Rabbit and human liver contain a novel pentacyclic triterpene ester with acyl-CoA: cholesterol acyltransferase inhibitory activity.

I Tabas,L L Chen,J W Clader,A T Mcphail,D A Burnett,P Bartner,P R Das,B N Pramanik,M S Puar,S J Feinmark

doi:10.1016/s0021-9258(19)39036-2

Abstract

Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) catalyzes the intracellular fatty acid esterification of cholesterol and is thought to play a key role in lipoprotein metabolism and atherogenesis. Herein we describe the purification and characterization of a novel pentacyclic triterpene ester from rabbit liver that has ACAT inhibitory activity. The inhibitor was purified by a combination of silicic acid chromatography and preparative thin layer chromatography. The compound inhibited both rabbit and rat liver microsomal ACAT activity with an IC50 = 20 microM. The lipid did not inhibit fatty acid incorporation into triglycerides, diglycerides, monoglycerides, or phospholipids nor did it inhibit plasma lecithin:cholesterol acyltransferase activity. However, rat liver microsomal acyl-CoA:retinol acyltransferase activity was inhibited by the terpene ester. Kinetic data are consistent with a mechanism in which ACAT is inhibited by the compound in an irreversible manner. The subcellular fractionation pattern of both ACAT activity and the ACAT inhibitor were similar in rabbit liver (both were approximately equally distributed in membranes that pelleted at 10,000 X g and 100,000 X g). A lipid with similar properties to the rabbit liver inhibitor was found in many other rabbit tissues, including adrenal and spleen, as well as in human liver. Rat liver did not contain this lipid. Structural analysis by NMR, mass spectrometry, and x-ray crystallography indicated that the rabbit liver inhibitor was a fatty acid ester (mostly stearate) of a pentacyclic triterpene acid. The carbon skeleton of the triterpene moiety is a new member of the olean-12-ene triterpene family. Both the negatively charged carboxylic acid group of the triterpene moiety and the esterified fatty acid group were necessary for the ACAT-inhibitory activity of the triterpene ester. Lastly, we present preliminary data which, together with the structural homology of the rabbit triterpene with known plant compounds, suggest the hypothesis that the triterpene moiety of the rabbit ACAT inhibitor arises from dietary absorption of a plant triterpene.

Highlights

A (CoA):cholesterol acyltransferase (ACAT) catalyzes the intracellular fatty acid esterification of cholesterol and is thought to play a key role in lipoprotein metabolism and atherogenesis
Preliminary fractionation experiments revealed that several components of the lipid extract possessed ACAT inhibitory activity
We have discovered that rabbit liver contains significant amounts of a lipid of novel structure, a fatty acid ester of a pentacyclic triterpene acid

Summary

RESULTS

Purification and Characterization of an ACAT Inhibitor from Rabbit Liver Lipid Extract-A lipid extract of rabbit liver was found to possess ACAT inhibitory activity (above). Preliminary fractionation experiments revealed that several components of the lipid extract possessed ACAT inhibitory activity. The purification of one of these components was pursued since its ACAT-inhibitory activity was relatively potent and since initial structural work (see below) indicated that it had an unusual and probably novel structure. The lipid extract was subjected to the fractionation procedure schematized in Fig. 1 (top) and described in detail under “Experimental Procedures.”. 3, 5-7, 11, and 12, and Tables II and III) are presented in miniprint at the end of this paper. Miniprint is read with the aid of a standard magnifying glass.

Procedures”

DISCUSSION

11. International