Acyl-coenzyme A:cholesterol Acyltransferase Activity Research Articles

Estrogen Decreases Atherosclerosis in Part by Reducing Hepatic Acyl-CoA:Cholesterol Acyltransferase 2 (ACAT2) in Monkeys

Estrogens decrease atherosclerosis progression, mediated in part through changes in plasma lipids and lipoproteins. This study aimed to determine estrogen-induced changes in hepatic cholesterol metabolism, plasma lipoproteins, and the relationship of these changes to atherosclerosis extent. Ovariectomized monkeys (n=34) consumed atherogenic diets for 30 months which contained either no hormones (control, n=17) or conjugated equine estrogens (CEE, n=17) at a human dose equivalent of 0.625 mg/d. Hepatic cholesterol content, low-density lipoprotein (LDL) receptor expression, cholesterol 7 alpha-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity, and expression levels were determined. CEE treatment resulted in lower plasma concentrations of very-low- and intermediate- density lipoprotein cholesterol (V+IDLC; P=0.01), smaller LDL particles (P=0.002), and 50% lower hepatic cholesterol content (total, free, and esterified; P<0.05 for all). Total ACAT activity was significantly lower (P=0.01), explained primarily by reductions in the activity of ACAT2. Estrogen regulation of enzymatic activity was at the protein level as both ACAT1 and 2 protein, but not mRNA levels, were lower (P=0.02 and <0.0001, respectively). ACAT2 activity was significantly associated with hepatic total cholesterol, plasma V+IDLC cholesterol, and atherosclerosis. Atheroprotective effects of estrogen therapy may be related to reduced hepatic secretion of ACAT2-derived cholesteryl esters in plasma lipoproteins.

Preventive Effects of Heregulin-β 1 on Macrophage Foam Cell Formation and Atherosclerosis

Rationale : Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. Objective : The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. Methods and Results : Plasma heregulin-β 1 levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3±0.3, 2.0±0.4 versus 7.6±1.4, 8.2±1.2 ng/mL; P &lt;0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-β 1 levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-β 1 was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-β 1 , but not heregulin-α, significantly reduced acetylated low-density lipoprotein–induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-β 1 significantly decreased endocytic uptake of [ 125 I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-β 1 into ApoE −/− mice significantly suppressed the development of atherosclerotic lesions. Conclusions : This study provided the first evidence that heregulin-β 1 inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

AM-251 and SR144528 are acyl CoA:cholesterol acyltransferase inhibitors

Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2 +/+ and CB2 −/− peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have anti-atherosclerotic activities independent of their affect on cannabinoid signaling.

Novel N-terminal Cleavage of APP Precludes Aβ Generation in ACAT-Defective AC29 Cells

A common pathogenic event that occurs in all forms of Alzheimer's disease is the progressive accumulation of amyloid beta-peptide (Abeta) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Abeta from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both alpha and the amyloidogenic beta cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Abeta generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Abeta generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment.

Androgen‐mediated cholesterol metabolism in LNCaP and PC‐3 cell lines is regulated through two different isoforms of acyl‐coenzyme A: Cholesterol Acyltransferase (ACAT)

The objective of this work was to determine the effect of an androgen agonist, R1881, on intracellular cholesterol synthesis and esterification in androgen-sensitive (AS) prostate cancer (LNCaP) cells. We investigated the activity and expression of cholesterol metabolism enzymes, HMG-CoA-reductase and ACAT in the LNCaP and PC-3 (androgen-independent control) models. Microsomal PC-3 HMG-CoA-reductase activity was increased with R1881 despite having similar cholesterol levels while increased cholesterol levels in microsomes from LNCaPs treated with R1881 (L+) were associated with increased HMG-CoA reductase activity. Increased intracellular cholesteryl esters (CE) found in (L+) were not associated with an increased ACAT1 activity. There was no effect from androgen treatment on ACAT1 protein expression in theses cells; however, ACAT2 expression was induced upon R1881 treatment. In contrast, we found an increase in the in vitro ACAT1 activity in PC-3 cells treated with androgen (P+). Only ACAT1 expression was induced in P+. We further assessed the expression of STAT1 alpha, a transcriptional activator that modulates ACAT1 expression. STAT1 alpha expression and phosphorylation were induced in P+. To determine the role of the AR on ACAT1 expression and esterification, we treated PC-3 cells overexpressing the androgen receptor with R1881 (PAR+). AR expression was decreased in PAR+ cells; ACAT1 protein expression and cholesterol ester levels were also decreased, however, ACAT2 remained unchanged. STAT1 alpha expression was decreased in PAR+. Overall, these findings support the importance of cholesterol metabolism regulation within prostate cancer cells and unravel a novel role for STAT1 alpha in prostate cancer metabolism.

Effects of trans-10,cis-12 conjugated linoleic acid on cholesterol metabolism in hypercholesterolaemic hamsters

Conjugated linoleic acid (CLA) has received great attention in recent years because of its pleiotropic biological activities, but considerably fewer studies have been published addressing its role in serum lipids and atherosclerosis compared to other topics covered. The aim of the present study was to assess the effects of the trans-10,cis-12 isomer of CLA on cholesterolaemia and on several metabolic pathways involved in cholesterol metabolism in hamsters. Animals were fed atherogenic diets supplemented with 0.5% linoleic acid, 0.5% trans-10,cis-12 CLA or 1.0% trans-10,cis-12 CLA, for 6 weeks. Serum lipoproteins were separated by FPLC. Cholesterol in serum and liver, as well as triacylglycerols and phospholipids in liver were assessed by spectrophotometry. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR), acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolase (CEH) activities were measured by radiometry, and LDL receptors were determined by Western blot. trans-10,cis-12 CLA feeding did not modify food intake nor final body weight. Although serum total cholesterol remained unchanged, when cholesterol fractions were analyzed a significant decrease in VLDL-cholesterol was observed in CLA-fed animals, without changes in HDL-cholesterol or LDL-cholesterol. trans-10,cis-12 CLA decreased cholesterol ester content and increased free cholesterol in liver. The activity of HMGCoAR was not modified. In contrast, ACAT activity was reduced by both CLA doses and CEH was increased by the high CLA dose. LDL receptors were significantly reduced by trans-10,cis-12 feeding when expressed as arbitrary units per mg of protein, however, the total receptor mass remained unchanged. These results suggest that, under the present experimental conditions, trans-10,cis-12 CLA feeding reduces cholesterol esterification in liver and decreases the minority serum VLDL-cholesterol fraction, but it does not show a hypocholesterolaemic effect. A dose-response effect was not observed.

Knockdown of ACAT-1 reduces amyloidogenic processing of APP

Previous studies have shown that acyl-coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell-based and animal models of Alzheimer’s disease. Here we report that ACAT-1 RNAi reduced cellular ACAT-1 protein by ∼50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Aβ secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Aβ generation.

Glabrol, an acyl-coenzyme A: Cholesterol acyltransferase inhibitor from licorice roots

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) esterifies free cholesterol in the liver and the intestine. It has relations with production of lipoproteins and accumulation of cholesteryl esters of the atheroma. Therefore, ACAT inhibitors may act as antihypercholesterolemic and antiatherosclerotic agents. One isoprenyl flavonoid was isolated from ethanol extract of licorice roots. On the basis of spectral evidences, the compound was identified as glabrol ( 1). Compound 1 inhibited rat liver microsomal ACAT activity with an IC 50 value of 24.6 μM and decreased cholesteryl ester formation with an IC 50 value of 26.0 μM in HepG2 cells. In addition, 1 showed a non-competitive type of inhibition against ACAT.

Genetics and molecular biology: macrophage ACAT depletion – mechanisms of atherogenesis

Genetics and molecular biology: macrophage ACAT depletion – mechanisms of atherogenesis

Acyl-coenzyme A:cholesterol acyltransferase inhibition ameliorates proteinuria, hyperlipidemia, lecithin-cholesterol acyltransferase, SRB-1, and low-denisty lipoprotein receptor deficiencies in nephrotic syndrome.

Nephrotic syndrome (NS) is associated with hyperlipidemia, altered lipid regulatory enzymes and receptors, and increased risk of progressive renal and cardiovascular diseases. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes intracellular esterification of cholesterol and plays an important role in production of apolipoprotein B-containing lipoproteins, regulation of cholesterol-responsive proteins, and formation of foam cells. Because hepatic ACAT-2 is markedly upregulated in NS, we tested the hypothesis that inhibition of ACAT may improve cholesterol metabolism in NS. Rats with puromycin-induced NS were treated with either the ACAT inhibitor CI-976 or placebo for 2 weeks. Normal rats served as controls. Plasma lipids, renal function, and key lipid regulatory factors were measured. Untreated NS rats showed heavy proteinuria; hypoalbuminemia; elevated plasma cholesterol, triglyceride, LDL, VLDL, and total cholesterol-to-HDL cholesterol ratio; increased hepatic ACAT activity, ACAT-2 mRNA, and ACAT-2 protein; and reduced LDL receptor, HDL receptor, otherwise known as scavenger receptor B-1 (SRB-1) and plasma lecithin-cholesterol acyltransferase (LCAT). ACAT inhibitor reduced plasma cholesterol and triglycerides, normalized total cholesterol-to-HDL cholesterol ratio, and lowered hepatic ACAT activity without changing ACAT-2 mRNA or protein. This was accompanied by near normalizations of plasma LCAT, hepatic SRB-1, and LDL receptor and a significant amelioration of proteinuria and hypoalbuminemia. Pharmacological inhibition of ACAT reverses NS-induced LDL receptor, HDL receptor, and LCAT deficiencies; improves plasma lipid profile; and ameliorates proteinuria in nephrotic animals. Further studies are needed to explore the effect of ACAT inhibition in nephrotic humans.

Acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) is responsible for elevated intestinal ACAT activity in diabetic rats.

Diabetes-induced dyslipidemia is seen in streptozotocin-induced diabetic rats. This is caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. Because two ACAT isozymes (ACAT-1 and ACAT-2) were identified, in the present study we determined which ACAT isozyme was involved in the elevated intestinal ACAT activity in diabetic rats. We cloned a full-length cDNA of rat ACAT-2. Its overexpression in ACAT-deficient AC29 cells demonstrated that the ACAT activity is derived from the cloned cDNA, and a 45-kDa protein of rat ACAT-2 cross-reacts with an anti-human ACAT-2 antibody. The tissue distribution of rat ACAT-2 mRNA revealed its restricted expression to liver and small intestine. Immunohistochemical analyses using an anti-human ACAT-2 antibody demonstrated that ACAT-2 is localized in villus-crypt axis of rat small intestine. The intestinal ACAT activity in diabetic rats was significantly immunodepleted by an anti-ACAT-2 antibody but not by an anti-ACAT-1 antibody. Finally, intestinal ACAT-2 in diabetic rats significantly increased at both protein and mRNA levels as compared with that in control rats. Our data demonstrate that ACAT-2 isozyme is responsible for the increased intestinal ACAT activity of diabetic rats, suggesting an important role of ACAT-2 for dyslipidemia in diabetic patients. Diabetic rats exhibit dyslipidemia caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. We determined which ACAT isozyme (ACAT-1 or ACAT-2) was involved in the elevated intestinal ACAT activity in diabetic rats. We demonstrated an important role of ACAT-2, implicating its involvement in dyslipidemia in diabetic patients.

Cholesterol absorption and hepatic acyl-coenzyme A:cholesterol acyltransferase activity play major roles in lipemic response to dietary cholesterol and fat in laboratory opossums*1

Partially inbred lines of laboratory opossums differ considerably in their low-density lipoprotein (LDL) cholesterol responses to dietary cholesterol and fat. Genetic analysis suggested that a single major gene is responsible for the variation in LDL cholesterol on the high cholesterol and high fat (HCHF) diet. We measured cholesterol absorption and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in intestine and liver to narrow the search for the major gene. We measured plasma lipoproteins and percent cholesterol absorption by the fecal isotope ratio method in high and low responding lines of opossums on basal and HCHF diets. We also measured lipids in liver and ACAT activity in liver and intestine on the HCHF diet. High and low lines exhibited no differences in percent cholesterol absorption on the basal diet. However, high responding opossums had significantly higher percent cholesterol absorption, hepatic free and esterified cholesterol, and hepatic ACAT activity than low responding opossums on the HCHF diet. Hepatic ACAT activity but not the intestinal ACAT activity was associated with hepatic cholesterol concentration and percent cholesterol absorption. Cholesterol absorption is a major determinant of diet-induced hyperlipidemia in opossums. Hepatic ACAT activity but not the intestinal ACAT may also play a role in diet-induced hyperlipidemia in opossums.

Cholesterol absorption and hepatic acyl-coenzyme A:cholesterol acyltransferase activity play major roles in lipemic response to dietary cholesterol and fat in laboratory opossums

Partially inbred lines of laboratory opossums differ considerably in their low-density lipoprotein (LDL) cholesterol responses to dietary cholesterol and fat. Genetic analysis suggested that a single major gene is responsible for the variation in LDL cholesterol on the high cholesterol and high fat (HCHF) diet. We measured cholesterol absorption and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in intestine and liver to narrow the search for the major gene. We measured plasma lipoproteins and percent cholesterol absorption by the fecal isotope ratio method in high and low responding lines of opossums on basal and HCHF diets. We also measured lipids in liver and ACAT activity in liver and intestine on the HCHF diet. High and low lines exhibited no differences in percent cholesterol absorption on the basal diet. However, high responding opossums had significantly higher percent cholesterol absorption, hepatic free and esterified cholesterol, and hepatic ACAT activity than low responding opossums on the HCHF diet. Hepatic ACAT activity but not the intestinal ACAT activity was associated with hepatic cholesterol concentration and percent cholesterol absorption. Cholesterol absorption is a major determinant of diet-induced hyperlipidemia in opossums. Hepatic ACAT activity but not the intestinal ACAT may also play a role in diet-induced hyperlipidemia in opossums.

Inhibitory effects of Polygonum cuspidatum water extract (PCWE) and its component rasveratrol on acyl-coenzyme A–cholesterol acyltransferase activity for cholesteryl ester synthesis in HepG2 cells

The pharmacological effects of Polygonum cuspidatum water extract (PCWE) on lipid biosynthesis were investigated in cultured human hepatocyte HepG2 cells. The addition of PCWE (5 and 20 μg/ml), which had no effect on cell proliferation and cellular protein content, caused a marked decrease in the cellular cholesterol content, particularly, the cholesteryl ester content following 24 h of incubation. The incorporation of 14C-oleate into the cellular cholesteryl ester fraction was also reduced remarkably during incubation for 6 and 24 h. The effect of PCWE on acyl-coenzyme A–cholesterol acyltransferase (ACAT) activity were studied in vitro to explore the mechanism by which PCWE inhibits cholesterol ester formation. The data confirmed that PCWE, in a dose dependent manner, remarkably inhibits ACAT activity. Among the main active chemicals of P. cuspidatum, resveratrol, a kind of flavonoid, decreased ACAT activity in a dose-dependent manner from the level of 10 −3 M. Theses results strongly suggest that PCWE reduces the cholesteryl ester formation in human hepatocytes by inhibiting ACAT.

Up-regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in nephrotic syndrome

We have previously demonstrated that hypercholesterolemia in rats with puromycin-induced nephrotic syndrome (NS) is associated with up-regulation of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and relative down-regulation of cholesterol 7alpha-hydroxylase (Ch-7alpha), which represent the rate-limiting steps in cholesterol biosynthesis and catabolism. Expression of HMG-CoA reductase is inhibited and Ch-7alpha is augmented by intracellular free cholesterol, which is avidly esterified by acyl-CoA:cholesterol acyltransferase (ACAT). Therefore, we hypothesized that NS may result in up-regulation of hepatic ACAT. Hepatic tissue ACAT mRNA (Northern blot), protein (Western blot) and enzymatic activity were determined in rats with puromycin-induced NS, placebo-treated control rats and Nagase hypoalbuminemic (NAG) rats. The NS group exhibited heavy proteinuria, hypoalbuminemia, normal creatinine clearance, severe hypercholesterolemia and hypertriglyceridemia. Despite severe hypoalbuminemia, NAG rats with inherited hypoalbuminemia exhibited only a mild elevation of plasma cholesterol and triglycerides. Severe hypercholesterolemia in the NS group was coupled with depressed liver tissue free cholesterol concentration and marked increases in hepatic ACAT mRNA, protein and enzymatic activity. In contrast, ACAT mRNA and protein contents of the liver were normal and ACAT activity was mildly elevated in the NAG group. NS results in marked up-regulation of hepatic ACAT, which is primarily due to proteinuria and not hypoalbuminemia, since the latter alone, as seen in NAG rats, does not significantly impact ACAT expression. Elevated ACAT in NS can contribute to dysregulation of cholesterol biosynthesis and catabolism by limiting the normal cholesterol signaling involved in regulation of these processes.

Mutant Acyl-coenzyme A:Cholesterol Acyltransferase 1 Devoid of Cysteine Residues Remains Catalytically Active

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in the early stages of atherosclerosis. ACAT1 is an integral membrane protein with multiple transmembrane domains. Human ACAT1 contains nine cysteine residues; its activity is severely inhibited by various thiol-specific modification reagents including p-chloromercuribenzene sulfonic acid, suggesting that certain cysteine residue(s) might be near or at the active site. We constructed various ACAT1 mutants that contained either single cysteine to alanine substitution at various positions, contained a reduced number of cysteines, or contained no cysteine at all. Each of these mutants retained 20% or more of the wild-type ACAT activity. Therefore, cysteine is not essential for ACAT catalysis. For the cysteine-free enzyme, its basic kinetic properties and intracellular localization in Chinese hamster ovary cells were shown to be very similar to those of the wild-type enzyme. The availability of the cysteine-free ACAT1 will facilitate future ACAT structure function studies. Additional studies show that Cys467 is one of the major target sites that leads to p-chloromercuribenzene sulfonic acid-mediated ACAT1 inactivation, suggesting that Cys467 may be near the ACAT active site(s).

Regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) synthesis, degradation, and translocation by high-density lipoprotein(2) at a low concentration.

(,Although plasma HDL(2) cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL(2) at a low concentration (< or = 40 microg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL(2) on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL(2) at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL(2), however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL(2) promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL(2) is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.

The developmental expression of acyl-coenzyme A:cholesterol acyltransferase in the yolk sac membrane, liver, and intestine of developing embryos and posthatch turkeys

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesterol esters (CE) from free cholesterol and fatty acyl-coenzyme A. This experiment was conducted to study the ontogeny of ACAT activity in the yolk sac membrane, liver, and intestine during embryonic development and early posthatch growth of turkeys. The ACAT activity was measured on tissue samples collected at 3-d intervals from embryonic Day (ED 13) 13 through 6 d posthatch (PD 6). The ACAT activity (pmol/mg microsomal protein per min) in the yolk sac membrane increased form 840 pmol at ED 13 to 2,497 pmol at ED 22, and subsequently declined to a very low level by PD 3. The high level of enzyme activity at ED 22 is concomitant with the large quantity of CE formed within the yolk sac membrane at this developmental age. Liver ACAT activity increased from 60 pmol at ED 13 to 242 to 243 pmol at ED 25 and PD 3, followed by a decline to 130 pmol by PD 6, mirroring the peak in hepatic CE concentration. This suggests that even during incubation, the liver plays a significant role in lipid metabolism. Intestinal ACAT specific activity increased from 14 pmol (ED 16) to 44 pmol (ED 25), and then declined to 23 pmol by hatch (ED 28), with no further decline through PD 6. Total intestinal ACAT activity (pmol per intestine/min) increased, however, from ED 16 through PD 6. This increase in activity suggests that the total capacity for cholesterol esterification increases during the course of incubation and shortly after hatching.

Immunological Quantitation and Localization of ACAT-1 and ACAT-2 in Human Liver and Small Intestine

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

Impact of subdermal norgestrel on hepatic acyl-coenzyme A:cholesterol- acyltransferase (ACAT) activity: possible antiatherogenic effect

The impact of subdermally placed ethinyl estradiol, norgestrel, and the combination of the two on cholesterol metabolism as measured by hepatic acyl:cholesterol-acyltransferase (ACAT) activity was examined in the rat model. A total of 48 rats were assigned to one of 6 groups, receiving either 0.1 mg or 1.0 mg of ethinyl estradiol daily, 1.0 or 10 mg of norgestrel daily, and combinations of either 0.1 mg ethinyl estradiol/1.0 mg norgestrel or 1.0 mg ethinyl estradiol/10 mg norgestrel daily. All drugs were administered through subdermally placed time release capsules. The administration of norgestrel only in either 1.0 mg or 10 mg resulted in significantly lower rates of ACAT activity (0.77 ± 0.566 and 0.91 ± 0.239 pmol/mg/min, respectively). The combination of 1.0 ethinyl estradiol and 10 mg norgestrel resulted in a significant increase in ACAT activity to 2.17 ± 0.873. This combination also resulted in significantly greater weight loss at the conclusion of treatment [247.83 ± 6.2 g (pre) vs. 205.50 ± 10.6 (post)]. There were no other differences in ACAT activity between groups and no other differences in weight, both between groups and pre- and post-treatment within groups. In summary, subdermally placed norgestrel resulted in a significant lowering of ACAT activity not seen with either administration of ethinyl estradiol alone or the combination of ethinyl estradiol and norgestrel in doses ranging from 0.1 to 1.0 mg of ethinyl estradiol and 1.0 to 10.0 mg of norgestrel. Significantly increased ACAT activity for the combination of 1.0 ethinyl estradiol and 10 mg norgestrel over either ethinyl estradiol or norgestrel alone or a lower dose combination suggests a dose-related threshold and drug-drug interaction for this effect. These results suggest that subdermally placed norgestrel may result in significantly lower ACAT activity and may have a potential role as an antiatherogenic treatment.