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(Circulation. 2004;110:2017-2023.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

ACAT2 Is Localized to Hepatocytes and Is the Major Cholesterol-Esterifying Enzyme in Human Liver

Paolo Parini, MD, PhD; Matthew Davis, MS; Aaron T. Lada, PhD; Sandra K. Erickson, PhD; Teresa L. Wright, PhD; Ulf Gustafsson, MD; Staffan Sahlin, MD; Curt Einarsson, MD; Mats Eriksson, MD; Bo Angelin, MD; Hiroshi Tomoda, PhD; Satoshi mura, PhD; Mark C. Willingham, MD; Lawrence L. Rudel, PhD

From the Metabolism Unit (P.P., M.E., B.A.), Center for Metabolism and Endocrinology, Department of Medicine, and the Molecular Nutrition Unit, Center for Nutrition and Toxicology, Novum, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden; the Department of Pathology (P.F., M.D., A.T.L., M.C.W., L.L.R.), Wake Forest University School of Medicine, Winston-Salem, NC; the Department of Medicine and Liver Center (S.K.E., T.L.W.), University of California, and the Veterans Administration Medical Center, San Francisco, Calif; the Department of Surgery (U.G., S.S.), Karolinska Institute at Danderyd Hospital, Danderyd, Sweden; the Center for Gastroenterology (C.E.), Department of Medicine, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden; the Kitasato Institute for Life Sciences (H.T., S.O.), Kitasato University, Toyko, Japan; and the Department of Biochemistry (L.L.R.), Wake Forest University School of Medicine, Winston-Salem, NC.

Correspondence to Lawrence L. Rudel, Department of Pathology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157. E-mail lrudel{at}wfubmc.edu

Received January 23, 2004; revision received May 12, 2004; accepted May 21, 2004.

	Abstract

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
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.

Key Words: Key Words: • cholesterol • genes • lipids • metabolism

	Introduction

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
A balance between the availability of free and esterified cholesterol is critically important for cell function. Two different acyl-coenzyme A:cholesterol acyltransferase (ACAT; EC2.3.1.26) genes,* ACAT1 and ACAT2, encoding enzymes responsible for intracellular esterification of cholesterol have been identified and cloned.^1–3 These genes have unique tissue distributions, with ACAT2 expression predominantly in the liver and intestine and ACAT1 in most tissues of the body.^1–3 Various roles for these enzymes in the maintenance and regulation of cholesterol homeostasis have been suggested.^4–8 ACAT2 expression in the liver and intestine^1–3 is consistent with its proposed roles in lipoprotein assembly and secretion of cholesteryl esters,⁵ which occur principally in hepatocytes and enterocytes, the 2 main lipoprotein-producing cell types in the body. Studies in mouse models of atherosclerosis showed reduced amounts of atherosclerosis when ACAT2 was deficient,⁹ with little or no reduction when ACAT1 was deficient.^10–12 Thus, a specific role for ACAT2 in determining plasma lipoprotein cholesteryl ester transport and atherogenesis is consistent with these observations.

Although ACAT activity is present in human liver,^13,14 the gene product(s) responsible for this activity remains uncertain. Data have been published suggesting that human liver differs from mouse and nonhuman primate liver with regard to ACAT expression.^6,15 Human liver ACAT2 mRNA abundance on commercially prepared human tissue blots was low, except in fetal liver.² ACAT2 expression in 2 human liver surgical specimens was minimal as assessed by activity after immunodepletion and Western blotting, whereas ACAT1 was reported to be highly expressed in human liver and in isolated human hepatocytes.¹⁵ On the basis of these data, Chang and colleagues^6,15 concluded that ACAT1 was the major ACAT isoenzyme in human liver, although attempts to confirm this by immunohistochemistry did not allow clear interpretation of the findings.¹⁵ Thus, the role(s) of ACAT1 and ACAT2 in human liver remains uncertain.

To rationally develop prevention and/or treatment paradigms for human diseases related to dysregulation of hepatic cholesterol metabolism, the expression and regulation of ACAT1 and ACAT2 in human liver need clarification. Given the present uncertain status of this issue, the usefulness and applicability of surrogate models such as the mouse and nonhuman primate also remain in question. The present studies were initiated to detail the cellular localization and relative degree of expression of these 2 enzymes in adult human liver. Our results show that the cellular pattern of ACAT2 expression in hepatocytes and of ACAT1 expression in Kupffer cells in human liver share many similarities with those in both nonhuman primates and mice. Furthermore, the findings suggest that the primary ACAT in hepatocytes (and enterocytes), ACAT2, may be a preferred target for treatments designed to prevent hypercholesterolemia and related diseases, such as premature coronary heart disease. Although ACAT inhibitors, most without selectivity for ACAT1 versus ACAT2, have not yet been identified that are effective in plasma cholesterol lowering in humans,¹⁶ the present findings suggest that an inhibitor specific for ACAT2 might be more successful.

	Methods

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Liver Collection
For mRNA comparisons and Western blot analysis, human liver samples from the Tissue Bank of the University of California San Francisco (UCSF) Liver Center Clinical and Translational Research Core were used. All samples were from patients (6 female and 4 male; age range, 10 to 58 years) undergoing liver transplantation and were obtained promptly after removal of the diseased liver. The UCSF Human Subjects Institutional Review Board approved all protocols for the acquisition and use of liver samples.

For immunohistochemical analyses and ACAT enzymatic assay, human liver samples were collected from 14 patients (9 female and 5 male; age range, 28 to 68 years) who underwent laparoscopic cholecystectomy because of gallstone disease. All gave their informed consent before the study, which was approved by the Ethics Committee on Human Research of the Karolinska Institute.

African green monkey liver biopsies were taken surgically from feral adult males that had been fed a saturated fat-enriched, cholesterol-free diet. The liver sample collection was approved by the Wake Forest University School of Medicine Animal Care and Use Committee.

To avoid ex vivo alterations, all liver samples were immediately flash-frozen in LN₂ at the time of collection and maintained at –80°C until analyses were performed.

RNA Preparation and mRNA Determination
Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Quantification of specific mRNAs was performed by the SYBR Green real-time polymerase chain reaction (PCR; see Appendix). Data are expressed in arbitrary units that were normalized by correction for the signal obtained in the same cDNA preparation for glyceraldehyde 3-phosphate dehydrogenase mRNA.

Isolation of Microsomes and Enzymatic Analyses
Liver samples (50 to 150 mg) were homogenized in 3-mL ice-cold buffer containing 0.1 mol/L K₂HPO₄, 0.25 mol/L sucrose, and 1 mmol/L EDTA, pH 7.4. A protease inhibitor cocktail (Sigma) was added to the buffer before homogenization. The homogenate was then centrifuged for 15 minutes at 12 000g (4°C) to remove cell debris. The resulting supernatant was centrifuged for 60 minutes at 100 000g. The microsomal pellet from this spin was resuspended in 0.1 mol/L K₂HPO₄ at pH 7.4 and immediately frozen at –80°C.

For Western blot analyses, 100 µg of microsomal protein was analyzed after suspension in protein solubilization buffer (120 mmol/L Tris, pH 6.8; 20% glycerol, 4% sodium dodecyl sulfate, and bromophenol blue). Dithiothreitol was added to a final concentration of 100 mmol/L, and samples were incubated at 37°C for 30 minutes. Separation and blotting were performed as described earlier with the antibodies made against monkey enzyme N-terminal sequences.¹⁷ Detection was accomplished by chemiluminescence, with exposure times being adjusted to maximize band intensity.

Total ACAT enzymatic activity was determined with hepatic microsomes as previously described,¹⁸ except preincubation included a cholesterol-saturated solution of ß-hydroxypropyl cyclodextrin for 30 minutes before addition of ¹⁴[C]oleoyl Co-A. In separate tubes, pyripyropene A, a specific ACAT2 inhibitor,¹⁹ was included in the preincubation and reaction mixture at a concentration of 5 µmol/L to separately identify ACAT1 (uninhibited) and ACAT2 (total–ACAT1) activities.

Tissue Immunostaining
Frozen liver biopsy cryostat sections were fixed in acetone for 10 minutes and blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS) for 10 minutes. The first primary antibody solution, rabbit anti-ACAT1 or ACAT 2 (10 µg/mL) in PBS, was added to the samples and incubated for 1 hour at room temperature for ACAT1 or overnight at 4°C for ACAT2. Samples were washed 3 times in PBS, and affinity-purified, rhodamine-labeled goat anti-rabbit IgG (25 µg/mL, Jackson Immuno Research) was added, followed by incubation for 30 minutes. The samples were then washed, and the second primary antibody solution, mouse monoclonal anti-human CD68 (1:200 dilution, Laboratory Vision), was applied for 1 hour and then washed. Fluorescein isothiocyanate (FITC)–labeled goat anti-mouse IgG was applied for 30 minutes, followed by washing with PBS and fixation with 3.7% formaldehyde for 10 minutes. Nuclear staining was performed with 4',6-diamidino-2-phenylindole diacetate in methanol for 5 minutes. Sections were mounted with a 10% PBS–90% glycerol solution and visualized by fluorescence microscopy. For colocalization of ACAT2 and protein disulfide isomerase (PDI) expression, immunostaining was performed as described, except that liver sections were prefixed in 3.7% formaldehyde for 10 minutes and incubated in 3 mol/L urea for 1 hour at 90°C. The second primary antibody, a monoclonal mouse anti-human PDI (a gift from S-Y. Cheng, National Cancer Institute, National Institutes of Health, Bethesda, Md), was used at a concentration of 10 µg/mL.

	Results

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Comparison of Gene Expression in Primate Livers
Expression of a variety of genes, including those previously documented to be hepatocyte- or Kupffer cell-specific, was assessed in both human and African green monkey livers by real-time PCR. The patterns in human and monkey livers were generally comparable (Table 1). Of importance, ACAT1 and ACAT2 were readily detectable in both. ACAT2 and DGAT1 were expressed at lower levels than most of the other genes studied in both humans and monkeys. ACAT1 mRNA levels were similar in livers of humans and monkeys, whereas ACAT2 was lower in human livers. Albumin mRNA and apoB mRNA levels were both higher in monkeys. Considering that the monkey liver samples were from healthy animals whereas the human liver samples were from transplant patients with liver disease, an explanation of apparent differences is difficult, and overinterpretation should be avoided.

View this table: [in this window] [in a new window]   TABLE 1. Hepatic Expression of mRNA (Arbitrary Units) for Kupffer Cell-Specific* and Hepatocyte-Specific Genes in Humans and African Green Monkeys

Immunologic Identification of ACAT1 and ACAT2 Proteins
Western blots of microsomes prepared from 10 human transplant recipient livers and from HepG2 cells showed an immunoreactive band at 48 to 50 kDa for ACAT1 (Figure 1A), although 1 sample, No. 9, had an unusual banding pattern. When the same blot was stripped and reprobed with ACAT2-specific antibodies (Figure 1B), all samples showed an immunoreactive band near 48 kDa. The intensity of the immunostaining pattern was different among individual samples; those with higher signals for ACAT1 did not necessarily show higher ACAT2. Of note, all human liver samples examined were positive for both enzymes, consistent with the finding of mRNA for both enzymes in each of the tissue specimen in this and other studies.²⁰

View larger version (43K): [in this window] [in a new window]   Figure 1. ACAT1 and ACAT2 proteins in human liver microsomes. Protein (200 µg) solubilized from microsomes prepared from 10 different human liver samples (number [nr.] 1–10) and from HepG2 cells (lane 1) was loaded and electrophoretically separated. After transfer onto nitrocellulose filter, samples were incubated with anti-monkey ACAT1 antibody. Filter was then stripped and thereafter incubated with anti-monkey ACAT2 antibody. A, ACAT1 protein expression. B, ACAT2 protein expression. All other abbreviations are as defined in text.

ACAT2 Enzymatic Activity in Liver Microsomes
ACAT activity was measured in microsomal fractions from 4 different pools of human liver biopsies taken from gallstone patients and from liver biopsies from 5 vervet monkeys (Table 2). Pyripyropene A, a highly selective ACAT2 inhibitor¹⁹ originally isolated as a fungal metabolite,²¹ was used to differentiate between ACAT1 and ACAT2 activities. Total ACAT activity in human liver microsomes was between 3- and 17-fold lower than that in monkey liver microsomes (Table 2). The level of ACAT1 activity, ie, the activity remaining after treatment with pyripyropene A, was similar among samples in both species. In all but 1 of the human liver pools, ACAT2 activity accounted for >50% of total ACAT activity. Thus, in contrast to ACAT1, ACAT2 activity was the predominant ACAT enzymatic activity in 3 of 4 human liver samples, although it varied widely among individual samples and between species.

View this table: [in this window] [in a new window]   TABLE 2. Total ACAT, ACAT1, and ACAT2 Activities (nmol/min per Milligram) Assayed in Liver Microsomes From Humans and Monkeys With Pyripyropene A (PPPA, 5 µmol/L)

In the last column of Table 2, mRNA estimates for ACAT2 were made for the same liver preparations. The correlation between ACAT2 mRNA and activity was not significant (r=0.5), an outcome suggesting that major factors responsible for the differences among individuals were not transcriptional.

Distribution of ACAT1 and ACAT2 in Liver Cells
We investigated the cell-specific distribution of the ACATs in human liver collected from gallstone patients. Immunostaining with ACAT1 antibody showed a clear, positive signal in cells, often with the stellate pattern typical of Kupffer cells, located among the chords of hepatocytes (Figure 2A2 and 2B2). Immunostaining of the same section with an antibody raised against CD68, a specific marker for macrophages (Kupffer cells), showed that the cells positive for ACAT1 also were positive for CD68 (Figure 2A3 and 2B3), as confirmed by overlay (Figure 2A4 and 2B4). Negative controls (omission of the primary antibodies) showed only light, nonspecific staining (Figure 2C2–2C4 and Figure 2D2–2D4).

View larger version (61K): [in this window] [in a new window]   Figure 2. Immunolocalization of ACAT1 and CD68. Liver biopsies were collected from female (A and C) and male (B and D) patient. Sections were fixed in acetone. A1–D1, Phase-contrast images of sections. A2 and B2, Rhodamine-channel images of sections stained with anti-ACAT1 antibody. A3 and B3, FITC-channel images of sections stained with anti-CD68 antibody. A4 and B4, Overlays of rhodamine-channel (red) and FITC-channel (green) images showing colocalization of ACAT1 and CD68 protein expression. C2–C4 and D2–D4, Negative controls in which incubations with different primary antibodies were omitted. Abbreviations are as defined in text.

Immunostaining of liver sections with ACAT2 antibody showed a strong, positive signal from all hepatocytes (Figure 3A2 and 3B2). Immunostaining of the same section with CD68 antibody (Figure 3A3 and 3B3) and the overlay (Figure 3A4 and 3B4) showed that ACAT2 and CD68 had separate patterns; ie, they did not colocalize. Controls showed little staining (Figure 2C2–2C4 and 2D2–2D4). Immunolocalization of ACAT2 (Figure 3C2 and 3D2) and PDI, a marker for the endoplasmic reticulum (Figure 3C3 and 3D3), was determined in liver sections fixed in 3.7% formaldehyde and incubated in 3 mol/L urea. Overlay of these sections showed colocalization of ACAT2 and PDI (Figure 3C4 and 3D4), thus confirming that ACAT2 is principally within the endoplasmic reticulum in hepatocytes. Little nonspecific staining was seen in control sections (Figure 3E2–3E4 and 3F2–3F4).

View larger version (70K): [in this window] [in a new window]   Figure 3. Immunolocalization of ACAT2, CD68, and PDI. Liver biopsies were collected from female (A, C, E) and male (B, D, F) patient. Sections were fixed either in acetone (A and B) or in formaldehyde followed by urea (C–F). A1–F1, Phase-contrast images. A2 and B2, Rhodamine-channel images of sections stained with anti-ACAT2 antibody. A3 and B3, FITC-channel images of sections stained with anti-CD68 antibody. A4 and B4, Overlays of rhodamine-channel (red) and FITC-channel (green) images showing localization of ACAT2 and CD68 protein expression in different cell types. C2 and D2, Rhodamine-channel images of sections stained with anti-ACAT2 antibody. C3 and D3, FITC-channel images of sections stained with anti-PDI antibody. C4 and D4, Overlays of rhodamine-channel (red) and FITC-channel (green) images showing colocalization of ACAT2 and PDI in hepatocytes. E2–E4 and F2–F4, Negative controls of sections fixed in formaldehyde in which primary antibodies were omitted. Abbreviations are as defined in text.

	Discussion

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
ACAT2 was present in all human liver samples studied (Figure 1 and Table 2); was highly variable among individuals, suggesting that it is a regulated enzyme; and was responsible for >50% of the ACAT activity in a majority of human liver samples studied (Table 2). ACAT2 expression was confined to hepatocytes in human liver (Figure 3), whereas ACAT1 was found in Kupffer cells only (Figure 2). These findings are similar to those made earlier in nonhuman primates.²² Based on these data, ACAT2 is the major ACAT enzyme in human hepatocytes, a finding consistent with the hypothesis that ACAT2 participates in the assembly and secretion of cholesteryl esters in lipoproteins in humans.⁵ Together with observations in apoE^–/- mice showing atherosclerosis protection with ACAT2 gene deletion⁹ and in monkeys showing a high correlation between hepatic cholesteryl ester secretion and coronary artery atherosclerosis,²⁰ ACAT2 is identified as an important regulator of susceptibility to the development of atherosclerosis.

Before this study, identification of ACAT2 as the principal ACAT enzyme in human liver and hepatocytes had not been made. The histochemical demonstration that ACAT2 is located exclusively in hepatocytes whereas ACAT1 is primarily found in Kupffer cells required fully specific, immunopurified antibodies²²; the present data are the first for which these conditions were met for human liver. The tissue used for immunohistologic identification was freshly isolated from live donors and immediately snap-frozen in LN₂ after removal. These conditions appear to be required for adequate preservation of ACAT immunodetectability. ACAT2 is an enzyme expressed in low amounts and lability, such as might occur in cadaveric tissue, as used previously,¹⁵ and may preclude accurate identification. Although ACAT1 had been identified immunohistochemically in isolated human hepatocytes¹⁵ and its mRNA in isolated monkey hepatocytes,¹ the significance of these observations relative to the in vivo condition is unclear. It is possible that ACAT1 expression in hepatocytes is below the limits of detection by immunohistochemistry on whole-liver sections. Alternatively, expression of ACAT1 may be induced by hepatocyte isolation procedures. HepG2 cells, a hepatoblastoma cell line with many fetal liver characteristics, express both enzymes, as confirmed here (Figure 1); however, this may reflect HepG2 cell characteristics rather than those of normal, highly differentiated human hepatocytes. Clearly, additional studies are needed to define the factors controlling expression of these 2 genes.

Comparison of mRNA expression patterns of genes involved in hepatic lipid metabolism in both humans and African green monkeys indicates that expression of ACAT2, in relation to ACAT1, was relatively higher in nonhuman primate liver (Table 1). The data in Table 2 show that ACAT2 activity is higher than ACAT1 activity in monkeys and most humans, despite the significantly higher amounts of ACAT1 mRNA compared with ACAT2 mRNA. The observation that more ACAT2 enzyme activity was associated with a lower mRNA level for ACAT2 may be a reflection of a relatively higher stability of the ACAT2 protein compared with ACAT1 protein. In Chinese hamster ovary cell transfection studies, we noted that the ACAT2 protein expressed in these cells had a half-life >6 hours, whereas the ACAT1 protein had a half-life of only 20 to 30 minutes (R. Temel and L.L. Rudel, unpublished observations). It may be that factors responsible for higher ACAT2 activities in the liver are related to greater stability of the enzyme.

African green monkeys had higher total ACAT activity in the liver, principally due to the high amount of ACAT2 activity, whereas ACAT1 activity levels were similar in human and monkey livers. To the extent that ACAT2 is the cholesterol-esterification enzyme in hepatocytes, the variability in ACAT2 activity may be related to the highly variable rates of hepatic cholesteryl ester secretion in nascent VLDL of monkeys.²³ It would be interesting to learn whether there is an association of ACAT2 activity with the variable hepatic VLDL apoB secretion noted for humans.²⁴ The fact that liver cholesterol concentration in humans is similar to that of African green monkeys^25–27 suggests that hepatic cholesteryl ester secretion proportional to that seen in the monkeys²³ may also occur in humans.

In comparisons between different species of nonhuman primates, the responses to dietary cholesterol were proportional to the degree of upregulation of the ACAT2 gene, suggesting that hepatic ACAT2 expression levels are correlated with dietary cholesterol sensitivity.¹⁷ The differences in hepatic expression and activity level of ACAT2 between monkeys and humans may in part reflect the higher dietary cholesterol responsiveness typical of nonhuman primates. The extent of coronary artery atherosclerosis in nonhuman primates was correlated with the amounts of cholesteryl esters secreted from the liver in response to dietary cholesterol.²³ This suggests that the lower ACAT2 activity observed in human livers may represent an advantage for delayed development of atherosclerosis. The atheroprotective effect of ACAT2 deficiency in mice and the observation of a positive association between plasma levels of cholesteryl oleate, the principal product of hepatic ACAT2 in monkeys,²³ and a higher risk for complications of coronary heart disease in humans with higher percentages in plasma of cholesteryl oleate and lower percentages of cholesteryl linoleate^28–31 further support this hypothesis.

The effectiveness of pyripyropene A, previously shown to be a specific ACAT2 inhibitor,¹⁹ in the human liver microsomes of this study suggests that it or similar compounds could be effective in the treatment and prevention of hypercholesterolemia and atherosclerosis and might complement drugs presently in use, eg, statins, which work through different molecular mechanisms.

In conclusion, the unequivocal identification of ACAT2 mRNA expression in adult human liver and of ACAT2 as the primary isoform within hepatocytes of adult human liver implies that this ACAT enzyme is a key factor in cholesterol transport and lipoprotein metabolism in humans. The findings lend strong support to the suggestion of a critical role for ACAT2 in the maintenance of cholesterol homeostasis at the level of the liver in human beings.

	Appendix 1

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
RNA Preparation and mRNA Determination
Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. One microgram total RNA was transcribed into cDNA by random-hexamer priming and Omniscript (Qiagen). Quantification of specific mRNAs was performed by SYBR Green real-time PCR in an ABI PRISM 7000 thermocycler (PE Applied Biosystems) under standard manufacturer’s conditions. Whenever possible, primer sets (Table 3) were designed to cross intron-exon junctions to prevent amplification of genomic DNA. Primers were based on human sequences, and amplification efficiencies for all genes were similar for both species. Data are expressed in arbitrary units that were normalized by correction for the signal obtained in the same cDNA preparation for glyceraldehyde 3-phosphate dehydrogenase mRNA.

View this table: [in this window] [in a new window]   TABLE 3. Primer Sequences for SYBR Green Real-Time PCR

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-49373, HL-24736, and DK-26743 (UCSF Liver Center) and by the Swedish Medical Research Council (71XD-14847, 03X-7137, K2002–72X-04793); by the Åke Wiberg, Tore Nilsson, Ruth and Richard Julin, Thuring, Fernström, and Throne Holst Foundations; by the Swedish Medical Association; and by a grant from Pfizer. We thank Tigist Belaye and Steven Lear for excellent technical support.

	Footnotes

 
*By convention with the National Center for Biotechnology Information, official names of the genes for these enzymes have been recommended to be sterol-O-acyltransferase 1 and 2 (soat1 and soat2).

	References

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 

1. Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Shelness G, Rudel LL. Identification of a form of acyl-CoA: cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J Biol Chem. 1998; 273: 26747–26754.[Abstract/Free Full Text]
   
2. Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A: cholesterol acyltransferase-related enzymes. J Biol Chem. 1998; 273: 26765–26771.[Abstract/Free Full Text]
   
3. Cases S, Novak S, Zheng Y-W, Myers HM, Lear SR, Sande E, Welch CB, Lusis AJ, Spencer TA, Krause BR, et al. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase: its cloning, expression, and characterization. J Biol Chem. 1998; 273: 26755–26764.[Abstract/Free Full Text]
   
4. Buhman KK, Chen HC, Farese RV Jr. The enzymes of neutral lipid synthesis. J Biol Chem. 2001; 276: 40369–40372.[Free Full Text]
   
5. Rudel LL, Lee RG, Cockman TL. Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis. Curr Opin Lipidol. 2001; 12: 121–127.[CrossRef][Medline] [Order article via Infotrieve]
   
6. Chang T-Y, Chang CCY, Lin S, Yu C, Li B-L, Miyazaki A. Roles of acyl-coenzyme A: cholesterol acyltransferase-1 and -2. Curr Opin Lipidol. 2001; 12: 289–296.[CrossRef][Medline] [Order article via Infotrieve]
   
7. Meiner VL, Cases S, Myers HM, Sande ER, Bellosta S, Schambelan M, Pitas RE, McGuire J, Herz J, Farese RV Jr. Disruption of the acyl-CoA: cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci U S A. 1996; 93: 14041–14046.[Abstract/Free Full Text]
   
8. Buhman KK, Accad M, Novak S, Choi RS, Wong JS, Hamilton RL, Turley S, Farese RV Jr. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT 2-deficient mice. Nat Med. 2000; 6: 1341–1347.[CrossRef][Medline] [Order article via Infotrieve]
   
9. Willner EL, Tow B, Buhman KK, Wilson M, Sanan DA, Rudel LL, Farese RV Jr. Deficiency of acyl CoA: cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2003; 100: 1262–1267.[Abstract/Free Full Text]
   
10. Yagyu H, Kitamine T, Osuga J, Tozawa R, Chen Z, Kaju Y, Oka T, Perrey S, Tamura Y, Ohashi K, et al. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia. J Biol Chem. 2000; 275: 21324–21330.[Abstract/Free Full Text]
    
11. Accad M, Smith SJ, Newland DL, Sanan DA, King LE Jr, Linton MF, Fazio S, Farese RV Jr. Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA: cholesterol acyltransferase 1. J Clin Invest. 2000; 105: 711–719.[Medline] [Order article via Infotrieve]
    
12. Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese RV Jr. Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages. J Clin Invest. 2001; 107: 163–171.[Medline] [Order article via Infotrieve]
    
13. Erickson SK, Cooper AD. Acyl-coenzyme A: cholesterol acyltransferase in human liver: in vitro detection and some characteristics of the enzyme. Metabolism. 1980; 29: 991–996.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Einarsson K, Benthin L, Ewerth S, Hellers G, Stahlberg D, Angelin B. Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes. J Lipid Res. 1989; 30: 739–746.[Abstract]
    
15. Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee C-YG, Strom S, Kashyap R, et al. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. 2000; 275: 28083–28092.[Abstract/Free Full Text]
    
16. Sliskovic DR, Picard JA, Krause BR. ACAT inhibitors: the search for a novel and effective treatment of hypercholesterolemia and atherosclerosis. Prog Med Chem. 2002; 39: 121–171.[Medline] [Order article via Infotrieve]
    
17. Rudel LL, Davis M, Sawyer J, Shah R, Wallace J. Primates highly responsive to dietary cholesterol upregulate hepatic ACAT2 while less responsive primates do not. J Biol Chem. 2002; 277: 31401–31406.[Abstract/Free Full Text]
    
18. Carr TP, Parks JS, Rudel LL. Hepatic ACAT activity in African green monkeys is highly correlated to plasma LDL cholesteryl ester enrichment and coronary artery atherosclerosis. Arterioscler Thromb. 1992; 12: 1274–1283.[Abstract/Free Full Text]
    
19. Lada AT, Davis M, Kent C, Chapman J, Tomoda H, Omura S, Rudel LL. Identification of ACAT1- and ACAT2- specific inhibitors using a novel, cell-based fluorescent assay: evidence defining uniqueness for individual ACATs. J Lipid Res. 2004; 45: 378–386.[Abstract/Free Full Text]
    
20. Smith JL, Rangaraj K, Simpson R, Maclean DJ, Nathanson LK, Stuart KA, Scott SP, Ramm GA, de Jersey J. Quantitative analysis of expression of ACAT genes in human tissues by real-time PCR. J Lipid Res. 2004; 45: 686–696.[Abstract/Free Full Text]
    
21. Tomoda H, Tabata N, Yang DJ, Namatame I, Tanaka H, Omura S, Kaneko T. Pyripyropenes, novel ACAT inhibitors produced by Aspergillus fumigatus,4: structure elucidation of pyripyropenes M to R. J Antibiot (Tokyo). 1996; 49: 292–298.[Medline] [Order article via Infotrieve]
    
22. Lee RG, Willingham MC, Davis MA, Skinner KA, Rudel LL. Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates. J Lipid Res. 2000; 41: 1991–2001.[Abstract/Free Full Text]
    
23. Rudel LL, Haines J, Sawyer JK, Shah R, Wilson MS, Carr TP. Hepatic origin of cholesteryl oleate in coronary artery atherosclerosis in African green monkeys; enrichment by dietary monounsaturated fat. J Clin Invest. 1997; 100: 74–83.[Medline] [Order article via Infotrieve]
    
24. Turner PR, Miller NE, Cortese C, Hazzard W, Coltart J, Lewis B. Splanchnic metabolism of plasma apolipoprotein B. J Clin Invest. 1981; 67: 1678–1686.[Medline] [Order article via Infotrieve]
    
25. Frantz ID Jr, Carey JB Jr. Cholesterol content of human liver after feeding of corn oil and hydrogenated coconut oil. Proc Soc Exp Biol Med. 1961; 106: 800–801.
    
26. Kwiterovich PO Jr, Sloan HR, Fredrickson DS. Glycolipids and other lipid constituents of normal human liver. J Lipid Res. 1970; 11: 322–330.[Abstract]
    
27. Salen G, Nicolau G, Shefer S, Mosbach EH. Hepatic cholesterol metabolism in patients with gallstones. Gastroenterology. 1975; 69: 676–684.[Medline] [Order article via Infotrieve]
    
28. Lewis B. Composition of plasma cholesterol ester in relation to coronary-artery disease and dietary fat. Lancet. 1958; 2: 71–73.[CrossRef][Medline] [Order article via Infotrieve]
    
29. Logan RL, Thomson M, Riemersma RA, Oliver MF, Olsson AG, Rossner S, Callmer E, Walldius G, Kaijser L, Carlson LA, et al. Risk factors for ischaemic heart-disease in normal men aged 40. Lancet. 1978; 1: 949–955.[Medline] [Order article via Infotrieve]
    
30. Kirkeby K, Ingvaldsen P, Bjerkedal I. Fatty acid composition of serum lipids in men with myocardial infarction. Acta Med Scand. 1972; 192: 513–519.[Medline] [Order article via Infotrieve]
    
31. Kingsbury KJ, Brett C, Stovold R, Chapman A, Anderson J, Morgan DM. Abnormal fatty acid composition and human atherosclerosis. Postgrad Med J. 1974; 50: 425–440.[Medline] [Order article via Infotrieve]
    

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