimental period. Phosphatidylserine and sphingomyelin slightly decreased with time. Fatty acid composition changes were expressed by a saturated acid decrease and monounsaturated acid increase. Arachidonic acid content was also reduced. A similar pattern appeared in the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. Delta 9-Desaturase activity was enhanced as early as 48 h after cholesterol administration, whereas delta 5- and delta 6-desaturases were depressed during the same period and this enzymatic behaviour remained after 21 days of diet administration. The microsomal membrane was rigidized, as demonstrated by the increase of the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.