In hibition of acyl-CoA : cholesterol acyltransferase decreases apolipoprotein B-1 00-containing lipoprotein secretion from HepG 2 cells

There is evidence that the overproduction of apoB-loocontaining lipoproteins by the liver is the underlying event in some forms of dyslipoproteinemia. This metabolic status is associated to an increased risk of deveIoping premature coronary artery disease CAD. The conclusions from previous studies suggested that the availability to the hepatocytes of cholesterol that is readily esterified is an important determinant for VLDL and LDL secretion. In the present study, we set out to investigate the effect of the specific stimulation and inhibition of the rate-limiting enzyme of the cholesterol esterification, acyl-CoAcholesterol acyltransferase (ACAT, E.C. 2.3.1.26), on the lipid and on the apoB-100 secretion rate from a human hepatoma cell line (HepC2). When the specific ACAT inhibitor FCE 27677 ( lo5 M) was added to the cultures, a decrease of the cellular cholesteryl ester content and at the same time a significant reduction of the neutral lipids and of the apoB-100 secretion rate were noticed. The stimulation of ACAT by 25-hydroxycholesterol (20 pg/ml) caused a 4fold increase of the cellular cholesteryl ester content and a 2-fold increase of the lipoprotein secretion rate. FCE 27677 (10” M to lo-’ M) prevented the effects elicited by the oxysterol. On the contrary, lovastatin (104 M) and gemfibrozil(106 M) had no effect. The analysis of the lipid and of the apolipoprotein composition of the lipoproteins secreted in the medium revealed that ACAT inhibition had the dual effect of both decreasing the number of apoB-100containing lipoproteins secreted as well as their cholesteryl ester load. UM Altogether, these data support the idea of a close relationship between ACAT activation, leading to increased cholesteryl ester availability, and apoB-100-containing lipoprotein secretion. It is speculated that ACAT inhibitors may prove useful for the treatment of human dyslipoproteinemias caused by the hepatic overproduction of apoB-1 OOcontaining lipoproteins. -Musmti, R. , L. Giorgini, P. Lovisolo, A. Pirillo, A. Chiari, and G. Ghiselli. Inhibition of acyl-CoAcholesterol acyl transferase decreases apolipoprotein B-100containing lipoprotein secretion from HepG2 cells. ]. Lipid Res. 1996. 37: 1-14. Supplementary key words hepatocytes ACAT ACAT inhibitors lipid synthesis cholesteryl esters apolipoprotein B-100 dys l ip proteinemia Whereas much is known about the plasma metabolism of apolipoprotein B-100 (apoB-loo), the mechanisms involved in the regulation of its synthesis are incompletely understood (1). ApoB-100 is the only form of apoB that is synthesized by the liver (2) and is an essential structural component of VLDL and LDL as it is required for the intracellular assembly and the secretion of these lipoproteins (3). The elevation of the concentration of LDL cholesterol as well as of apoB-100 is regarded as an important risk factor €or coronary artery disease (CAD) (4), therefore the factors that control the rate of secretion of apoB-100-containing lipoproteins by the liver are of considerable importance. The intracellular assembly of the apoB-100-containing lipoproteins proceeds through a series of steps compartmentalized at specific intracellular sites (5). Soon after translation, the nascent apolipoprotein is combined with phospholipids and becomes enriched with triglycerides and cholesteryl esters as it moves across the endoplasmic reticulum (6). Subsequently, the bulk of phospholipids and cholesterol is added at the Golgi apparatus (7). At the same time, apoB-100 undergoes a number of modifications including proteolytic Abbreviations: ACAT, acy1CoA:cholesterol acyltransferase (E.C. 2.3.1.26); VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins: BSA, bovine serum albumin; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; MTT, 3-(4,5-dimethylthiazol-2-y1>2,5diphenyltetrazo lium bromide; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid. ’To whom correspondence should be addressed. ‘Present address: Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Box 575, Husargatan 3, S-75 123 Uppsala, Sweden. Journal of Lipid Research Volume 37,1996 1 by gest, on Jauary 2, 2018 w w w .j.org D ow nladed fom cleavage (8), glycosylation (9), and fatty acid acylation (10, 11) which appears necessary for the intracellular trafficking and the secretion of the nascent lipoprotein. At least under certain circumstances, the lipid supply appears to be the major determinant for the apolipoprotein synthesis and secretion. In this regard there is evidence that lipoprotein cholesterol may be of particular importance in promoting the synthesis of the apoB1OOcontaining lipoproteins. In fact, unlike fatty acids, whose flux to the liver mainly affects the lipoprotein triglyceride output (12-19), the availability to the hepatocyte of lipoprotein cholesterol, either LDL, betaVLDL, or chylomicron remnants (20-23), has been shown to enhance the secretion of VLDL and LDL particles. Interestingly, this stimulating effect is not mimicked by non-lipoprotein cholesterol (23), apparently because it is not as good a substrate for esterification as the lipoprotein cholesterol internalized via a receptor-mediated mechanism (21, 23). In support of this idea, maneuvers to increase the cholesteryl ester content of HepG2 cells lead to stimulation of the d < 1.063 g/ml lipoprotein secretion (23,24). Furthermore, in humans, increased apoB-100 production has been documented in patients with cholesteryl ester storage disease (25) and in those with betasitosterolemia (26). However, an unequivocal demonstration that cholesterol esterification rate in the hepatocyte directs the secretion of the apoB-100-containing lipoprotein has not yet been obtained. In this study, the effect of the stimulation and of the specific inhibition of acylCoA.cholestero1 acyltransferase (E.C. 2.3.1.26, ACAT), the rate-limiting enzyme of cholesterol esterification, on the lipids and on the apoB-100 secretion rate by a human hepatoma cell line (HepG2) has been investigated. Results are consistent with the view that the activity of ACAT is a major determinant for the hepatic lipoprotein production. MATERIALS AND METHODS