Ceramide-mediated gut dysbiosis enhances cholesterol esterification and promotes colorectal tumorigenesis in mice.

Colorectal cancer (CRC) severely threatens human health and life span. An effective therapeutic strategy has not been established because we do not clearly know its pathogenesis. Here, we report that ceramide and sterol O-acyltransferase 1 (SOAT1) have roles in both spontaneous and chemical-induced intestinal cancers. We first found that miRNA-148a deficiency dramatically increased mouse gut dysbiosis through upregulating ceramide synthase 5 (Cers5) expression, which promoted ceramide synthesis afterward. The newly generated ceramide further promoted both azoxymethane/dextran sodium sulfate-induced (AOM/DSS-induced) and ApcMin/+ spontaneous intestinal tumorigenesis via increasing mouse gut dysbiosis. Meanwhile, increased level of ceramide correlated with the significant enhancements of both ß-catenin activity and colorectal tumorigenesis in a TLR4-dependent fashion. Next, we found a direct binding of ß-catenin to SOAT1 promoter to activate transcriptional expression of SOAT1, which further induced cholesterol esterification and colorectal tumorigenesis. In human patients with CRC, the same CERS5/TLR4/ß-catenin/SOAT1 axis was also found to be dysregulated. Finally, the SOAT1 inhibitor (avasimibe) showed significant levels of therapeutic effects on both AOM/DSS-induced and ApcMin/+ spontaneous intestinal cancer. Our study clarified that ceramide promoted CRC development through increasing gut dysbiosis, further resulting in the increase of cholesterol esterification in a SOAT1-dependent way. Treatment with avasimibe to specifically decrease cholesterol esterification could be considered as a clinical strategy for effective CRC therapy in a future study.

Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFß pathway in endodermal epithelial cells of Japanese quail.

Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFß pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions.

High-Fructose Diet-Induced Hypertriglyceridemia Is Associated With Enhanced Hepatic Expression of ACAT2 in Rats.

High levels of fructose induce hypertriglyceridemia, characterized by excessive levels of triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL); however, the underlying mechanisms are poorly understood. The aim of this short communication was to examine hepatic changes in the expression of genes related to cholesterol metabolism in rats with hypertriglyceridemia induced by high-fructose or high-glucose diets. Rats were fed a 65 % (w/w) glucose diet or a 65 % (w/w) fructose diet for 12 days. Serum levels of triglycerides, total cholesterol, and VLDL+LDL-cholesterol, hepatic levels of triglycerides and cholesterol, and ACAT2 expression at the gene and protein levels were significantly higher in the fructose diet group compared to the glucose diet group. The hepatic levels of Abcg5/8 were lower in the fructose group than in the glucose group. Serum high-density lipoprotein (HDL)-cholesterol and hepatic expression levels of Hmgcr, Ldlr, Acat1, Mttp, Apob, and Cyp7a1 did not differ significantly between groups. These findings suggest that high-fructose diet-induced hypertriglyceridemia is associated with increased hepatic ACAT2 expression.

Targeted Knockdown of Hepatic SOAT2 With Antisense Oligonucleotides Stabilizes Atherosclerotic Plaque in ApoB100-only LDLr-/- Mice.

OBJECTIVE: To test the hypothesis that the attenuation of cholesterol oleate packaging into apoB-containing lipoproteins will arrest progression of pre-existing atherosclerotic lesions. APPROACH AND RESULTS: Atherosclerosis was induced in apoB-100 only, LDLr(-/-) mice by feeding a diet enriched in cis-monounsaturated fatty acids for 24 weeks. A subset of mice was then euthanized to quantify the extent of atherosclerosis. The remaining mice were continued on the same diet (controls) or assigned to the following treatments for 16 weeks: (1) a diet enriched in n-3 polyunsaturated fatty acids, (2) the cis-monounsaturated fatty acid diet plus biweekly injections of an antisense oligonucleotide specific to hepatic sterol-O-acyltransferase 2 (SOAT2); or (3) the cis-monounsaturated fatty acid diet and biweekly injections of a nontargeting hepatic antisense oligonucleotide. Extent of atherosclerotic lesions in the aorta was monitored morphometrically in vivo with magnetic resonance imaging and ex vivo histologically and immunochemically. Hepatic knockdown of SOAT2 via antisense oligonucleotide treatment arrested lesion growth and stabilized lesions. CONCLUSIONS: Hepatic knockdown of SOAT2 in apoB100-only, LDLr(-/-) mice resulted in remodeling of aortic atherosclerotic lesions into a stable phenotype, suggesting SOAT2 is a viable target for the treatment of atherosclerosis.

Hepatitis C virus infection mediates cholesteryl ester synthesis to facilitate infectious particle production.

Cholesterol is a critical component of the hepatitis C virus (HCV) life cycle, as demonstrated by its accumulation within infected hepatocytes and lipoviral particles. To cope with excess cholesterol, hepatic enzymes ACAT1 and ACAT2 produce cholesteryl esters (CEs), which are destined for storage in lipid droplets or for secretion as apolipoproteins. Here we demonstrate in vitro that cholesterol accumulation following HCV infection induces upregulation of the ACAT genes and increases CE synthesis. Analysis of human liver biopsy tissue showed increased ACAT2 mRNA expression in liver infected with HCV genotype 3, compared with genotype 1. Inhibiting cholesterol esterification using the potent ACAT inhibitor TMP-153 significantly reduced production of infectious virus, but did not inhibit virus RNA replication. Density gradient analysis showed that TMP-153 treatment caused a significant increase in lipoviral particle density, suggesting reduced lipidation. These data suggest that cholesterol accumulation following HCV infection stimulates the production of CE, a major component of lipoviral particles. Inhibition of CE synthesis reduces HCV particle density and infectivity, suggesting that CEs are required for optimal infection of hepatocytes.

Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells.

Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats.

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2 weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.

Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system.

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker's yeast Saccharomyces cerevisiae. First we demonstrate that a quadruple knockout yeast strain deficient in storage lipids has a reduced growth rate in a medium supplemented with fatty acids. This phenotype is rescued by restoring TAG biosynthesis and can be thus used to select yeast cells expressing a recombinant TAG-SE. In the second method, the activity of the recombinant enzyme is measured in a fluorescent in situ assay using Nile red dye that is specific for neutral lipids. Correlation between Nile red fluorescence and enzyme activity is demonstrated with several mutants of a TAG synthesizing enzyme. This yeast live-cell-based assay is rapid, inexpensive, sensitive, and is amenable to high-throughput applications. The methods can be used for a variety of applications such as isolation of novel genes, directed evolution, gene-specific drug screening and will facilitate novel approaches in the research of TAG-SE.

Pyripyropenes, fungal sesquiterpenes conjugated with alpha-pyrone and pyridine moieties, exhibits anti-angiogenic activity against human umbilical vein endothelial cells.

In the course of our search for anti-angiogenic substances, pyripyropenes A (1), B (2), and D (3) were re-discovered as selective anti-proliferative substances against human umbilical vein endothelial cells (HUVECs) from a marine-derived fungus of Aspergillus sp. Pyripyropenes showed potent anti-proliferative activity against HUVECs with IC(50) values of the range of 0.1-1.8 muM, which were cytostatic at 0.05 to 20 muM. The selective index was more than 55-fold in comparison with those of several tumor cell lines. Compound 1 inhibited vascular endothelial growth factor (VEGF)-induced migration and tubular formation of HUVECs, while 1 showed no effect on the VEGF-induced phosphorylations of extracellular signal-regulated kinase (ERK)1/2, p38, and Akt. Pyripyropenes were originally isolated as an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT-2). While, the expression level of ACATs between HUVECs and other tumor cell lines did not correspond to the selective index of the anti-proliferative activity of compound 1. Moreover, ACATs inhibitor, 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), showed growth inhibitory activity with only poor selectivity (2.4-fold) between HUVECs and human epidermoid carcinoma KB3-1 cells.

Control of ACAT2 liver expression by HNF4{alpha}: lesson from MODY1 patients.

OBJECTIVE: ACAT2 is thought to be responsible for cholesteryl ester production in chylomicron and VLDL assembly. Recently, we identified HNF1alpha as an important regulator of the human ACAT2 promoter. Thus, we hypothesized that MODY3 (HNF1alpha gene mutations) and possibly MODY1 (HNF4alpha, upstream regulator of HNF1alpha, gene mutations) subjects may have lower VLDL esterified cholesterol. METHODS AND RESULTS: Serum analysis and lipoprotein separation using size-exclusion chromatography were performed in controls and MODY1 and MODY3 subjects. In vitro analyses included mutagenesis and cotransfections in HuH7 cells. Finally, the relevance in vivo of these findings was tested by ChIP assays in human liver. Whereas patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol, as well as of VLDL triglyceride. Mutagenesis revealed one important HNF4 binding site in the human ACAT2 promoter. ChIP assays and protein-to-protein interaction studies showed that HNF4alpha, directly or indirectly (via HNF1alpha), can bind to the ACAT2 promoter. CONCLUSIONS: We identified HNF4alpha as an important regulator of the hepatocyte-specific expression of the human ACAT2 promoter. Our results suggest that the lower levels of esterified cholesterol in VLDL- and LDL-particles in patients with MODY1 may-at least in part-be attributable to lower ACAT2 activity in these patients.

The optional long 5'-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay.

We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG(11397-1399) initiation codon, and also a minor 56 kDa isoform using the upstream in-frame GGC(1274-1276) initiation codon. The GGC(1274-1276) codon is located at the optional long 5'-untranslated region (5'-UTR, nt 1-1396) of the mRNAs. The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes, 7 and 1. In the current work, we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG(1397-1399)codon, mainly by promoting its mRNA decay. The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG(1397-1399) codon, meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'- UTR dramatically reduced the contents of its linked mRNAs. Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency. We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription. The results showed that this 5'-UTR promoted its mRNA decay. Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that, in both the cytoplasm and the nucleus of cells, the optional long 5'-UTR-linked mRNAs decayed faster than those without the link. Overall, our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

Chlamydia pneumoniae induces macrophage-derived foam cell formation by up-regulating acyl-coenzyme A: cholesterol acyltransferase 1.

In macrophages, the accumulation of cholesteryl esters synthesized by acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) plays a crucial role in foam cell formation, a hallmark of early atherosclerotic lesions. It is suggested that Chlamydia pneumoniae (C. pneumoniae) induces foam cell formation. However, the mechanism of foam cell formation induced by C. pneumoniae has not been fully elucidated. In this study, we found that C. pneumoniae increased the expression of acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) mRNA and protein in a dose-dependent manner in THP-1-derived macrophages exposed to low density lipoprotein (LDL). In addition, C. pneumoniae dose-dependently suppressed the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) mRNA and protein. Rosiglitazone, a specific PPAR gamma agonist, not only dose-dependently alleviated the down-regulation of PPAR gamma expression by C. pneumoniae infection, but also dose-dependently inhibited the C. pneumoniae-induced ACAT1 expression. Furthermore, higher doses of rosiglitazone (10 and 20 microM) suppressed the C. pneumoniae-induced foam cell formation from morphological (Oil red O staining) and biochemical (zymochemistry method) criteria. These results first demonstrate that C. pneumoniae induces macrophage-derived foam cell formation by up-regulating ACAT1 expression via PPAR gamma-dependent pathway, which may contribute to its pro-atherogenic properties.

Increased NPC1L1 and ACAT2 expression in the jejunal mucosa from Chinese gallstone patients.

The incidence of cholesterol gallstones is a very common disease. The aim of this study is to probe for underlying intestinal molecular defects associated with development of gallstones. Twelve Chinese patients with cholesterol gallstone disease (GS) and 31 gallstone-free (GSF) patients were investigated. Quantitation of mRNA levels for individual genes in mucosal biopsies from jejunum was carried out with real-time PCR. The frequency of two SNPs in the ABCG8 gene (Y54C and T400K) was determined by allelic discrimination. The intestinal mRNA expression of NPC1L1 and ACAT2 were significantly higher in GS than GSF (P<0.05). No differences were observed concerning the levels for plasma lipids, plant sterols and 7alpha-hydroxy-4-cholesten-3-one between GS and GSF. No correlations were observed between patients carrying the different genotypes for Y54C or T400K and their mRNA levels for ABCG5 or ABCG8. The increased NPC1L1 and ACAT2 mRNA levels in gallstone patients might indicate an upregulated absorption and esterification of cholesterol in the small intestine.

Cholesterol regulates ACAT2 gene expression and enzyme activity in human hepatoma cells.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the synthesis of cholesteryl esters from cholesterol and long-chain fatty acids. The two ACAT enzymes, ACAT1 and ACAT2, lack sterol regulatory elements in their promoters and have not been thought to be transcriptionally regulated by cellular cholesterol. However, Cynomolgus monkeys respond to high-cholesterol diet with increased hepatic ACAT2 mRNA expression. Also, a decrease in hepatic ACAT2 mRNA expression has been observed during statin treatment in humans. Thus, we hypothesized that cholesterol may exert transcriptional regulation on the human ACAT2 gene. To test this, we studied two human hepatoma cell lines (HuH7 and HepG2) under conditions of cholesterol loading or depletion and analyzed ACAT gene expression, enzymatic activity, and cellular cholesterol mass. We show a dose-dependent increase of ACAT2 mRNA expression, an increased enzymatic activity of ACAT2, and increased esterified cholesterol mass upon cholesterol loading. These results suggest that human ACAT2 is transcriptionally regulated by cholesterol.

ACAT2 stimulates cholesteryl ester secretion in apoB-containing lipoproteins.

Previous studies in nonhuman primates revealed a striking positive correlation between liver cholesteryl ester (CE) secretion rate and the development of coronary artery atherosclerosis. CE incorporated into hepatic VLDL is necessarily synthesized by ACAT2, the cholesterol-esterifying enzyme in hepatocytes. We tested the hypothesis that the level of ACAT2 expression, in concert with cellular cholesterol availability, affects the CE content of apolipoprotein B (apoB)-containing lipoproteins. In a model system of lipoprotein secretion using COS cells cotransfected with microsomal triglyceride transfer protein and truncated forms of apoB, ACAT2 expression resulted in a 3-fold increase in microsomal ACAT activity and a 4-fold increase in the radiolabeled CE content of apoB-lipoproteins. After cholesterol-cyclodextrin (Chol-CD) treatment, CE secretion was increased by 27-fold in ACAT2-transfected cells but by only 7-fold in control cells. Chol-CD treatment also caused the percentage of CE in the apoB-lipoproteins to increase from 3% to 33% in control cells and from 16% to 54% in ACAT2-transfected cells. In addition, ACAT2-transfected cells secreted 3-fold more apoB than control cells. These results indicate that under all conditions of cellular cholesterol availability tested, the relative level of ACAT2 expression affects the CE content and, hence, the potential atherogenicity, of nascent apoB-containing lipoproteins.

Serotonin acts as an up-regulator of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-macrophages.

Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. Serotonin (5-HT), a potent vasoconstrictor that is released from activated platelets, increases uptake of oxidized low-density lipoprotein (LDL) by macrophages, leading to foam cell formation, and contributes to the development of atherosclerotic plaque. However, it is not yet known whether 5-HT affects ACAT-1 expression in human monocyte-macrophages as the molecular mechanism of enhanced foam cell formation by 5-HT remains unclear. We examined the effects of 5-HT on ACAT-1 expression during differentiation of cultured human monocytes into macrophages. Expression of ACAT-1 protein but not 5-HT2A receptor increased in a time-dependent manner. 5-HT increased ACAT activity in a concentration-dependent manner after 7 days in primary monocyte culture. Immunoblotting analysis showed that 5-HT at 10 microM increased ACAT-1 protein expression level by two-fold, and this effect was abolished completely by a 5-HT2A receptor antagonist (sarpogrelate), its major metabolite (M-1), a G protein inactivator (GDP-beta-S), a protein kinase C (PKC) inhibitor (rottlerin), a Src family inhibitor (PP2), or a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD98059). Northern blotting analysis indicated that among the four ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the levels of the 2.8- and 3.6-kb transcripts were selectively up-regulated by approximately 1.7-fold by 5-HT (10 microM). The results of the present study suggested that 5-HT may play a crucial role in macrophage-derived foam cell formation by up-regulating ACAT-1 expression via the 5-HT2A receptor/G protein/c-Src/PKC/MAPK pathway, contributing to the progression of atherosclerotic plaque.

Acyl-coenzyme A binding protein expression alters liver fatty acyl-coenzyme A metabolism.

Although studies in vitro and in yeast suggest that acyl-CoA binding protein ACBP may modulate long-chain fatty acyl-CoA (LCFA-CoA) distribution, its physiological function in mammals is unresolved. To address this issue, the effect of ACBP on liver LCFA-CoA pool size, acyl chain composition, distribution, and transacylation into more complex lipids was examined in transgenic mice expressing a higher level of ACBP. While ACBP transgenic mice did not exhibit altered body or liver weight, liver LCFA-CoA pool size increased by 69%, preferentially in saturated and polyunsaturated, but not monounsaturated, LCFA-CoAs. Intracellular LCFA-CoA distribution was also altered such that the ratio of LCFA-CoA content in (membranes, organelles)/cytosol increased 2.7-fold, especially in microsomes but not mitochondria. The increased distribution of specific LCFA-CoAs to the membrane/organelle and microsomal fractions followed the same order as the relative LCFA-CoA binding affinity exhibited by murine recombinant ACBP: saturated > monounsaturated > polyunsaturated C14-C22 LCFA-CoAs. Consistent with the altered microsomal LCFA-CoA level and distribution, enzymatic activity of liver microsomal glycerol-3-phosphate acyltransferase (GPAT) increased 4-fold, liver mass of phospholipid and triacylglyceride increased nearly 2-fold, and relative content of monounsaturated C18:1 fatty acid increased 44% in liver phospholipids. These effects were not due to the ACBP transgene altering the protein levels of liver microsomal acyltransferase enzymes such as GPAT, lysophosphatidic acid acyltransferase (LAT), or acyl-CoA cholesterol acyltransferase 2 (ACAT-2). Thus, these data show for the first time in a physiological context that ACBP expression may play a role in LCFA-CoA metabolism.

The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A:cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.

ACAT2 is localized to hepatocytes and is the major cholesterol-esterifying enzyme in human liver.

BACKGROUND: Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification. METHODS AND RESULTS: In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined. CONCLUSIONS: The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.

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

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