Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by relieving product inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.     

Characterization of a second sterol‐esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl‐CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester‐synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT‐like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum‐associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl‐CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol‐auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.     

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.     

Human Acyl-CoA：cholesterol Acyltransferase （ACAT） and its Potential as a Target for Pharmaceutical Intervention against Atherosclerosis

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl estersfrom cholesterol and long-chain fatty-acyl-coenzyme A.At the single-cell level,ACAT serves as a regulatorof intracellular cholesterol homeostasis.In addition,ACAT supplies cholesteryl esters for lipoproteinassembly in the liver and small intestine.Under pathological conditions,the accumulation of cholesterylesters produced by ACAT in macrophages contributes to foam cell formation,a hallmark of the earlystage of atherosclerosis.Several reviews addressing various aspects of ACAT and ACAT inhibitors areavailable [1-8].This review briefly outlines the current knowledge on the biochemical properties of humanACATs,and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventionsagainst atherosclerosis.     

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-l-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner. Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1 gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex, which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.     

Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay: individual ACAT uniqueness

Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 are enzymes responsible for the formation of cholesteryl esters in tissues. While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. In this study, NBD-cholesterol was used to screen for specific inhibitors of ACAT1 and ACAT2. Incubation of AC29 cells, which do not contain ACAT activity, with NBD-cholesterol showed weak fluorescence when the compound was localized in the membrane. When AC29 cells stably transfected with either ACAT1 or ACAT2 were incubated with NBD-cholesterol, the fluorescent signal localized to the nonpolar core of cytoplasmic lipid droplets was strongly fluorescent and was correlated with two independent measures of ACAT activity. Several compounds were found to have greater inhibitory activity toward ACAT1 than ACAT2, and one compound was identified that specifically inhibits ACAT2. The demonstration of selective inhibition of ACAT1 and ACAT2 provides evidence for uniqueness in structure and function of these two enzymes. To the extent that ACAT2 is confined to hepatocytes and enterocytes, the only two cell types that secrete lipoproteins, selective inhibition of ACAT2 may prove to be most beneficial in the reduction of plasma lipoprotein cholesterol concentrations.     

Acyl-coenzyme A:cholesteryl acyltransferase 2.

In addition to acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1), an enzyme in the endoplasmic reticulum of cells found ubiquitously throughout the body, data recently obtained in at least three mammalian species, including nonhuman primates, mice and humans, demonstrate the presence of an additional ACAT (EC 2.1.3.26), termed ACAT2, which is localized to the endoplasmic reticulum of liver and intestine. Data suggest that ACAT2 may be the enzyme responsible for cholesteryl ester secretion into apolipoprotein B-containing lipoproteins. We have hypothesized that oversecretion of cholesteryl esters produced by the action of hepatic ACAT2 could account for the increased atherogenicity associated with cholesteryl ester-enriched LDL in nonhuman primates. In such cases, ACAT2 is an appealing target for therapy to reduce coronary heart disease.     

Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis.

Two ACAT sharing protein sequence homology near their C termini have been identified. Both proteins may span the endoplasmic reticulum (ER) membrane several times. There is good evidence implicating the role of ACAT1 in macrophage foam cell formation, and ACAT2 in intestinal cholesterol absorption. On the other hand, the functional roles of ACAT1 and ACAT2 in the VLDL or chylomicron assembly process are less clear. It is possible that both enzymes are able to form lipid droplets (which are present in the cytoplasm), and participate in lipoprotein assembly (which occurs in the ER lumen). To link the site of ACAT catalysis with its function, we propose that part of the ACAT catalytic site may reside within the lipid bilayer, allowing catalysis to be completed within the plane of the membrane. Cholesteryl esters (CE) produced in situ may burst into cytoplasmic lipid droplets, carrying phospholipid monolayers as their outer coats. In cells engaged in lipoprotein assembly and secretion, CE in the bilayer may be recognized by the specific protein microsomal triacylglycerol transfer protein (MTP), reaching out from the lumenal side of the membrane. MTP then lipidates the growing apolipoprotein B (apoB) chain with CE and TG during the early stages of apoB lipoprotein assembly.     

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

The participation of sterol carrier protein2 in cholesterol esterification in rat adrenal microsomes

The effect of sterol carrier protein2 (SCP2) purified from rat liver on the formation of cholesterol esters by acyl-CoA: cholesterol acyl-transferase (ACAT: EC 2.3.1.26) in rat adrenal microsomes was studied. The rate of incorporation of [1-14C]oleoyl-CoA into cholesteryl oleate was determined in the presence or absence of exogenously added cholesterol or SCP2, or both. The addition of SCP2 had no effect on the formation of cholesterol esters from endogenous cholesterol by ACAT in rat adrenal microsomes. In contrast, the formation of cholesterol esters from exogenous cholesterol by ACAT was dose-dependently increased by the addition of SCP2. These experiments showed that SCP2 had an enhancing effect on cholesterol esterification by ACAT in rat adrenal microsomes most likely by modulating the availability of exogenous cholesterol and that SCP2 may participate in the formation of cholesterol esters in the rat adrenal gland.     

Overexpression of human diacylglycerol acyltransferase 1, acyl-coa:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells

The relative importance of each core lipid in the assembly and secretion of very low density lipoproteins (VLDL) has been of interest over the past decade. The isolation of genes encoding diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) provided the opportunity to investigate the effects of isolated increases in triglycerides (TG) or cholesteryl esters (CE) on apolipoprotein B (apoB) lipoprotein biogenesis. Overexpression of human DGAT1 in rat hepatoma McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of TG. These effects were associated with decreased intracellular degradation and increased secretion of newly synthesized apoB as VLDL. Similarly, overexpression of human ACAT1 or ACAT2 in McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of CE. This led to decreased intracellular degradation and increased secretion of VLDL apoB. Overexpression of ACAT2 had a significantly greater impact upon assembly and secretion of VLDL from liver cells than did overexpression of ACAT1. The addition of oleic acid (OA) to media resulted in a further increase in VLDL secretion from cells expressing DGAT1, ACAT1, or ACAT2. VLDL secreted from DGAT1-expressing cells incubated in OA had a higher TG:CE ratio than VLDL secreted from ACAT1- and ACAT2-expressing cells treated with OA. These studies indicate that increasing DGAT1, ACAT1, or ACAT2 expression in McA-RH7777 cells stimulates the assembly and secretion of VLDL from liver cells and that the core composition of the secreted VLDL reflects the enzymatic activity that is elevated.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.     

The essentiality of arachidonic acid and docosahexaenoic acid

MCF-10A breast epithelial cells treated with docosahexaenoic acid (DHA) or oleic acid (OA) accumulated cytoplasmic lipid droplets containing both triacylglycerol and cholesteryl esters (CE). Interestingly, total CE mass was reduced in cells treated with DHA compared to cells treated with OA, and the CEs were rich in n-3 fatty acids. Thus, we hypothesized that DHA may be, in addition to a substrate, an inhibitor of cholesterol esterification in MCF-10A cells. We determined that the primary isoform of acyl-CoA: cholesterol acyltransferase expressed in MCF-10A cells is ACAT1. We investigated CE formation with DHA, OA, and the combination in intact cells and isolated microsomes. In both cells and microsomes, the rate of CE formation was faster and more CE was formed with OA compared to DHA. DHA substantially reduced CE formation when given in combination with OA. These data suggest for the first time that DHA can act as a substrate for ACAT1. In the manner of a poor substrate, DHA also inhibited the activity of ACAT1, a universally expressed enzyme involved in intracellular cholesterol homeostasis, in a cell type that does not secrete lipids or express ACAT2.     

人细胞内胆固醇酯化酶ACAT

酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一催化游离胆固醇与长链脂肪酸形成胆固醇酯的酶,在维持细胞内胆固醇、脂肪酸等脂质代谢平衡中起着极重要的作用。人类一些重要疾病如心脑血管病的动脉粥样硬化、神经系统的阿尔茨海默症、消化吸收系统的胆囊病变等都与ACAT紧密关联。本文主要从人ACAT的表达、功能效应、抑制剂应用前景等三方面介绍有关的研究进展。     

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.     

Effects of substrate composition on the esterification and hydrolysis activity of lysosomal acid sterol ester hydrolase

Lipid microemulsions with various core and surface lipid compositions were prepared by co-sonication of cholesteryl esters, triolein (TO), egg phosphatidylcholine (egg PC), and cholesterol. The heterogeneous emulsion particle mixture was purified by gel filtration and particles with the size and general organization of low density lipoproteins were obtained. These lipid microemulsion particles were used for studies of the cellular metabolism of lipoprotein-derived cholesterol and cholesteryl esters as catalyzed by the enzyme acid sterol ester hydrolase (EC 3.1.1.13). The hydrolysis of cholesteryl oleate (CO) was more than twice and that of cholesteryl linoleate (CL) more than three times faster than the hydrolysis of cholesteryl stearate (CS) over the temperature range 25-39.6 degrees C. Both the synthesis and hydrolysis of cholesteryl esters were insensitive to the physical state of the microemulsion cores. The synthesis of cholesteryl esters by this enzyme was also insensitive to the ratios of cholesterol and egg PC in the microemulsion surface layers. Incorporation of triolein into the microemulsion cholesteryl ester core slightly increased the rate of cholesteryl ester synthesis. A decreasing fatty acyl chain length (C18:0 to C14:0) and an increasing degree of unsaturation (C18:0 to C18:2) enhanced the synthesis rate. It is suggested that the hydrolysis and synthesis of cholesteryl esters in microemulsions (and lipoproteins) take place only in the particle surface layer and that the rate of catalysis is directly dependent on the amount of substrate in this surface layer.     

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.