ACAT Inhibitor Research Articles

Potential antiatherosclerotic agents. 6. Hypocholesterolemic trisubstituted urea analogues.

The discovery that a series of N,N-dialkyl-N'-arylureas were inhibitors of the ACAT enzyme has led to a structure-activity study involving the systematic modification of three sites of the urea backbone. This study culminated in the selection of N'-(2,4-dimethylphenyl)-N-benzyl-N-n-butylurea (115) for more extensive biological evaluation. ACAT inhibitors are seen as potentially beneficial agents against hypercholesterolemia and atherosclerosis.

Synthesis and secretion of apoE in thioglycolate-elicited mouse peritoneal macrophages: effect of cholesterol efflux.

ApoE synthesis and secretion, as a function of cellular cholesterol content and cholesterol efflux, was studied in thioglycolate-elicited mouse peritoneal macrophages. As expected, loading elicited macrophages with cholesterol induced a 5-fold increase in apoE secretion and a 2.5-fold increase in cellular apoE content over a 5-h period. Treatment of cholesterol-loaded cells with HDL3 further increased apoE secretion 1.7-fold and decreased cellular cholesterol by 20%. Treatment of cholesterol-loaded cells with HDL3 and SAH 58.035 (an ACAT inhibitor) increased apoE secretion 2.4-fold and decreased cellular cholesterol content by 35%. Treatment of the cells with the ACAT inhibitor alone suppressed apoE secretion by 40% but did not change cellular cholesterol content. Northern blot analysis of RNA indicated that cholesterol loading increased apoE mRNA 2-fold. ApoE mRNA levels were not further affected by treatment with HDL3 and/or the ACAT inhibitor. Cholesterol-loaded cells, in the absence of HDL3, secreted apoE into the media in two fractions as determined by column chromatography: a large molecular weight complex, (larger than HDL), and an essentially lipid-free protein. In the presence of HDL3, the cells secreted apoE in three fractions: a large molecular weight complex, an essentially lipid-free protein, and over 50% of apoE associated with HDL. In the process, HDL3 became larger and eluted in a position identical to that of HDL2. A small amount of HDL3-derived material was also transformed to an LDL-size particle. Incubation of HDL3 in the absence of cholesterol-loaded cells did not produce these changes. It is concluded that cholesterol-loading increases apoE mRNA content and apoE synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of sterol synthesis. Exogenous oleate reduces the inhibitory effect of 5α-Cholest-8(14)-en-3β-ol-15-one on the growth of CHO-K1 cells

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one, a hypocholesterolemic agent and a potent inhibitor of sterol biosynthesis, inhibited the growth of CHO-K1 cells incubated in medium containing fetal calf serum. The concentration of the oxysterol required to inhibit growth by 50% was 13 microM. Sodium oleate (82 microM) reduced the inhibitory effects of the sterol, and increased the concentration of the 15-ketosterol required to cause a 50% inhibition of growth to 25 microM. The ACAT inhibitor N'-(2,4-difluoro-phenyl)-N-[4-(2,2-dimethylpropy)-phenyl]-methyl)- N- heptylurea (5 microM) abolished the effect of sodium oleate, and reduced the concentration of the 15-ketosterol required to inhibit growth by 50% to 5 microM.

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 2. Identification of a coenzyme A regulatory site by p-mercuribenzoate modification.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26, ACAT) is the major intracellular cholesterol-esterifying activity in vascular tissue and is potentially a key regulator of intracellular cholesterol homeostasis during atherogenesis. We have previously reported inhibition of microsomal ACAT by histidine and sulfhydryl-selective chemical modification reagents and present here a more detailed analysis of the effect of sulfhydryl modification on ACAT activity. This analysis indicated two effects of sulfhydryl modification on ACAT activity. Modification of aortic microsomes with relatively low concentrations of p-mercuribenzoate (PMB) (100-200 microM) identified an inhibitory coenzyme A binding site on ACAT which contains a modifiable sulfhydryl group. This site binds CoA tightly (Ki = 20 microM), and PMB modification prevented subsequent ACAT inhibition by CoA without itself inhibiting enzyme activity. At higher concentrations (1-2 mM), PMB inhibited ACAT activity, indicating the presence of a modifiable sulfhydryl group necessary for cholesterol esterification by ACAT. Modification of both sites by PMB was reversible by thiols, and protection against modification was afforded in both cases by oleoyl-CoA, indicating that these sites may also bind oleoyl-CoA. Thus, at least two sulfhydryl groups influence ACAT activity: one is necessary for cholesterol esterification by ACAT, and one is at or near an inhibitory CoA binding site, which may be occupied at intracellular concentrations of CoA.

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. II. Characterization of intracellular morphologic changes.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on cholesterol-loaded macrophages were compared at the ultrastructural level. The Ca++ antagonist induced the formation of membrane-surrounded "lamellar bodies" originating from lysosomes. The macrophages secreted these lamellar bodies, which were rich in phospholipids and cholesterol, into the culture medium even in the absence of cholesterol acceptors. In contrast, the ACAT inhibitor induced the formation of lamellar bodies originating from lipid droplets, which were also surrounded by membranes. There is strong evidence that these latter membranes were newly synthesized at the margin of the lipid droplets by the endoplasmic reticulum. The lamellar bodies descending from lipid droplets after ACAT inhibitor treatment were not secreted by the cells. They were stored in the cytoplasmic compartment in the absence of high density lipoproteins (HDL). When HDL were added to the medium, the lamellar bodies specifically interacted with endosomes containing the internalized HDL particles and disappeared concomitantly with an enhanced HDL-mediated cholesterol efflux. From the biochemical data in the accompanying article and the morphological data here, we conclude that macrophages release cholesterol by two major pathways: 1) an HDL-independent secretion of lamellar bodies containing cholesterol, which originate from lysosomes, and 2) an HDL-dependent release of cholesterol via the formation of lamellar bodies descending from lipid droplets, which intracellularly fuse with HDL-containing endosomes. The cholesterol-enriched HDL are then secreted as intact lipoproteins.

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. I. Characterization of cellular lipid metabolism.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on the cholesterol metabolism of cholesterol-loaded macrophages were compared. We demonstrated that apolipoprotein A-I containing high density lipoproteins (HDL) bind to specific receptor sites on macrophages, are internalized, take up cholesterol, and are then released from the cells as native lipoproteins. The ACAT inhibitor enhances HDL receptor activity and promotes HDL-mediated cholesterol efflux from cultured mouse peritoneal macrophages. In contrast, the Ca++ antagonist increases acetyl LDL-mediated cholesterol influx, abolishes the increase in HDL binding induced by cholesterol accumulation, enhances apo E synthesis, and promotes cholesterol efflux by a mechanism independent of the presence of HDL in the surrounding medium. Concomitantly, a decrease in nucleoside transporter activity, an increase in intracellular ATP hydrolysis, adenosine and cyclic AMP concentration, and a stimulation of the activities of acid and neutral cholesteryl ester hydrolase and ACAT indicated that protein kinase A-catalyzed phosphorylation reactions might be involved in the increase in cholesterol efflux. The Ca++ antagonist-induced efflux occurred only with lysosomal-associated cholesterol, while the ACAT inhibitor acted on the formation of cytoplasmic lipid droplets. The secreted lipoprotein particles contained 68% unesterified cholesterol and 21% phospholipids, 8% esterified cholesterol, and 3% triglycerides. The phospholipid components were: 72% phosphatidylcholine, 22% sphingomyelin, and 6% phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine. We conclude that macrophages release cholesterol in two ways: 1) an HDL-mediated release of unesterified cholesterol increasing upon ACAT inhibition, and 2) an HDL-independent secretion of cholesterol which can be amplified by Ca++ antagonists.

Mechanism of the Inhibition of Cholesterol Absorption by DL-Melinamide: Inhibition of Cholesterol Esterification

In order to elucidate the mechanism of action of DL-melinamide [DL-MA, N-(α-methylbenzyl)linoleamide], an inhibitor of cholesterol absorption, the effect of DL-MA on esterification of cholesterol in the mucosa of rabbit small intestine was studied. DL-MA inhibited acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) activity in the mucosal microsomes, with 50% inhibition occurring at approximately 0.5 μM. On the other hand, DL-MA had no effect on the cholesterol esterase (EC 3.1.1.13) activity in the mucosal cytosol. Kinetic studies indicate that DL-MA is an uncompetitive inhibitor of ACAT. D-MA, one of the two optical isomers of DL-MA, was found to be a more effective inhibitor of ACAT than L-MA, another isomer. This finding indicates that the inhibition of cholesterol absorption by DL-MA depends on the inhibition of ACAT by this compound, in view of the fact that D-MA is a more effective inhibitor of cholesterol absorption than L-MA.

Role of acyl CoA:cholesterol acyltransferase in cholesterol absorption and its inhibition by 57-118 in the rabbit.

Esterification of cholesterol in rabbit small intestine mucosal microsomes by acyl CoA:cholesterol acyltransferase (ACAT, Ec 2.3.1.26) and mucosal cytosol by cholesterol esterase (EC 3.1.1.13) was studied. Compound 57-118. N-(1-oxo-9-octadecenyl)-DL-tryptophan(Z)ethyl ester, an inhibitor of cholesterol absorption, was found to inhibit in vitro ACAT in mucosal microsomes at concentrations of 2-20 nmol/0.5 ml incubation mixture, but had no effect on cholesterol esterase in the cytosol at similar concentrations. A kinetic analysis using a Lineweaver-Burk plot indicates that 57-118 acts as a competitive inhibitor of ACAT. An ex vivo study in the rabbit where 57-118 was given by gavage at a dose of 200 mg/kg also showed inhibition of ACAT but not of cholesterol esterase. High performance liquid chromatography determination of 57-118 in various subcellular fractions demonstrated the presence of this substance after oral administration in concentrations in mucosal microsomes equivalent to those required to show inhibition of ACAT in vitro. These data support the work of Norum et al. (1979, Eur. J. Clin. Invest. 9: 55-62) indicating mucosal ACAT plays a significant role in cholesterol absorption.

Effect of chlorpromazine on lipid metabolism in aortas from cholesterol-fed rabbits and normal rats, in vitro: Inhibition of sterol esterification and modification of phospholipid synthesis

Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes and in intact arterial tissue from the rat and cholesterol-fed rabbit in vitro. In isolated rabbit arterial microsomes, CPZ resulted in a concentration-dependent inhibition of ACAT with 50% inhibition of [1- 14C]oleoylCoA incorporation into [ 14C]cholesteryl esters occurring at 0.1 m M CPZ. CPZ also effectively inhibited the incorporation of [ 14C]oleate into triglycerides without affecting incorporation into diglycerides. Additionally, CPZ altered the pattern of arterial phospholipids synthesized from [1- 14C]oleate. Incorporation into phosphatidylcholine was depressed while incorporation into phosphatidylinositol was increased. Since diglyceride synthesis appreared to be unaffected by CPZ, a redirection of phosphatidic acid into the CDP-diglyceride pathway of glycerolipid synthesis does not adequately account for the effect of CPZ on arterial phospholipid and triglyceride synthesis in these experiments.