Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a approximately 36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.     

Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035

Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]pro panamide) has been found to inhibit the accumulation of cholesteryl esters in both rat hepatoma (Fu5AH) cells and arterial smooth muscle cells in culture. To explore the specificity of 58-035, we have studied the esterification of cholesterol, retinol, and glycerides by the Fu5AH cell and by isolated membranes. Exposure of Fu5AH to cholesterol/phospholipid dispersions and 58-035 (greater than 100 ng/ml) for 24 h resulted in greater than 95% inhibition of cholesterol esterification while cellular free cholesterol increased slightly. Inhibition was also rapid; incorporation of [3H]oleate into cholesteryl [3H]oleate equaled only 12% of control value after 30 min with 58-035 at 5 micrograms/ml. In contrast, there was no decrease in [3H]oleate incorporation into phospholipids or diglycerides, nor was the esterification of [3H]retinol inhibited by 58-035. In microsomal fractions, acyl-CoA:cholesterol acyltransferase could be inhibited completely by 58-035, while activities of acyl-CoA: retinol acyltransferase and triglyceride synthesis proceeded at 75-100% of control values. These observations that 58-035 is highly selective allow the inference that acyl-CoA:cholesterol acyltransferase is a separate microsomal enzyme whose activity can be modulated independently from acyl-CoA:retinol acyltransferase and other cellular acyltransferases.     

A simple method for reconstitution of CHO cell and human fibroblast acyl coenzyme A: cholesterol acyltransferase activity into liposomes

A new method for reconstituting acyl coenzyme A: cholesterol acyltransferase (ACAT) activity from either Chinese hamster ovary (CHO) or human fibroblast cell extracts into cholesterol-phosphatidylcholine liposomes is described. The method is rapid (less than 60 min) and easy to perform. The procedure involves solubilizing the cell extracts with deoxycholate followed by dilution into preformed liposomes. Ficoll gradient analysis demonstrated that, after reconstitution, almost all of the detectable ACAT activity co-migrated with the liposomes. Exogenous cholesterol in the liposomes was absolutely necessary for providing ACAT activity, but not for incorporation of the ACAT enzyme into the vesicle bilayer. Human fibroblast cell extracts prepared from cells grown in medium containing 10% fetal calf serum were found to contain a 10-fold higher microsomal ACAT activity compared to extracts from cells grown in 10% delipidated fetal calf serum. In contrast, when the ACAT activity from these extracts was measured using the reconstitution assay, there was no difference in the specific activities. These results support our previous work (Doolittle, G. M., and T. Y. Chang. 1982. Biochim. Biophys. Acta. 713: 529-537; and Chang, C. C. Y., et al. 1986. Biochemistry. 25: 1693-1699), and suggest that cholesterol regulates ACAT activity in CHO cells and human fibroblasts by mechanism(s) other than modulation of the amount of enzyme.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase

Growth of a temperature-sensitive general fatty acid synthesis mutant of Escherichia coli K12 at its restrictive temperature in the presence of exogenous palmitate results in lysis of the bacterium. Under these conditions, palmitate is incorporated into membrane phospholipid to a high level. Mutants of bacteria restricting this incorporation (having a palmitate-resistant phenotype) have been isolated and one such mutant, strain L8-2/3, has been further characterized. This mutant has lowered acyl-CoA synthetase (fadD) activity (25-33% of normal) and consequently is defective in fatty acid uptake. This lowered uptake could explain the palmitate-resistant phenotype of strain L8-2/3. However, both in vivo (fatty acid composition and positional distribution data) and in vitro (acyltransferase activity measurements) experiments suggest that this mutant is also altered in its acyltransferase activities. The mutation(s) of strain L8-2/3 appears to allow increased (approximately 2-fold) incorporation of myristate (and possible unsaturated fatty acids) into position 2 of 1-acyl-sn-glycerol 3-phosphate but normal palmitate incorporation into the same position. The incorporation of palmitate, myristate, and oleate into position 1 of sn-glycerol 3-phosphate by strain L8-2/3 is also higher than that observed with the parent, strain L8-2. Replacing the partially defective fadD gene of strain L8-2/3 with a wild type allele conferred on this strain the palmitate sensitivity and the acyltransferase activity of the parent strain L8-2. This finding, taken together with other data, suggests that acyl-CoA synthetase interacts with the acyltransferase(s) in some manner to influence the fatty acid specificity of the acyltransferase.     

Sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase from the corn earworm, Heliothis zea

The enzymatic activity and sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase (ACAT) were measured in microsomes of cells from Heliothis zea. Under standard assay conditions, the specific enzymatic activity of ACAT was highest in the intestine followed by the fat body and ovary (380.7, 30.7, 8.3 pmol/min per mg, respectively). The structure of the exogenous sterol used in the ACAT assay affected its rate of esterification. The relative rates of esterification of analogs of cholesterol with various modifications of the side chain were: 24-H greater than 24 alpha-CH3 greater than delta 22 greater than delta 24 greater than 24 alpha-C2H5 greater than 24 beta-CH3, delta 22-24 beta-CH3 and delta 22-24 alpha-C2H5. The number and position of double bonds in the B-ring of the sterol nucleus greatly affected the rate of esterification of sterols by ACAT. The average relative rates of esterification of sterols with differences in their B-rings were: delta 7 much greater than delta 8 greater than delta 0 greater than delta 5 greater than delta 5.7. The presence of a 9,14-cyclopropane group and/or methyl groups at the C-4 and 14 positions prevented significant esterification of such sterols. The formation of cholesteryl and lathosteryl esters was partially inhibited in microsomes from the intestine, fat body, and ovary by the addition of the ACAT inhibitor, 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide (Sandoz Compound 58-035).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

And then there were acyl coenzyme A:cholesterol acyl transferase inhibitors

PURPOSE OF REVIEW: The reputation of acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitors has changed profoundly from promising new drugs for cardiovascular prevention to drugs without clinical benefits or possibly even with adverse effects. RECENT FINDINGS: ACAT inhibitors decrease the intracellular conversion of free cholesterol into cholesteryl ester in a number of tissues, including intestine, liver and macrophages. In contrast to promising results in experimental animal models, all subsequent clinical studies in humans with ACAT inhibitors failed to show lipid profile changes as well as reductions in surrogate markers for coronary artery disease. In fact, there was even a tendency towards an increase in atheroma burden in the most recent and well executed clinical trials. In addition, the inhibition of this pivotal enzyme in cholesterol esterification may interfere with reverse cholesterol transport. SUMMARY: In our opinion, the consistent negative findings in recent clinical trials have virtually eliminated the chances for this class of drugs to be introduced for cardiovascular prevention. Possible strategies focused on selective ACAT 2 inhibition or the combination of ACAT inhibitors with compounds that stimulate reverse cholesterol transport may prove to have clinical benefit. This will have to await further clinical research in humans, however, as, obviously, rodent models cannot provide reliable data as to the efficacy of this class of drugs in humans.     

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acylation of acylglycerols by acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1). Functional importance of DGAT1 in the intestinal fat absorption

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of the four intestinal membrane bound acyltransferases implicated in dietary fat absorption. Recently, it was found that, in addition to acylating diacylglycerol (DAG), DGAT1 also possesses robust enzymatic activity for acylating monoacylglycerol (MAG) (Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005) J. Lipid Res. 46, 1502-1511). In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 microm), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50-200 microm) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC(50) of human DGAT1: 16.6+/-4.0 nM (MAG as substrate) and 1499+/-318 nM (DAG as substrate); IC(50) values of human DGAT2, MGAT2, and MGAT3 are >30,000 nM). Using this pharmacological tool, we have shown that approximately 76 and approximately 89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by approximately 50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.     

Functional size of acyl coenzyme A:diacylglycerol acyltransferase by radiation inactivation

Rat liver acyl coenzyme A:diacylglycerol acyltransferase, an intrinsic membrane activity associated with the endoplasmic reticulum, catalyzes the terminal and rate-limiting step in triglyceride synthesis. This enzyme has never been purified nor has its gene been isolated. Inactivation by ionizing radiation and target analysis were used to determine its functional size in situ. Monoexponential radiation inactivation curves were obtained which indicated that a single-sized unit of 72 +/- 4 kDa is required for expression of activity. The size corresponds only to the protein portion of the target and may represent one or several polypeptides.     

Role of the intestinal acyl-CoA:cholesterol acyltransferase activity in the hyperresponse of diabetic rats to dietary cholesterol.

Contrary to normal rats, diabetic rats are known to develop marked hypercholesterolemia when fed a cholesterol-enriched diet. The triggering factor involved in this hyperresponse has not been identified. With the aim of clarifying the role of the intestinal acyl-CoA:cholesterol acyltransferase (ACAT), we studied the effects of a high fat diet and the changes of intestinal ACAT activity during the early development of streptozotocin-diabetes in rats. Feeding diabetic rats with a diet enriched in cholesterol and saturated fat produced an increase in plasma and in tissue cholesterol as early as 3 days after streptozotocin injection in the absence of hyperphagia. Under these experimental conditions, treatment with insulin or with the ACAT inhibitor CL-277082 significantly reduced the plasma cholesterol to levels measured in nondiabetic rats fed the same high fat diet. An increase in [14C]cholesterol in plasma very low density lipoprotein was observed after oral administration of labeled cholesterol to 3-day diabetic rats. In parallel experiments, the direct measurement of small intestine microsomal ACAT activity revealed an increase, averaging 288% in diabetic rats 3 days after diabetes induction. This change in ACAT activity occurred simultaneously with an increase in plasma glucagon and was normalized by insulin treatment. The induction of intestinal ACAT activity in diabetic rats, its modulation by insulin, and the hypocholesterolemic effects of insulin or CL-277082 treatment clearly indicate that ACAT activity plays a major role in the initiation of diabetes-associated hypercholesterolemia.     

Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase

During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.     

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.     

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.     

Regulation of rat hepatic cholesterol metabolism. Effects of lipoprotein composition on acyl coenzyme A:cholesterol acyltransferase in vivo and in the perfused liver and on hepatic cholesterol secretion

Lipoproteins that are removed from the circulation by the liver can deliver both cholesterol and triglycerides to the hepatocyte. Relative proportions of these lipids may vary in lipoproteins and, thus, their uptake may have differing effects on cholesterol homeostasis. To study this, lipoproteins containing the same amounts of cholesterol but different amounts of triglyceride were administered to intact rats or to an isolated perfused rat liver. The responses of acyl coenzyme A:cholesterol acyltransferase (ACAT), very low density lipoprotein (VLDL) triglyceride and cholesterol secretion, and biliary cholesterol content were examined after 2 hr. Administration of triglyceride-rich chylomicrons (average triglyceride:cholesterol = 136.5 by mass) in vivo or their remnants (average triglyceride:cholesterol = 32.7 by mass) to the perfused liver resulted in an 80% decrease in ACAT activity. In the perfused liver system, VLDL cholesterol and triglyceride secretion was increased while biliary cholesterol content decreased. Administration of standard chylomicrons (average triglyceride:cholesterol = 33.9 by mass) or their remnants (average triglyceride:cholesterol = 11.4 by mass) lowered ACAT activity by 24% in vivo, but had no significant effect on any of the parameters measured in the perfused liver system. Administration of cholesterol-rich VLDL (average triglyceride:cholesterol = 0.47 by mass) in vivo increased ACAT activity 1.4-fold, but administration of their remnants (average triglyceride:cholesterol = 0.17 by mass) had little effect on any of the parameters measured in the perfused liver. Thus, the lipid composition of lipoproteins removed by the liver elicited acute responses by parameters important in the maintenance of hepatic cholesterol homeostasis. These responses reflected the net effects of both the cholesterol and the triglyceride contents of the particles.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.