Differences in the potency of ACAT inhibitors in two assay systems

Abstract

Acyl CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to prevent cholesterol absorption in the intestine [l], cholesterol storage and apo B secretion from the liver [2], and cholesterol accumulation in the aortic artery [3,4]. Compounds have been established as ACAT inhibitors by their ability to inhibit incorporation of labelled fatty acyl CoA into cholesteryl esters i n microsomal preparations. This assay involves an unknown amount of cholesterol and modulators of the enzyme may be present [5,6]. Therefore in order to assess the action of ACAT inhibitors i n a more controlled environment, a reconstitution assay was set up.Firstly, the enzyme was solubilised using detergent and then it was reconstituted into liposomes providing a defined lipid environment. The method was based on that of Cadigan and Chang for human skin fibroblasts and Chinese hamster ovary cells 151. The assay showed protein and time dependence. The kinetics of compounds in the two assays were compared.Liver microsomes were prepared from male Sprague-Dawley rats which had been fed 2% cholesterol to stimulate ACAT activity. They were stored freeze-dried and stored at -2OOC and resuspended in 0.05M KP04/0.05M NaF buffer (pH 7.4) as required. Liposomes were made by the cholestyramine method [7] and contained cholesterol and phosphatidylcholine at a molar ratio of 0.3. Solubilisation was carried out using 3% DOC and solubilised protein was then diluted 1 in 240 into liposomes to effectively dilute out the detergent which would otherwise inhibit ACAT activity.Assays contained 3 . 3~9 reconstituted protein, 5pg fatty acid-free BSA, 1mM DTT in 50mM Tris, 1mM EDTA buffer (pH 7.7) in a final volume of 0.1 ml. Compounds were added in dimethylsulphoxide (DMSO) and after a 5 minute preincubation, the reaction was started by the addition of 1 nmoie [14C]oleoyl CoA and incubated at 37OC for 10 minutes. Reactions were stopped using 1 ml of a propan-2-olfheptane mix (4:l) containing [3H]cholesteryl oleate (approximately 20,000 dpm/ml) as a recovery marker. Lipids were extracted with 0.4ml heptane, 0.6ml water and 0.4ml of the top layer was removed and reaction products were separated using silica gel columns [8]. Activities were expressed as a percentage of the DMSO control. Microsomal assays were carried out as described previously [9].The results show that octimibate is active as an inhibitor of ACAT activity in both assay systems ( Fig. l(a)). However, CI 976 ( Fig. l(b)) does not inhibit in the reconstituted system and DuP 128 shows an intermediate response (Fig. l(c)). Table 1 shows differences in iC50s. (M) and a reconstituted ( R ) assay. The ratio of the two IC50s is also indicated ( R / M ) . octimibate 45 40 1 CI 976 0 . 1 3 14 1 0 8 DuP 128 0 . 0 0 3 5 0.11 3 1 Table 2. Effect of adding an incubation mixture for a reconstitution assay containing CI 976 to a microsomal a s s a y . T h e c o n c e n t r a t i o n o f C I 9 7 6 i n t h e reconstitution assay ( R ) and the final concentration i n t h e m i c r o s o m a l a s s a y (M)...

160 The effect of ACAT inhibition on VLDL secretion in rats fed a diet rich in fish-oil

“…Second, whether the inhibitor potentiated the suppressive effect of fish-oil feeding on VLDL secretion. YM17E was chosen as it is a potent inhibitor of Acyl-coAcholesterol acyltransferase (ACAT) [9].…”

“…Second, whether the inhibitor potentiated the suppressive effect of fish-oil feeding on VLDL secretion. YM17E was chosen as it is a potent inhibitor of Acyl-coAcholesterol acyltransferase (ACAT) [9].Male Wistar rats were fed for two weeks with either a fish-oil [FO] diet or a normal low-fat [LF] diet. The FO diet consisted of 20% (v/w) Maxepa mixed with the LF diet.…”