Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.     

Regulation of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in the rat adrenal gland.

The activities of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in rat adrenal gland were measured at various time intervals over 24 h. The activity of cholesterol esterase displayed diurnal rhythm, with a major peak at the onset of darkness coinciding with the peak in the diurnal rhythm of plasma corticosterone concentration. The activity of acyl-CoA : cholesterol acyltransferase also exhibited a characteristic diurnal rhythm, with the minimum activity occurring 3 h after the onset of darkness. The profile of the rhythm exhibited by the activity of the esterifying enzyme was similar to the mirror image of the pattern of diurnal rhythm in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Microsomal non-esterified cholesterol showed a gradual decline with a significant decrease in concentration at the onset of darkness, thus suggesting that diurnal removal of cholesterol in the environment of the esterifying enzyme and hydroxymethylglutaryl-CoA reductase leads to such diurnal decrease or increase in the activities of these two enzymes. Acute administration of corticotropin led to a 3-fold increase in the activity of cholesterol esterase, a 50% decrease in the activity of acyl-CoA : cholesterol acyltransferase and a 2-fold increase in the activity of hydroxymethylglutaryl-CoA reductase. Corticotropin administration also resulted in a significant decrease in microsomal non-esterified cholesterol and increase in plasma corticosterone concentration. These observations suggest that corticotropin plays an important part in generating the diurnal rhythm in the activities of the three enzymes.     

Upregulation of acyl-CoA: cholesterol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with profound abnormalities of lipid metabolism and accelerated arteriosclerotic cardiovascular disease. In a recent study, we found marked downregulation of hepatic lecithin-cholesterol acyltransferase, or LCAT, expression, which can account for impaired HDL maturation and depressed HDL cholesterol concentration in CRF. Here, we report on the effect of CRF on acyl-CoA:cholesterol acyltransferase (ACAT) expression. ACAT is an intracellular enzyme that catalyzes esterification of free cholesterol to cholesterol ester for storage or secretion. ACAT plays a major role in hepatic production and release of VLDL, intestinal absorption of cholesterol, foam cell formation, and atherogenesis. We examined hepatic expression of ACAT-1 and ACAT-2 mRNA (Northern blot) and protein (Western blot) abundance and total ACAT activity in male CRF rats (6 wk after 5/6 nephrectomy) and sham-operated controls. The CRF animals showed a significant reduction in creatinine clearance, marked hypertriglyceridemia, modest hypercholesterolemia, and significant upregulation of hepatic tissue ACAT-2 protein and mRNA abundance. In contrast, hepatic ACAT-1 mRNA and protein abundance were unaffected by CRF. Upregulation of ACAT-2 expression was accompanied by a significant increase in hepatic ACAT activity and a significant decrease in hepatic microsomal and whole liver free cholesterol concentration. Thus CRF results in significant upregulation of hepatic ACAT-2 (but not ACAT-1) expression and ACAT activity, which may, in part, contribute to the associated lipid disorders.     

Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.     

Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro.

The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.     

Induction of intracellular acyl-CoA:cholesterol acyltransferase activity in glioblastoma cells by lidocaine

The perturbation of cellular cholesteryl ester biosynthesis in glioblastoma C-6 cells by lidocaine was investigated. Lidocaine specifically inhibited the incorporation of radioactive oleic acid into cellular cholesteryl ester but had no significant effect on the incorporation of oleic acid into phosphatidylcholine. Oxygenated cholesterol-enhanced cholesteryl ester formation was less sensitive to lidocaine inhibition. Several other local anesthetics were compared. Lidocaine altered cholesteryl ester formation in time- and dose-dependent manners. Lidocaine was a powerful inhibitor initially and its potency declined with time. Lidocaine was capable of directly inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) activity in broken cell homogenates. The lidocaine-mediated inhibition of cellular cholesteryl ester formation triggered an enhanced intracellular ACAT activity that was not fully expressed in the presence of lidocaine. The activation of ACAT activity by lidocaine might represent a compensatory mechanism by which the inhibitory effect of lidocaine was partially overcome with time.     

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.     

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.     

Role of the intestinal acyl-CoA:cholesterol acyltransferase activity in the hyperresponse of diabetic rats to dietary cholesterol.

Contrary to normal rats, diabetic rats are known to develop marked hypercholesterolemia when fed a cholesterol-enriched diet. The triggering factor involved in this hyperresponse has not been identified. With the aim of clarifying the role of the intestinal acyl-CoA:cholesterol acyltransferase (ACAT), we studied the effects of a high fat diet and the changes of intestinal ACAT activity during the early development of streptozotocin-diabetes in rats. Feeding diabetic rats with a diet enriched in cholesterol and saturated fat produced an increase in plasma and in tissue cholesterol as early as 3 days after streptozotocin injection in the absence of hyperphagia. Under these experimental conditions, treatment with insulin or with the ACAT inhibitor CL-277082 significantly reduced the plasma cholesterol to levels measured in nondiabetic rats fed the same high fat diet. An increase in [14C]cholesterol in plasma very low density lipoprotein was observed after oral administration of labeled cholesterol to 3-day diabetic rats. In parallel experiments, the direct measurement of small intestine microsomal ACAT activity revealed an increase, averaging 288% in diabetic rats 3 days after diabetes induction. This change in ACAT activity occurred simultaneously with an increase in plasma glucagon and was normalized by insulin treatment. The induction of intestinal ACAT activity in diabetic rats, its modulation by insulin, and the hypocholesterolemic effects of insulin or CL-277082 treatment clearly indicate that ACAT activity plays a major role in the initiation of diabetes-associated hypercholesterolemia.     

Characterization of acyl-CoA:cholesterol acyltransferase from neonatal chick liver

Endogenous cholesterol esterification in chick liver microsomes was catalyzed by acyl-CoA:cholesterol acyltransferase using palmitoyl-CoA as substrate. An acyl-CoA hydrolase activity was also found in our microsomal preparations. Acyltransferase activity was stable after microsomes storage at -40 degrees C for 6 weeks and increased linearly with the preincubation time between 0 and 45 min. In our assay conditions, cholesteryl ester formation was linear up to 0.3 mg of microsomal protein in the reaction vial and 10 min of incubation. Maximal activity was found in reactions carried out in the presence of 1-2 mM dithiothreitol and 1.2 mg of bovine serum albumin, while acyl-CoA hydrolase was clearly inhibited by increasing albumin amounts.     

Saucerneol B derivatives as human acyl-CoA: cholesterol acyltransferase inhibitors

A series of 2a-i were prepared from a lead compound, saucerneol B (1) for evaluating their acyl-CoA: cholesterol acyltransferase inhibitory activities. Compounds 2a-g exhibited the high specificity of hACAT-1 than hACAT-2, whereas 2h and 2i showed very weak inhibitory activities in both hACAT-1 and hACAT-2. Saucerneol B (1) exhibited strong cholesterol-lowering effect in high cholesterol-fed mice.     

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.     

Novel 3,5-diaryl pyrazolines as human acyl-CoA:cholesterol acyltransferase inhibitors

A series of pyrazoline derivatives were prepared for evaluating their acyl-CoA:cholesterol acyltransferase activities. 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-5-(multi-substituted 4-hydroxyphenyl)-2-pyrazolines 4a-i were shown in vitro inhibitory activity on hACAT-1 and -2.     

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.     

Inactivation of acyl-CoA:cholesterol acyltransferase by ATP+Mg2+ in rat liver microsomes

The molecular modulation of acyl-CoA:cholesterol acyltransferase (EC 2.3.2.26) was studied in the microsomes of rat liver. Acyl-CoA: cholesterol acyltransferase was specifically inactivated by ATP and ADP, requiring Mg2+ as a cofactor. The inactivation was not due to substrate diminution nor to inhibition by the activity of acyl-CoA hydrolase, which was not affected by Mg2+ or ATP+Mg2+. Enhancement of inactivation of acyl-CoA: cholesterol acyltransferase by ATP+Mg2+, NaF and a heat-labile cytosolic factor (or factors) is consistent with a protein-kinase catalyzed phosphorylation being involved in the short term regulation of this enzyme.     

Influence of diets on acyl-CoA:cholesterol acyltransferase and on acyl-CoA:retinol acyltransferase in villous and crypt cells from rat small intestinal mucosa and in the liver

Cholesterol and retinol are both esterified with long-chain fatty acid within the mucosal cells of the small intestine. The reactions are catalyzed by microsomal acyl-CoA:cholesterol and acyl-CoA:retinol acyltransferases (EC 2.3.1.26, and EC 2.3.1.-, respectively). To gain more insight into the physiological importance of these acyltransferases, they were studied in villous and crypt cells from rats either fasting or on diets which varied in fat and cholesterol content. Both enzymes had a higher activity in villous than in crypt cells. The activities in villous cells varied with feeding and fasting and the composition of diet when the animals were killed postprandially. Acyl-CoA:cholesterol acyltransferase activity went up upon cholesterol feeding whereas retinol acyltransferase in the mucosa was reduced by high-fat diets. The liver cholesterol acyltransferase activity varied with diet, it increased with both cholesterol and fat feeding, whereas retinol acyltransferase activity remained relatively constant. The results obtained suggest that different diets are of importance for cholesterol and retinol acyltransferase activities both in the intestinal mucosa and in the liver. The variation in activities of the two acyltransferases suggests that they may be different enzymes.