A:cholesterol Acyltransferase Research Articles

ACAT2 negatively modulated by FOXA2 suppresses ferroptosis to expedite the aggressive phenotypes of endometrial cancer cells.

Endometrial cancer (EC) remains a prevalent gynecological disease with a continuously rising incidence and fatality rate. Acyl coenzyme A: cholesterol acyltransferase 2 (ACAT2) has been commonly perceived as a tumor promoter in multiple human malignancies. This study was conducted to specify the role and mechanism of ACAT2 in EC, which has not been covered. The expression and prognostic significance of ACAT2 in EC samples were respectively analyzed by the ENCORI and Kaplan-Meier plotter databases. RT-qPCR and western blot examined ACAT2 and forkhead box protein A2 (FOXA2) expression in EC cells. The CCK-8 method, colony formation, and EdU staining assays detected cell proliferation. The cell cycle was detected by flow cytometry analysis. Wound healing and Transwell assays, respectively, estimated cell migration and invasion. The thiobarbituric acid reactive species (TBARS) method and BODIPY 581/591 C11 probe detected lipid peroxidation levels. FerroOrange staining estimated intracellular iron level. Western blot examined the expression of epithelial-mesenchymal transition (EMT) and ferroptosis-associated proteins. The human TFDB database predicted the binding of FOXA2 with the ACAT2 promoter, which was substantiated by ChIP and luciferase reporter assays. As a result, ACAT2 expression was increased in EC tissues and cells and associated with poor survival outcomes in EC patients. ACAT2 deletion might hinder EC cell proliferation, migration, invasion, and EMT while stimulating cell cycle arrest. Moreover, ACAT2 silencing promoted the ferroptosis of EC cells. Also, FOXA2 inactivated the transcription of ACAT2 through binding with the ACAT2 promoter. FOXA2 interference could promote the proliferation, migration, invasion, EMT, cell cycle, and ferroptosis of ACAT2-silenced EC cells, which was partially reversed by the ferroptosis activator erastin. Conclusively, ACAT2 transcriptionally inactivated by FOXA2 might contribute to the malignant progression of EC via the inhibition of ferroptosis.

A study on cholesterol-cholesteryl ester metabolic homeostasis and drug intervention in hyperlipidemic hamsters using UHPLC-MS/MS

Hyperlipidemia is a global metabolic disorder characterized by dysregulation of lipid metabolism. This dysregulation is closely associated with the altered homeostasis of cholesterol-cholesteryl ester (CE) metabolism in systemic circulation, and some organs. Additionally, the relationship between oxidized cholesteryl ester (oxCE) and the disease has also gained attention. Currently, there is a lack of comprehensive research on the alterations in cholesterol-CE metabolism in the context of hyperlipidemia, as well as the characteristics of lipid-lowering agents in regulating this metabolic state. Therefore, 40 oxCEs were identified in the hamster liver sample, and novel ultrahigh performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) methods were established for simultaneous analysis of cholesterol, 57 CEs, and 40 oxCEs in the serum, liver, adipose tissue, and intestine of hyperlipidemic hamsters. This study investigated the metabolic alterations between cholesterol-CE/oxCE in hyperlipidemic hamsters and those treated with lipid-lowering agents, including the Niemann-Pick-C1 like-1 protein (NPC1L1) inhibitor ezetimibe and the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe. The study findings demonstrate metabolic disorders in cholesterol-CE/oxCE homeostasis in hyperlipidemic hamsters. Lipid-lowering agent therapy can improve the metabolic dysregulation caused by hyperlipidemia, with distinct characteristics: ezetimibe is more effective in reducing cholesterol, while avasimibe is more effective in reducing CEs/oxCEs. Eight potential biomarkers indicating the dysregulation of cholesterol-CE metabolism caused by hyperlipidemia and its improvement by lipid-lowering agents have been identified in the serum. This study offers new insights into the hyperlipidemia pathophysiology and the mechanisms of lipid-lowering agents from a novel perspective on cholesterol-CE/oxCE metabolic homeostasis.

A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection.

Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM's sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.

Role of sortilin 1 (SORT1) on fatty acid-mediated cholesterol metabolism in primary calf hepatocytes.

Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism-related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum β-hydroxybutyrate concentration <0.6 mM) and cows diagnosed with clinical ketosis (serum β-hydroxybutyrate concentration >3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid-mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35-45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA.

Targeting the Cholesterol Paradigm in the Risk Reduction for Atherosclerotic Cardiovascular Disease: Does the Mechanism of Action of Pharmacotherapy Matter for Clinical Outcomes?

Hypercholesterolemia is a well-established risk factor for atherosclerotic cardiovascular disease (ASCVD). Low-density lipoprotein cholesterol (LDL-C) has been labeled as "bad" cholesterol and high-density lipoprotein cholesterol (HDL-C) as "good" cholesterol. The prevailing hypothesis is that lowering blood cholesterol levels, especially LDL-C, reduces vascular deposition and retention of cholesterol or apolipoprotein B (apoB)-containing lipoproteins which are atherogenic. We review herein the clinical trial data on different pharmacological approaches to lowering blood cholesterol and propose that the mechanism of action of cholesterol lowering, as well as the amplitude of cholesterol reduction, are critically important in leading to improved clinical outcomes in ASCVD. The effects of bile acid sequestrants, fibrates, niacin, cholesteryl ester transfer protein (CETP) inhibitors, apolipoprotein A-I and HDL mimetics, apoB regulators, acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, statins, and proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors, among other strategies are reviewed. Clinical evidence supports that different classes of cholesterol lowering or lipoprotein regulating approaches yielded variable effects on ASCVD outcomes, especially in cardiovascular and all-cause mortality. Statins are the most widely used cholesterol lowering agents and have the best proven cardiovascular event and survival benefits. Manipulating cholesterol levels by specific targeting of apoproteins or lipoproteins has not yielded clinical benefit. Understanding why lowering LDL-C by different approaches varies in clinical outcomes of ASCVD, especially in survival benefit, may shed further light on our evolving understanding of how cholesterol and its carrier lipoproteins are involved in ASCVD and aid in developing effective pharmacological strategies to improve the clinical outcomes of ASCVD.

Xuezhitong capsule, an extract of Allium macrostemon Bunge, exhibits reverse cholesterol transport and accompanies high-density lipoprotein levels to protect against hyperlipidemia in ApoE-/- mice.

Xuezhitong capsules (XZT) are derived from Xie Bai and used for abnormal lipid homeostasis treatment through maintained metabolic balance. However, their mechanisms are largely unknown. Here, we mainly assessed the contribution of reverse cholesterol transport (RCT) and the accompanying increase in the high-density lipoprotein (HDL) effects of XZT to cholesterol dysfunction amelioration in mice. We assessed serum lipids by using enzymatic kits. We observed atherosclerotic plaque formation by hematoxylin-eosin (HE) and Oil Red O staining. We studied the lipid metabolism, fatty acid synthase (FAS), HDL, low-density lipoprotein receptor (LDLR), triglyceride (TG) metabolic enzyme expression levels, and RCT function in various tissues upon stimulation with high-fat diet, XZT, and some positive drugs by ELISA. After 34 weeks of high-fat diet administration, blood lipids levels increased because attenuated by XZT treatment (800 and 1,600 mg/kg, i.g.). XZT improved the lipid metabolism instability, induced RCT activation, and subsequently increased the HDL levels in hyperlipidemic mice (P<0.05). FAS (P<0.05) and LDLR (P<0.01) levels also remarkably improved. The effects of XZT were closely associated with RCT activation and the accompanying increase in the HDL levels, as characterized by XZT-induced preservation in ATP-binding cassette transporter member 1 (ABCA1), scavenger receptor class B type 1 (SRB1), acyl coenzyme A: cholesterol acyltransferase (ACAT), lecithin cholesterol acyltransferase (LCAT), apolipoprotein A I (ApoA1) and apolipoprotein B (ApoB). However, XZT showed no effect on high fat diet-activated TG metabolic enzyme expression levels (P>0.05). XZT are promising drugs in balancing the cholesterol dysfunction from hyperlipidemia through RCT activation and accompanying increase in HDL levels.

Avasimibe: A novel hepatitis C virus inhibitor that targets the assembly of infectious viral particles

Direct-acting antivirals (DAAs), which target hepatitis C virus (HCV) proteins, have exhibited impressive efficacy in the management of chronic hepatitis C. However, the concerns regarding high costs, drug resistance mutations and subsequent unexpected side effects still call for the development of host-targeting agents (HTAs) that target host factors involved in the viral life cycle and exhibit pan-genotypic antiviral activity. Given the close relationship between lipid metabolism and the HCV life cycle, we investigated the anti-HCV activity of a series of lipid-lowering drugs that have been approved by government administrations or proven safety in clinical trials. Our results showed that avasimibe, an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), exhibited marked pan-genotypic inhibitory activity and superior inhibition against HCV when combined with DAAs. Moreover, avasimibe significantly impaired the assembly of infectious HCV virions. Mechanistic studies demonstrated that avasimibe induced downregulation of microsomal triglyceride transfer protein expression, resulting in reduced apolipoprotein E and apolipoprotein B secretion. Therefore, the pan-genotypic antiviral activity and clinically proven safety endow avasimibe exceptional potential as a candidate for combination therapy with DAAs. In addition, the discovery of the antiviral properties of ACAT inhibitors also suggests that inhibiting the synthesis of cholesteryl esters might be an additional target for the therapeutic intervention for chronic HCV infection.

Antihyperlipidaemic effects of oxyresveratrol-containing Ramulus mori ethanol extract in rats fed a high-cholesterol diet

The antihyperlipidaemic effect of an ethanol extract of Ramulus mori containing a high concentration of oxyresveratrol (ERMO) was investigated in a high-cholesterol diet (HCD)-fed rat model. Cholesterol metabolism-related genes and proteins, such as lecithin cholesterol acyltransferase (LCAT), low-density lipoprotein receptor (LDLR), cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and acyl coenzyme A cholesterol acyltransferase-2 (ACAT2) were analysed by quantitative real-time reverse-transcription polymerase chain reaction and western blot analysis. ERMO-treated HCD-fed rats had decreased body and liver weights and reduced serum lipid levels, atherogenic index (AI) and coronary artery risk index (CRI). ERMO treatment lowered serum cholesterol levels by increasing the expression of LCAT, CYP7A1 and LDLR and by decreasing HMGCR and ACAT2. Histological analysis of liver and aortic tissues revealed a significant decrease in lipid droplets and foam cell formation in HCD-induced hyperlipidaemic rats treated with ERMO. ERMO is therefore a potentially potent functional food for preventing hyperlipidaemia.

Functional characterization of two single nucleotide polymorphisms of acyl-coenzyme A:cholesterol acyltransferase 2

BackgroundAcyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays a critical role in the formation of cholesteryl esters from cholesterol and fatty acids, and is a potential target for treating hypercholesterolemia. We recently reported the significant effects of two human ACAT2 gene polymorphisms, 41A>G (Glu14Gly, rs9658625) and 734C>T (Thr254Ile, rs2272296), on plasma lipid levels and coronary artery disease susceptibility in a case–control association study. In the present study, we evaluated the possible biological influence of the two polymorphism using two approaches. MethodsIn the first approach, the functional impact of the two polymorphisms was predicted in-silico using available web-based software, and in the second approach, the varying functions of the two polymorphisms were characterized in in vitro experiments, using ACAT2-deficient AC-29 cells. ResultsOur results show that the enzymatic activity of mutant Glu14Gly is approximately two times higher than wildtype, and that this increase is primarily due to the increased expression and/or stability of the mutant ACAT2 protein. ConclusionsThese results suggest that the genetic variation at Glu14Gly is functionally important and may contribute to ACAT2 protein expression and stability.

Fatty acid moieties have little effect on cholesterol‐lowering potency of plant sterol esters

Plant sterol esters are the popular cholesterol‐lowering dietary supplements. In order to understand the role of fatty acid moieties in the cholesterol‐lowering activity of plant sterol esters, the present study was to: (i) compare the cholesterol‐lowering activity of sterol esters of sunflower oil (SSF) with that of sterol esters of canola oil (SCO); and (ii) to systematically investigate the effect of SSF and SCO on the gene expression of sterol transporters, enzymes, receptors and proteins involved in cholesterol metabolism in hamsters fed a high cholesterol diet. GC analysis showed that SSF and SCO had similar sterol profile but they had different fatty acid moieties. Results showed both SSF and SCO could equally reduce plasma cholesterol in a dose‐dependent manner. Both SSF and SCO could inhibit the cholesterol absorption and enhance the excretion of bile acids. In the similar ways, dietary SSF and SCO could up‐regulate hepatic cholesterol‐7α‐hydroxylase (CYP7A1) and down‐regulate intestinal sterol transporter Niemann‐Pick C1‐like 1 (NPC1L1), acyl coenzyme A: cholesterol acyltransferase 2 (ACAT2) and hepatic 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA reductase). It was concluded that the fatty acid moieties had little effect on the cholesterol‐lowering activity of SSF and SCO.Practical applications: Plant sterols are effective in reducing plasma cholesterol. Commercially, plant sterol esters of sunflower oil and plant sterol esters of canola oil are mostly used for this purpose. The present study found that SSF and SCO were equally effective in reducing plasma cholesterol in a dose‐dependent manner regardless the significant difference in their fatty acid compositions.Fatty acid moieties have little effect on cholesterol‐lowering potency of plant sterol esters.

Cholesteryl esters and ACAT

AbstractAcyl‐coenzyme A:cholesterol acyltransferase (ACAT) 1 and 2 are both involved in intracellular cholesterol esterification. The expression of acyl‐coenzyme A:cholesterol acyltransferase 1; (ACAT1) is ubiquitous, whereas, enterocytes and hepatocytes exclusively express acyl‐coenzyme A:cholesterol acyltransferase 2 (ACAT2). Studies in mice lacking ACAT1 showed extensive deposition of unesterified cholesterol in skin and brain, and larger atherosclerotic lesions compared to controls. ACAT2 deficiency in mice protected against atherosclerosis, diet‐induced hypercholesterolemia and cholesterol gallstone disease, and reduced hepatic lipid accumulation and decreased serum cholesterol levels. Two unspecific ACAT inhibitors have been tested in humans. In the face of the results in ACAT1 deficient mice, strongly arguing against an ACAT1 inhibition, it was not surprising that none of these inhibitors showed beneficial effects on the predefined primary endpoints of the studies. However, and still to be proven, ACAT2 inhibition might be a therapeutic option for the treatment and prevention of cardiovascular disease. In this review, we aim to outline some important aspects on the role of ACAT in cholesterol esterification and to reappraise ACAT2 as therapeutic target.

In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man.

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.

Changes in Esculeoside A Content in Different Regions of the Tomato Fruit during Maturation and Heat Processing

We previously demonstrated that esculeogenin A, a new aglycone of the tomato sapogenol esculeoside A, inhibits both acyl coenzyme A:cholesterol acyl-transferase (ACAT)-1 and -2 and ameliorates the pathogenesis of atherosclerosis in apoE deficient mice. Although we believe that daily intake of esculeoside A from tomato products can play a beneficial role in preventing the pathogenesis of atherosclerosis, the compound is not being used for preventive medicine due to the lack of information on methods for quantitative analysis and the content and stability of the compound in tomato products. In the present study, we report the development of a high-performance liquid chromatography (HPLC) method using an instrument equipped with a refractive index (RI) detector for esculeoside A quantification. We used this method to measure the changes in esculeoside A content during maturation, its distribution in the fruit body, and its stability during the heating process. The contents of esculeoside A in cherry tomatoes and Momotaro tomatoes were 21- and 9-fold, respectively, higher than that of lycopene, which is the most well-known compound in tomatoes. Furthermore, the esculeoside A content in pericarp wall was higher than in the whole tomato fruit and increased in a time-dependent manner during maturation. Although the melting point of purified esculeoside A was 225 °C, the esculeoside A in crude tomato extract decreased in a temperature-dependent manner. Degradation due to the heating process was inhibited under a pH of 9. These results demonstrated that the esculeoside A content differs in the various types of tomatoes, during maturation, and during the heating process used for preservation.

Pyripyropene A, an Acyl–Coenzyme A:Cholesterol Acyltransferase 2–Selective Inhibitor, Attenuates Hypercholesterolemia and Atherosclerosis in Murine Models of Hyperlipidemia

Pyripyropene A (PPPA) of fungal origin is the first compound that has been found to strongly and selectively inhibit acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) isozyme activity in vitro. The purpose of the present study was to investigate in vivo efficacy of the ACAT2-selective inhibitor in atherosclerosis. PPPA treatment (10 to 100 mg/kg) caused 30.5±4.7% to 55.8±3.3% inhibition of the cholesterol absorption from the mouse intestine. When PPPA (10 to 50 mg/kg per day) was orally administered to apolipoprotein E-knockout mice for 12 weeks, the levels of plasma cholesterol, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) and hepatic cholesterol content were lowered. Furthermore, the ratio of cholesteryl oleate (exclusively synthesized in hepatic ACAT2) to cholesteryl linoleate in VLDL- and LDL-derived cholesteryl ester decreased, indicating that hepatic ACAT2 activity was inhibited by PPPA. PPPA-treated mice had reduced atherogenic lesion areas that were lowered by 26.2±3.7% to 46±3.8% in the aortae and by 18.9±3.6% to 37.6±6.0% in the hearts. Our findings indicate that ACAT2-selective inhibition in the intestine and the liver can be effective against atherosclerosis and that PPPA appears to be a potential antiatherogenic lead compound. This study is the first demonstration of the in vivo efficacy of PPPA, an ACAT2-selective inhibitor, in atherosclerosis. PPPA-treated atherogenic mice showed a decrease in intestinal cholesterol absorption and cholesterol and cholesteryl oleate levels in both LDL and VLDL, resulting in protection of atherosclerosis development.

Rimonabant is a dual inhibitor of acyl CoA:cholesterol acyltransferases 1 and 2

Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC 50 = 2.9 ± 0.38 μM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC 50 = 1.5 ± 1.2 μM and 2.2 ± 1.1 μM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT.

Frequent Cholesterol Intake Up-regulates Intestinal NPC1L1, ACAT2, and MTP

Dietary cholesterol elevates plasma total cholesterol (TC) level. However, no study to date has examined how cholesterol intake frequency interacts with the gene of sterol transporters, receptors, and enzymes involved in cholesterol metabolism. Thirty-three hamsters were divided into three groups with the control hamsters being given daily 9 mg of cholesterol in the diet (CD), whereas the second group being gavage-administered 3 mg of cholesterol three times per day (C-3) and the third group being gavage-administered 9 mg of cholesterol one time per day (C-1). The experiment lasted for 6 weeks. The hamsters were killed under carbon dioxide suffocation. Data demonstrated that plasma TC, non-high-density lipoprotein cholesterol, and triacylglycerols were elevated with the increasing cholesterol intake frequency. Western blotting analyses revealed that the intake frequency had no effect on protein mass of hepatic sterol regulatory element binding protein-2, liver X receptor-alpha, 3-hydroxy-3-methylglutaryl-CoA reductase, LDL receptor, and cholesterol-7alpha-hydroxylase. However, the frequent cholesterol intake down-regulated the mRNA level of hepatic LDL receptor. In contrast, the frequent cholesterol intake up-regulated the mRNA levels of intestinal Niemann-Pick C1-like 1 (NPC1L1), acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2), and microsomal triacylglycerol transport protein (MTP). It was concluded that the cholesterol intake frequency-induced elevation in plasma TC was associated with greater cholesterol absorption, possibly mediated by up-regulation of NPC1L1, ACAT2, and MTP.

Inhibition of acyl‐coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Amyloid beta-peptide (Abeta) has a central role in the pathogenesis of Alzheimer's disease (AD). Cellular cholesterol homeostasis regulates endoproteolytic generation of Abeta from the amyloid precursor protein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that regulates subcellular cholesterol distribution, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Abeta generation in cell- and animal-based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live-cell imaging. This resulted in partial ER retention of APP and enhanced ER-associated degradation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors, and strongly correlated with reduced brain APP-C99 and cerebrospinal fluid Abeta levels in individual animals. Our results identify a novel ACAT-dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Abeta generation in vivo.

Nothing Captivating About Acyl–Coenzyme A:Cholesterol Acyltransferase Inhibition

Acyl–coenzyme A:cholesterol acyltransferase (ACAT) inhibition has been touted as a novel means to control cholesterol accumulation in human tissue.

ACAT Inhibition and Progression of Carotid Atherosclerosis in Patients With Familial Hypercholesterolemia

Inhibition of acyl coenzyme A:cholesterol acyltransferase (ACAT), an intracellular enzyme involved in cholesterol accumulation, with pactimibe was developed to assist in the prevention of cardiovascular disease. To evaluate the efficacy and safety of pactimibe in inhibition of atherosclerosis. A prospective, randomized, stratified, double-blind, placebo-controlled study (Carotid Atherosclerosis Progression Trial Investigating Vascular ACAT Inhibition Treatment Effects [CAPTIVATE]) of 892 patients heterozygous for familial hypercholesterolemia conducted at 40 lipid clinics in the United States, Canada, Europe, South Africa, and Israel between February 1, 2004, and December 31, 2005. Study was terminated on October 26, 2005. Participants received either 100 mg/d of pactimibe (n = 443) or matching placebo (n = 438), in addition to standard lipid-lowering therapy. Carotid atherosclerosis, assessed by ultrasound carotid intima-media thickness (CIMT), at baseline, 12, 18, and 24 months. Maximum CIMT was the primary end point and mean CIMT the secondary end point. Because pactimibe failed to show efficacy in the intravascular coronary ultrasound ACTIVATE study, the CAPTIVATE study was terminated prematurely after a follow-up of 15 months. After 6 months of treatment with pactimibe, low-density lipoprotein cholesterol increased by 7.3% (SD, 23%) compared with 1.4% (SD, 28%) in the placebo group (P = .001). The carotid ultrasonographic scans of the 716 patients with at least 2 scans and obtained at least 40 weeks apart were analyzed. Maximum CIMT measurements did not show a pactimibe treatment effect (difference, 0.004 mm; 95% confidence interval [CI], -0.023 to 0.015 mm; P = .64); however, the less variable mean CIMT measurement revealed an increase of 0.014 mm (95% CI, -0.027 to 0.000 mm; P = .04) in patients administered pactimibe vs placebo. Major cardiovascular events (cardiovascular death, myocardial infarction, and stroke) occurred more often in patients administered pactimibe vs placebo (10/443 [2.3%] vs 1/438 [0.2%]; P = .01). In patients with familial hypercholesterolemia, pactimibe had no effect on atherosclerosis as assessed by changes in maximum CIMT compared with placebo but was associated with an increase in mean CIMT as well as increased incidence of major cardiovascular events. clinicaltrials.gov Identifier: NCT00151788.

Appearance of the Ser‐His‐Asp catalytic triad at the putative active site domain of the acyl coenzyme A:cholesterol acyltransferase (ACAT) enzymes

To understand the molecular mechanism of cholesterol esterification reaction we sought to determine the active site residues of the ACAT enzymes using chemical modification and site directed mutagenesis approaches. The ACAT enzymes are 56% similar after N‐terminal 100 amino acids (AA). The sequence similarity within ACAT1 AA 386 to 462 and ACAT2 AA 364 to 440 is 86% and this region contains two conserved motifs FYXDWWN and HEY. We suspect this region has the putative active site domain of the enzymes due its high sequence conservation from yeast to humans. As these enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of S‐H‐D residues. Our results show that in ACAT1, S456, H460 and D400 are essential for activity of the enzyme. In contrast, in ACAT2, only H438 was necessary for enzymatic activity. The A2S434A mutant retained about 20% of the wild type activity whereas A2D378 mutants were not expressed after mutagenesis. Based on these data, we propose the active site may be different among the two ACAT enzymes. In ACAT1, a S‐H‐D catalytic triad serves as the active site whereas in ACAT2 there may be a H‐D catalytic diad in which the esterification reaction occurs in a substrate assisted manner with the 3β OH group of cholesterol acting as the nucleophile. (NIH:HL‐49373)