The process of cholesterol homeostasis in extrahepatic tissues such as the fibroblast involves the uptake of lipoproteins by cell surface receptors, lysosomal hydrolysis of lipoprotein-derived cholesteryl esters to yield free cholesterol, and reesterification of free cholesterol in the endoplasmic reticulum for storage in cytoplasmic lipid droplets. The re-esterification step is crucial to prevent excess free cholesterol from disrupting cell membranes and is carried out by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) ()(for a review, see (1) ). In the classic model for tissue cholesterol homeostasis, the cultured fibroblast, ACAT activity is up-regulated by low density lipoprotein (LDL), exogenous free cholesterol, and oxygenated sterols such as 25-hydroxycholesterol; thus, as the level of free cholesterol substrate in the cell increases, ACAT activity coordinately increases to maintain the level of free cholesterol within the cell in a fairly narrow range(2) .

ACAT has a variety of roles in other tissues as well. In the liver, ACAT-derived cholesteryl esters are secreted as a component of very low density lipoprotein (VLDL). In steroidogenic tissues, ACAT activity generates a storage pool of cholesteryl ester that is readily mobilized by hormone-sensitive cholesteryl ester hydrolase to produce free cholesterol for the synthesis of steroid hormones. Last, ACAT activity has been measured in arterial tissue from several animal species and is thought to play a crucial role in the development of atherosclerosis by contributing to the accumulation of cholesteryl ester in fatty streaks.

The regulation of ACAT at the molecular level has been difficult to examine, as the enzyme has never been purified to homogeneity. However, the recent isolation of a human ACAT cDNA clone (``K1'') from a human macrophage cDNA library (3) allowed the investigation of this enzyme at the molecular level. The predicted protein product of the cloned cDNA is a 64-kDa protein with at least two transmembrane domains and five sequences homologous to the ``signature sequences'' found in many enzymes involved in the catalysis of acyl adenylate formation with subsequent acyl thioester formation and acyl transfer (4) . ACAT-deficient CHO cells transfected with the cDNA clone regained the ability to esterify cholesterol and accumulated intracellular lipid droplets. Similarly, insect Sf 9 cells, which do not normally possess the ability to esterify cholesterol, gained this function when transfected with the human ACAT cDNA, and initial studies of its biological activity have shown that ACAT activity is up-regulated by cholesterol and 25-hydroxycholesterol in this system(5) .

Due to the limited range of experiments that can be carried out in human subjects, the use of animal models for the study of genetic contributions to cardiovascular disease has become widespread. Of the available animals, the mouse is particularly well suited for these studies, due in part to the abundance of information on the mouse genome and the availability of phenotypically distinct inbred strains. One of these strains, the C57BL/6J mouse, is susceptible to diet-induced atherosclerosis (6) and thus has been widely used as a model for the development and progression of arterial lesions. In order to define the role of ACAT in this process, we cloned the mouse equivalent of the previously reported human ACAT K1 clone. In this study we report the cDNA-derived mouse ACAT sequence, the tissue distribution of ACAT mRNA in C57BL/6J mice, and the regulation of its expression by dietary cholesterol. In addition, we have transfected ACAT-deficient CHO cells with our mouse ACAT clone to determine the modes of regulation of mouse ACAT activity in vitro.

EXPERIMENTAL PROCEDURES

Materials

Cloning of Mouse ACAT

Sequence and structure analysis of the cloned cDNA and its predicted protein product were carried out using the Genetics Computer Group (GCG) program package(7) .

Chromosomal Localization of the Mouse ACAT Gene

Animals and Diets

Plasma Lipoprotein Profiles of Chow and Fat-fed Mice

Fast protein liquid chromatography (FPLC) was performed by the method of Jiao et al.(9) . 160-200 µl of plasma from each mouse was applied onto two Superose 6 columns (Pharmacia Biotech Inc.) connected in series to a Beckman System Gold HPLC/FPLC system. The columns were eluted at a constant flow rate of 0.5 ml/min with 154 mM NaCl containing 0.02% NaN and 1 mM EDTA. Fifty 0.5-ml fractions from each plasma sample were collected and assayed for total cholesterol and triglycerides as above.

RNA Isolation

For RNA isolation from peritoneal macrophages, mice were sacrificed by cervical dislocation. 4-5 ml of sterile phosphate-buffered saline at 4 °C was injected into the peritoneum of each mouse and withdrawn approximately 5 min later. Cells from five mice were pooled and pelleted at 1500 rpm in a Sorvall RC-3B centrifuge at 4 °C for 10 min. The supernatant was discarded and the cell pellet lysed with 1 ml of Ultraspec RNA. The lysates were then processed as above.

For RNA isolation from CHO cells, culture medium was removed and the cells were lysed directly by the addition of 1 ml/75-cm Ultraspec RNA followed by brief tituration. The lysates were processed as above.

Northern Blot Analysis

RNase Protection Assay to Quantitate ACAT mRNA

Liver Microsomal ACAT Assays

Microsomal ACAT assays were performed as follows. 100 µg of mouse liver microsomal protein were brought up to 60 µl with ice-cold Buffer A in a 13 100-mm borosilicate tube and equilibrated to 37 °C in a water bath for 1 min. 40 µl of oleoyl-CoA substrate mixture (10-12 µCi/µmol [C]oleoyl-CoA, 170 µM oleoyl-CoA, 12.5 mg/ml fatty acid-free bovine serum albumin in 104 mM Tris, pH 7.8) were then added and the microsomes were further incubated at 37 °C for 5 min. The reaction was stopped by adding 1.5 ml of 2:1 methanol/chloroform containing 50 µg of unlabeled cholesteryl oleate and 3000 dpm of [H]cholesteryl oleate as a carrier for thin-layer chromatography and as an internal standard for recovery, respectively. Total lipids were extracted by the method of Bligh and Dyer(10) , dried under nitrogen, and separated by thin layer chromatography on Silica Gel G plates (Analtech, Inc., Newark, DE) using 85:20:1 heptane/ethyl ether/glacial acetic acid as the mobile phase. The cholesteryl ester bands were visualized by iodine vapor staining, blanched with gentle heating, and scraped into scintillation vials. 3.5-ml Econofluor-2 (DuPont NEN) was added to each band, and H/C cpm were quantitated using a Beckman LS 8000 scintillation counter.

CHO Cells Transfected with Mouse ACAT

To measure the rate of cholesteryl ester synthesis in intact CHO cells, the cells were passed into 100-mm culture dishes at a density of 3 10 cells per dish. After 24 h, the medium was changed to serum-free medium supplemented with 2% lipoprotein-deficient serum. 24 h later, the medium was removed and replaced with serum-free medium containing 2% lipoprotein-deficient serum alone or supplemented with 20 µg/ml human LDL. After another 24 h, the media were removed and 4 ml of fresh serum-free medium containing 40 µl of [H]oleic acid complexed to bovine serum albumin (12) was added to each dish. Following a 2-h incubation at 37 °C, the medium was removed and the cells harvested by scraping with a rubber policeman. The cells were pelleted, rinsed once with phosphate-buffered saline, and resuspended in 0.5 ml of phosphate-buffered saline. Protein content of the cell suspension and incorporation of [H]oleate into cholesteryl [H]oleate were measured as above, except that 20,000 dpm of [C]cholesteryl oleate was added to each sample prior to lipid extraction as the internal standard.

RESULTS

Cloning and Sequence of Mouse ACAT cDNA

Figure 1: a, sequence of the 3.7-kb mouse ACAT cDNA clone and alignment of mouse and human ACAT peptide sequences. The positions of two short open reading frames in the 5`-untranslated region and the AATAAA polyadenylation signal in the 3`-untranslated region are underlined. Amino acid residues of the human ACAT cDNA sequence (GenBank accession L21934) identical to those of the mouse ACAT cDNA sequence (GenBank accession L42293) are indicated by an asterisk (*). The residues comprising the two putative transmembrane domains are underlined, and the leucine residues predicted to be involved in the heptad motif are shown in bold type. Potential N-glycosylation sites on mouse ACAT are indicated by the brackets; regions homologous to the signature sequences are boxed. b, Kyte-Doolittle hydrophobicity plot of the deduced ACAT protein sequence. Domains that appear above the central line are predicted to be hydrophobic. The two putative transmembrane domains are represented by the brackets. The potential leucine heptad dimerization motifs are located in the region indicated by the dashed line. Signature sequences are indicated by the arrows.

Chromosomal Location of Mouse ACAT

Figure 2: Chromosomal localization of mouse ACAT. a, Southern blot analysis to detect ACAT restriction fragment length polymorphisms. EcoRI-digested mouse genomic DNAs from parental mouse strains and 94 backcrossed progeny were probed with a 1.6-kb fragment corresponding to the entire coding region of mouse ACAT. B, C57BL/6J DNA; S, M. spretus DNA; S/B, DNA from one of the (C57BL/6J X SPRET/Ei)F X SPRET/Ei backcrossed mice showing the heterozygous pattern. b, haplotype analysis of the backcross panel. The solid boxes represent the presence of a C57BL/6J allele, and the open boxes represent the presence of an M. spretus allele. The stippled box indicates an untyped locus. The number of offspring inheriting each haplotype is listed at the bottom of each column. c, partial map of mouse chromosome 1. The location of the ACAT gene (Acact) is shown relative to linked genes in the BSS map of mouse chromosome 1 (right) and the Chromosome Committee map of the same region of mouse chromosome 1 (left). Relative recombination distance in 10 centimorgans (cM) is as indicated.

Northern Blot and RNase Protection Assay of ACAT mRNA in Mouse Tissues

Figure 3: a, Northern blot of ACAT in various mouse tissues. Total RNA was isolated from the tissues shown as described under ``Experimental Procedures.'' 10 µg of total RNA from each tissue was probed with a 1.6-kb cDNA probe corresponding to the entire coding region of mouse ACAT. The upper panels show the results of a 4-day exposure to Kodak X-AR film; the bottom panels show the ethidium bromide-stained gels prior to transfer. The positions of RNA molecular weight markers are shown on the right. b, RNase protection assay for ACAT in various mouse tissues. 5 µg of total RNA from each mouse tissue shown was incubated with a P-labeled antisense RNA probe against mouse ACAT as described under ``Experimental Procedures,'' and protected fragments were separated by gel electrophoresis on a 6% acrylamide, 8 M urea gel. The results of an 8-h exposure to Kodak BioMax film are shown.

An RNase protection assay was also used to detect and quantitate ACAT mRNA in the various mouse tissues. Fig. 3b shows the results of a representative assay using total RNA from 4-5-week-old C57BL/6J mice. A protected fragment corresponding to the 5` end of the ACAT coding region was detected in every tissue examined. PhosphorImager quantitation of radioactivity in the ACAT protected fragment confirmed that ACAT mRNA was most abundant in preputial gland, ovary, and aorta, followed by adrenal, thymus, testis, and peritoneal macrophages; brain, adipose tissue, heart, and skeletal muscle showed the lowest levels of ACAT mRNA.

Effect of an Atherogenic Diet upon Hepatic and Intestinal ACAT mRNA Expression

Figure 4: Dietary regulation of ACAT mRNA in mouse liver, small intestine, and adrenal. Total RNA from liver and small intestine from each of five mice in each feeding group was isolated and assayed for ACAT mRNA by an RNase protection assay. For adrenal tissue, the glands from all five mice in each diet group were pooled prior to RNA isolation. ACAT mRNA is expressed as the band volume of the ACAT protected fragment normalized to the band volume of the GAPDH protected fragment from each individual sample, with the ACAT/GAPDH value for each tissue from chow-fed mice standardized to 1.00. Liver and intestine data were analyzed using Student's t test: *, p < 0.05;**, p < 0.001.

Effect of an Atherogenic Diet upon Plasma Lipids and Liver Microsomal ACAT Activity

Figure 5: Effect of the HF/HC diet on plasma lipoprotein profiles. Male C57BL/6J mice were fed either a standard chow diet or the HF/HC diet for 3 weeks. Plasma samples were fractionated by FPLC as described under ``Experimental Procedures,'' and total cholesterol (top) and triglycerides (bottom) in each fraction were quantitated by enzymatic assay. The results from one representative mouse on each diet are shown.

Cholesterol esterification activity in chow- and cholesterol-fed mouse liver microsomes was measured to determine whether the increases in hepatic ACAT mRNA observed in mice fed the atherogenic diet resulted in increased liver microsomal ACAT activity. In Experiment 1, ACAT activity in liver microsomes from cholesterol-fed mice increased to over twice the activity in chow-fed mouse liver microsomes (Table 1). A similar trend was observed in Experiment 2, although the increase in microsomal ACAT activity from cholesterol-fed mice was less than that seen in Experiment 1 and did not reach statistical significance (Table 1).

These findings suggest that ACAT activity in the liver is regulated at least in part by ACAT mRNA abundance. However, increasing the supply of cholesterol is also known to increase ACAT activity in liver microsomes from several species(22) . Hence we measured free cholesterol levels in liver microsomes from chow and cholesterol-fed mice and found a significant positive correlation between ACAT activity and free cholesterol in mouse liver microsomes. This was true whether the chow- and cholesterol-fed animals were analyzed separately or together as a group (Fig. 6). These data suggest that an increased cholesterol supply up-regulates ACAT activity in the liver. In contrast, no significant correlation was found between microsomal cholesteryl ester and ACAT activity (data not shown).

Figure 6: Correlation between ACAT activity and microsomal free cholesterol in mouse liver microsomes. ACAT activity and free cholesterol content were measured in liver microsomes from male C57BL/6J mice fed either a standard chow diet (closed circles) or a HF/HC diet (open circles) for 3 weeks as described under ``Experimental Procedures.'' Left and middle panels, linear regression of data from mice on the chow and HF/HC diets, respectively; right panel, linear regression of data from all mice.

Effect of an Atherogenic Diet on ACAT mRNA Expression in Mouse Aorta and Peritoneal Macrophages

Figure 7: Dietary regulation of ACAT mRNA in mouse aorta and peritoneal macrophages. Total RNA was obtained from the aortas of each of five mice in each diet group and assayed for ACAT and GAPDH mRNA using an RNase protection assay. For peritoneal macrophages, cells from all five mice in each diet group were pooled prior to RNA isolation. ACAT mRNA is expressed as the band volume of the ACAT protected fragment normalized to the band volume of the GAPDH protected fragment from each individual sample, with the ACAT/GAPDH value for each tissue from chow-fed mice in each experiment standardized to 1.00. t test analysis of the data using showed no significant difference in aorta ACAT mRNA between the diet treatments (p > 0.05).

Expression of Mouse ACAT cDNA in Transfected CHO Cells

Figure 8: a, Northern blot analysis of CHO cells transfected with mouse ACAT. 10 µg of total RNA from CHO cell lines mACAT1 through mACAT5, AC29, and 25-RA were electrophoresed through agarose, transferred to nylon, and probed with the P-labeled 1.6-kb mouse ACAT cDNA probe as described under ``Experimental Procedures'' (top panel). After exposure to Kodak BioMax film, the blot was stripped and reprobed with the P-labeled 0.3-kb mouse GAPDH probe (bottom panel). b, RNase protection assay of CHO cells transfected with mouse ACAT. 5 µg of total RNA from each cell line was incubated with the P-labeled mouse ACAT and GAPDH antisense RNA probes at 45 °C overnight. Protected fragments were separated by electrophoresis on a 6% acrylamide, 8 M urea gel, which was dried and exposed to Kodak BioMax film overnight.

Regulation of ACAT Activity in CHO Cells Expressing Mouse ACAT

DISCUSSION

ACAT is believed to play an important role in lipoprotein assembly. In vitro studies in cultured HepG2 cells have suggested that the cellular pool of cholesteryl esters drives lipoprotein secretion from these cells(23) , and inhibition of ACAT activity reduces the de novo production of VLDL from monkey (24) and pig (25) liver. In the small intestine, ACAT activity has been implicated in the process of dietary cholesterol absorption and in secretion of the absorbed cholesterol in the form of chylomicrons; ACAT inhibition in the rat (26) and hamster (27) blocked cholesteryl ester secretion in chylomicrons from the intestine.

ACAT also mediates lipid droplet accumulation in macrophages and smooth muscle cells in atheromatous lesions, as its activity is increased in atherosclerotic arterial tissue and in normal tissue which has been preincubated with lipoproteins(28) . The mouse ACAT clone reported here provides us with a unique opportunity to examine ACAT's contribution to each of these processes in a widely used animal model, the atherosclerosis-susceptible C57BL/6J mouse.

The mouse ACAT is predicted to contain 540 residues, 10 residues shorter than human ACAT (Fig. 1a). The mouse ACAT cDNA clone we have isolated is a true homolog of the human ACAT clone previously reported (3) because they show high sequence similarity and the genes are both located on chromosome 1 in a region known to be syntenic between human and mouse (Fig. 2). The major divergence between the two species occurs mainly in the extremely N-terminal region; in this part of the enzyme there are two deletions of six and four amino acids in the mouse sequence, and there are 12 substitutions in the first 35 residues of the mouse enzyme. There is much higher homology in the rest of the molecule. The two enzymes also share a number of putative functional sequence motifs, which include the so-called signature sequences characteristic of enzymes with acyltransferase activities, two transmembrane domains and two potential N-linked glycosylation sites. Like the human enzyme, mouse ACAT contains heptad leucine repeat sequences close to the N terminus (between residues 37 and 84). Such sequences are thought to mediate polypeptide dimerization(15) . The fact that the functional unit size of rat ACAT has been estimated to be between 170 and 210 kDa(29, 30) and the existence of these heptad leucine repeats suggest that mouse ACAT may exist in some form of homo- or heteromultimer.

The isolation of mouse ACAT cDNA enabled us to examine the tissue distribution of ACAT mRNA expression. Our Northern results using total RNA isolated from mouse tissues show that one primary mRNA species of approximately 3.9 kb hybridizes to our 1.6-kb mouse ACAT probe and is detectable in almost every tissue. The mRNA is fairly abundant in the ovary and adrenal, consistent with ACAT's role in the preservation of a sterol ester pool for steroid hormone biosynthesis and with its observed activity in these tissues from other species (31, 32, 33) . It is interesting that the mouse testis contains less ACAT mRNA than the other steroidogenic tissues and may explain the previous observation that, in contrast to rat adrenal and ovarian cells, mouse Leydig tumor cells contain little of their total cholesterol mass as cholesteryl esters(34) . ACAT mRNA was also detected in tissues with a significant resident population of macrophage-like cells (lung, spleen, and thymus) as well as in peritoneal macrophages, which have been well characterized with respect to activation of ACAT, intracellular accumulation of cholesteryl esters, and transformation into foam cells (for a review, see (35) ). The presence of ACAT mRNA in mouse aorta supports the previous observations of ACAT activity in this tissue in rats (36, 37) and further implies a role for this protein in the process of atherogenesis.

In tissues that express high amounts of ACAT, another major species of approximately 9.5 kb in length was detected by Northern hybridization to total RNA. This is reminiscent of the mRNA pattern found in Northern blots of human (3) and rabbit (38) poly(A) RNA, in which up to six major species were detected. Additionally, Northern analysis of RNA from human monocytes showed the presence of multiple ACAT mRNA species which were differentially regulated upon differentiation of these cells to macrophages(39) . However, only the 3.9-kb band was detected in most of the tissues with detectable ACAT expression, and this size approximates the length of our cDNA clone (3.7 kb not including the poly(A) tail).

One interesting feature of the ACAT tissue distribution is the high level of ACAT mRNA in the mouse preputial gland, which consistently displayed 3-5-fold more ACAT mRNA than adrenal and ovary. This tissue is a sebaceous structure that has been widely used as an experimental system to study cell differentiation. While most of the lipids associated with fully differentiated sebocytes are identified as waxes, the sterol ester content of these cells also increases as they mature(40) . Thus, increased ACAT activity may contribute to the sebocyte maturation process. Interestingly, increased metabolism of testosterone in male mouse preputial cells is highly correlated with the degree of sebocyte maturation(41) . Additionally, preputial gland size in both male and female rats is increased by progesterone, while estrogen stimulates lipid synthesis, including that of sterol esters, in female rat preputial gland cells(42) . At this point we do not know how steroid hormone administration affects mouse ACAT mRNA levels in vivo, but progesterone has been shown to inhibit ACAT activity in cultured fibroblasts(43) , rabbit ovary(44) , and rat liver microsomes(14, 45) . Thus it is possible that ACAT expression is steroid hormone-responsive in vivo and that hormonal regulation of ACAT is involved in the development of certain specialized cells such as the sebocyte.

Another interesting observation is the relative scarcity of ACAT mRNA in liver. Both rabbit (38) and human (3) liver express ACAT mRNA to a lesser extent than other tissues studied, and liver is one of the tissues in which ACAT is the least abundant in the mouse as well. This observation is not limited to the C57BL/6J strain, as similar results were obtained using tissues isolated from FVB mice (data not shown). ACAT expression may be limited to a subpopulation of hepatic cells; Pape and co-workers (38) showed that ACAT mRNA in rabbit liver is approximately 30-fold enriched in nonparenchymal cells, which comprise about 35% of the total hepatic cell population and include macrophage-like Kupffer cells. Although we did not do a similar separation of the total mouse liver cell population, the presence of substantial amounts of ACAT mRNA in peritoneal macrophages and macrophage-rich tissues such as lung, spleen, and thymus suggests that liver macrophages may account for the bulk of hepatic ACAT mRNA in the mouse as well.

A striking difference between mice and rabbits fed a high-fat, high-cholesterol diet is the response of plasma lipids to the dietary challenge. Total plasma cholesterol in cholesterol-fed rabbits rose to 26 times the chow-fed level(38) , compared to two to three times in the mice used in our experiments. Similarly, rabbit liver microsomal ACAT activity increased 17-fold, compared to 1.4-fold or less for mouse microsomal ACAT activity. Despite these widely different responses, cholesterol feeding resulted in similar responses of ACAT mRNA levels in mouse and rabbit tissues. Feeding rabbits a high-fat, high-cholesterol diet for 8 weeks increased ACAT mRNA about 2-fold in liver with no effect in the adrenal or small intestine(38) ; in our studies, hepatic ACAT mRNA increased 2-3-fold in mice fed the HF/HC diet, with adrenal and small intestinal ACAT mRNA showing no change or a slight decrease, respectively. However, ACAT mRNA also increased roughly 3-fold in the rabbit aorta in response to fat and cholesterol feeding, whereas we were only able to detect a modest, statistically nonsignificant increase in mouse aortic ACAT mRNA following administration of an atherogenic diet for 3 weeks. It is possible that a longer feeding period would induce greater accumulation of ACAT mRNA in mouse aorta and macrophages, although 3 weeks is sufficient to decrease high density lipoprotein levels (18) and increase total plasma cholesterol ( Table 1and (18) ) and VLDL ( Fig. 5and (18) ) levels in C57BL/6J mice, and by five weeks on an atherogenic diet, aortic lesions are already visible by light microsocopy(46) .

In order to address the role of cholesterol availability in the regulation of ACAT activity, two stable cell lines expressing mouse ACAT mRNA under the control of a consitutively expressed cytomegalovirus promoter were generated from an ACAT-deficient CHO cell line. These transfectants (mACAT1 and mACAT3) regained the ability to esterify cholesterol (Table 2), and this activity was up-regulated in both transfected cell lines by the addition of LDL to the culture medium (Table 3). The observation that cholesterol esterification is increased by nearly 200% in mACAT1 and 100% in mACAT3 in a system in which ACAT cannot be transcriptionally regulated indicates that the LDL-induced up-regulation of mouse ACAT activity in the transfected CHO cells occurs at the post-transcriptional level. This is supported by our in vivo studies in mouse liver microsomes, in which ACAT activity was highly correlated with microsomal free cholesterol content, suggesting that cholesterol availability is a major mode of regulation. However, we also observed that hepatic ACAT mRNA increased coordinately with liver microsomal ACAT activity upon feeding mice a high-fat, high-cholesterol diet. Thus we have used both in vivo and in vitro techniques to demonstrate that mouse ACAT activity is regulated in at least two ways: at the level of mRNA accumulation, and at the level of enzyme activity, most likely by cholesterol availability.

FOOTNOTES

*

This work was supported in part by Grants HL-16512 and HL-27341 (to L. C.) and HL-36709 (to T. Y. C.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L21934 [GenBank]and L42293[GenBank].

§

Supported by National Research Service Awards Grant HL-08827.

¶

To whom correspondence should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

()

The abbreviations used are: ACAT, acyl-coenzyme A:cholesterol acyltransferase; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HF/HC, high-fat, high-cholesterol; FPLC, fast protein liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; CHO, Chinese hamster ovary; kb, kilobase pair(s).

ACKNOWLEDGEMENTS

REFERENCES

1. Suckling, K. E., and Stange, E. F. (1985) J. Lipid Res. 26, 647-671 [Medline] [Order article via Infotrieve]
2. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47 [Free Full Text]
3. Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. (1993) J. Biol. Chem. 268, 20747-20755 [Abstract/Free Full Text]
4. Babbitt, P. C., Kenyon, G. L., Martin, B. M., Charest, H., Slyvestre, M., Scholten, J. D., Chang, K.-H., Liang, P.-H., and Dunaway-Mariano, D. (1992) Biochemistry 31, 5594-5604 [CrossRef][Medline] [Order article via Infotrieve]
5. Cheng, D., Chang, C. C. Y., Qu, X., and Chang, T.-Y. (1995) J. Biol. Chem. 270, 685-695 [Abstract/Free Full Text]
6. Paigen, B., Morrow, A., Brandon, C., Mitchell, D., and Holmes, P. (1985) Atherosclerosis 57, 65-73 [CrossRef][Medline] [Order article via Infotrieve]
7. GCG Sequence Analysis Software Package, Version 8.0 (1994) Genetics Computer Group, Inc., Madison, WI
8. Rowe, L. B., Nadeau, J. H., Turner, R., Frankel, W. N., Letts, V. A., Eppig, J. T., Ko, M. S. H., Thurston, S. J., and Birkenmeier, E. H. (1994) Mammalian Genome 5, 253-274
9. Jiao, S., Cole, T. G., Kitchens, R. T., Pfleger, B., and Schonfeld, G. (1990) Metabolism 39, 155-160 [CrossRef][Medline] [Order article via Infotrieve]
10. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
11. Cadigan, K. M., Heider, J. G., and Chang, T.-Y. (1988) J. Biol. Chem. 263, 274-282 [Abstract/Free Full Text]
12. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260 [Medline] [Order article via Infotrieve]
13. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [CrossRef][Medline] [Order article via Infotrieve]
14. Erickson, S. K., Shrewsbury, M. A., Brooks, C., and Meyer, D. J. (1980) J. Lipid Res. 21, 930-941 [Abstract]
15. Landschulz, W. H., Johnson, P. F., and McKnight, S. L. (1988) Science 240, 1759-1764 [Abstract/Free Full Text]
16. Seldin, M. F., Morse, H. C., LeBoeuf, R. C., and Steinberg, A. D. (1988) Genomics 2, 48-56 [CrossRef][Medline] [Order article via Infotrieve]
17. Lusis, A. J., Taylor, B. A., Wangenstein, R. W., and LeBoeuf, R. C. (1983) J. Biol. Chem. 258, 5071-5078 [Abstract/Free Full Text]
18. Paigen, B., Mitchell, D., Reue, K., Morrow, A., Lusis, A. J., Taylor, B. A., and LeBoeuf, R. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3763-3767 [Abstract/Free Full Text]
19. Paigen, B., Albee, D., Holmes, P. A., and Mitchell, D. (1987) Biochem. Genet. 25, 501-511 [CrossRef][Medline] [Order article via Infotrieve]
20. Chang, C. C. Y., Noll, W. W., Nutile-McMenemy, N., Lindsay, E. A., Baldini, A., Chang, W., and Chang, T. Y. (1994) Som. Cell Mol. Genet. 20, 71-74 [CrossRef][Medline] [Order article via Infotrieve]
21. Lusis, A. J. (1993) Trends Cardiovasc. Med. 3, 135-143
22. Chang, T.-Y., and Doolittle, G. M. (1983) in The Enzymes (Boyer, P., ed) 3rd Ed., Vol. 16, pp. 523-539, Academic Press, New York
23. Cianflone, K. M., Yasruel, Z., Rodriguez, M. A., Vas, D., and Sniderman, A. D. (1990) J. Lipid Res. 31, 2045-2055 [Abstract]
24. Carr, T. P., and Rudel, L. L. (1990) Arteriosclerosis 10, 823a
25. Huff, M. W., Telford, D. E., Barrett, P. H. R., Billheimer, J. T., and Gillies, P. J. (1994) Arterioscler. Thromb. 14, 1498-1508 [Abstract/Free Full Text]
26. Krause, B. R., Anderson, M., Bisgaier, C. L., Bocan, T., Bousley, R., DeHart, P., Essenburg, A., Hamelehle, K., Homan, R., Kieft, K., McNally, W., Stanfield, R., and Newton, R. S. (1993) J. Lipid Res. 34, 279-294 [Abstract]
27. Burrier, R. E., Smith, A. A., McGregor, D. G., Hoos, L. M., Zilli, D. L., and Davis, H. R. J. (1995) J. Pharmacol. Exp. Ther. 272, 156-163 [Abstract/Free Full Text]
28. Hashimoto, S., and Dayton, S. (1977) Atherosclerosis 28, 447-452 [CrossRef][Medline] [Order article via Infotrieve]
29. Billheimer, J. T., Cromley, D. A., and Kempner, E. S. (1990) J. Biol. Chem. 265, 8632-8635 [Abstract/Free Full Text]
30. Erickson, S. K., Lear, S. R., and McCreery, M. J. (1994) J. Lipid Res. 35, 763-769 [Abstract]
31. Tavani, D. M., Tanaka, T., Strauss, J. F. I., and Billheimer, J. T. (1982) Endocrinology 111, 794-800 [Abstract/Free Full Text]
32. Beins, D. M., Vining, R., and Balasubramanian, S. (1982) Biochem. J. 202, 631-637 [Medline] [Order article via Infotrieve]
33. Suckling, K. E., Tocher, D. R., Smellie, C. G., and Boyd, G. S. (1983) Biochim. Biophys. Acta 753, 422-429 [Medline] [Order article via Infotrieve]
34. Freeman, D. A., and Ascoli, M. (1982) J. Biol. Chem. 257, 14231-14238 [Abstract/Free Full Text]
35. Brown, M. S., and Goldstein, J. L. (1983) Ann. Rev. Biochem. 52, 223-261 [CrossRef][Medline] [Order article via Infotrieve]
36. Hashimoto, S., and Dayton, S. (1974) Proc. Soc. Exp. Biol. Med. 145, 89-92 [CrossRef][Medline] [Order article via Infotrieve]
37. Brecher, P. I., and Chobanian, A. V. (1974) Circ. Res. 35, 692-701 [Abstract/Free Full Text]
38. Pape, M. E., Schultz, P. A., Rea, T. J., DeMattos, R. B., Kieft, K., Bisgaier, C. L., Newton, R. S., and Krause, B. R. (1995) J. Lipid Res. 36, 823-838 [Abstract]
39. Wang, H., Benfield, P. A., Germain, S. J., and Gillies, P. J. (1994) Circulation 90, I-351
40. Wheatley, V. R., Potter, J. E. R., and Lew, G. (1979) J. Invest. Dermatol. 73, 291-296 [CrossRef][Medline] [Order article via Infotrieve]
41. Brind, J. L., Marinescu, D., Gomez, E. C., Wheatley, V. R., and Orentreich, N. (1984) J. Endocr. 100, 377-388 [Abstract/Free Full Text]
42. Alves, A. M., Thody, A. J., Fisher, C., and Shuster, S. (1986) J. Endocr. 109, 1-7 [Abstract/Free Full Text]
43. Goldstein, J. L., Faust, J. R., Dygos, J. H., Chorvat, R. J., and Brown, M. S. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1877-1881 [Abstract/Free Full Text]
44. Flint, A. P., Grinwich, D. L., and Armstrong, D. T. (1973) Biochem. J. 132, 313-321 [Medline] [Order article via Infotrieve]
45. Lichtenstein, A. H., and Brecher, P. (1980) J. Biol. Chem. 255, 9098-9104 [Free Full Text]
46. Stewart-Phillips, J. L., and Lough, J. (1991) Atherosclerosis 90, 211-218 [CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. R. Turkish and S. L. Sturley The genetics of neutral lipid biosynthesis: an evolutionary perspective Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E19 - E27. [Abstract] [Full Text] [PDF]

				S. N. Mathur, K. R. Watt, and F. J. Field Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid J. Lipid Res., February 1, 2007; 48(2): 395 - 404. [Abstract] [Full Text] [PDF]

				C.-L. E. Yen, C. H. Brown IV, M. Monetti, and R. V. Farese Jr. A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters J. Lipid Res., November 1, 2005; 46(11): 2388 - 2397. [Abstract] [Full Text] [PDF]

				C. Pramfalk, M. A. Davis, M. Eriksson, L. L. Rudel, and P. Parini Control of ACAT2 liver expression by HNF1 J. Lipid Res., September 1, 2005; 46(9): 1868 - 1876. [Abstract] [Full Text] [PDF]

				N. Rotllan, V. Ribas, L. Calpe-Berdiel, J. M. Martin-Campos, F. Blanco-Vaca, and J. C. Escola-Gil Overexpression of Human Apolipoprotein A-II in Transgenic Mice Does Not Impair Macrophage-Specific Reverse Cholesterol Transport In Vivo Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): e128 - e132. [Abstract] [Full Text] [PDF]

				J.-Y. Lee and T. P. Carr Dietary Fatty Acids Regulate Acyl-CoA:Cholesterol Acyltransferase and Cytosolic Cholesteryl Ester Hydrolase in Hamsters J. Nutr., December 1, 2004; 134(12): 3239 - 3244. [Abstract] [Full Text] [PDF]

				I. Namatame, H. Tomoda, S. Ishibashi, and S. Omura Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages PNAS, January 20, 2004; 101(3): 737 - 742. [Abstract] [Full Text] [PDF]

				G. Dagher, N. Donne, C. Klein, P. Ferre, and I. Dugail HDL-mediated cholesterol uptake and targeting to lipid droplets in adipocytes J. Lipid Res., October 1, 2003; 44(10): 1811 - 1820. [Abstract] [Full Text] [PDF]

				E. L. Willner, B. Tow, K. K. Buhman, M. Wilson, D. A. Sanan, L. L. Rudel, and R. V. Farese Jr. Deficiency of acyl CoA:cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice PNAS, February 4, 2003; 100(3): 1262 - 1267. [Abstract] [Full Text] [PDF]

				L. O'Rourke, L. M. Gronning, S. J. Yeaman, and P. R. Shepherd Glucose-dependent Regulation of Cholesterol Ester Metabolism in Macrophages by Insulin and Leptin J. Biol. Chem., November 1, 2002; 277(45): 42557 - 42562. [Abstract] [Full Text] [PDF]

				K. Liang and N. D. Vaziri Upregulation of acyl-CoA:cholesterol acyltransferase in chronic renal failure Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E676 - E681. [Abstract] [Full Text] [PDF]

				C. Xie, L. A. Woollett, S. D. Turley, and J. M. Dietschy Fatty acids differentially regulate hepatic cholesteryl ester formation and incorporation into lipoproteins in the liver of the mouse J. Lipid Res., September 1, 2002; 43(9): 1508 - 1519. [Abstract] [Full Text] [PDF]

				K. Kaneko, K. Uchida, T. Kobayashi, K. Miura, K. Tanokura, K. Hoshino, I. Kato, M. Onoue, and T. Yokokura Sex-Dependent Toxicity of a Novel Acyl-CoA:Cholesterol Acyltransferase Inhibitor, YIC-C8-434, in Relation to Sex-Specific Forms of Cytochrome P450 in Rats Toxicol. Sci., December 1, 2001; 64(2): 259 - 268. [Abstract] [Full Text] [PDF]

				W. M. Pandak, C. Schwarz, P. B. Hylemon, D. Mallonee, K. Valerie, D. M. Heuman, R. A. Fisher, K. Redford, and Z. R. Vlahcevic Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G878 - G889. [Abstract] [Full Text] [PDF]

				K. Jensen-Pergakes, Z. Guo, M. Giattina, S. L. Sturley, and M. Bard Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae J. Bacteriol., September 1, 2001; 183(17): 4950 - 4957. [Abstract] [Full Text] [PDF]

				K. K. Maung, A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T.-Y. Chang, and S. Horiuchi Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D3 or 9-cis-retinoic acid in undifferentiated THP-1 cells J. Lipid Res., February 1, 2001; 42(2): 181 - 187. [Abstract] [Full Text]

				L. Li and H. J. Pownall Regulation of Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) Synthesis, Degradation, and Translocation by High-Density Lipoprotein2 at a Low Concentration Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2636 - 2642. [Abstract] [Full Text] [PDF]

				C. G. Panousis and S. H. Zuckerman Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-{gamma} J. Lipid Res., January 1, 2000; 41(1): 75 - 83. [Abstract] [Full Text]

				J. Kusunoki, K. Aragane, T. Kitamine, H. Kozono, K. Kano, K. Fujinami, K. Kojima, T. Chiwata, and Y. Sekine Postprandial Hyperlipidemia in Streptozotocin-Induced Diabetic Rats Is Due to Abnormal Increase in Intestinal Acyl Coenzyme A:Cholesterol Acyltransferase Activity Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 171 - 178. [Abstract] [Full Text] [PDF]

				G. J. Schroepfer Jr. Oxysterols: Modulators of Cholesterol Metabolism and Other Processes Physiol Rev, January 1, 2000; 80(1): 361 - 554. [Abstract] [Full Text] [PDF]

				N. Sakashita, A. Miyazaki, M. Takeya, S. Horiuchi, C. C. Y. Chang, T.-Y. Chang, and K. Takahashi Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues Am. J. Pathol., January 1, 2000; 156(1): 227 - 236. [Abstract] [Full Text] [PDF]

				B.-L. Li, X.-L. Li, Z.-J. Duan, O. Lee, S. Lin, Z.-M. Ma, C. C. Y. Chang, X.-Y. Yang, J. P. Park, T. K. Mohandas, et al. Human Acyl-CoA:Cholesterol Acyltransferase-1 (ACAT-1) Gene Organization and Evidence That the 4.3-Kilobase ACAT-1 mRNA Is Produced from Two Different Chromosomes J. Biol. Chem., April 16, 1999; 274(16): 11060 - 11071. [Abstract] [Full Text] [PDF]

				J.-L. Escary, H. A. Choy, K. Reue, X.-P. Wang, L. W. Castellani, C. K. Glass, A. J. Lusis, and M. C. Schotz Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages J. Lipid Res., March 1, 1999; 40(3): 397 - 404. [Abstract] [Full Text]

				S. Cases, S. J. Smith, Y.-W. Zheng, H. M. Myers, S. R. Lear, E. Sande, S. Novak, C. Collins, C. B. Welch, A. J. Lusis, et al. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis PNAS, October 27, 1998; 95(22): 13018 - 13023. [Abstract] [Full Text] [PDF]

				R. A. Anderson, C. Joyce, M. Davis, J. W. Reagan, M. Clark, G. S. Shelness, and L. L. Rudel Identification of a Form of Acyl-CoA:Cholesterol Acyltransferase Specific to Liver and Intestine in Nonhuman Primates J. Biol. Chem., October 9, 1998; 273(41): 26747 - 26754. [Abstract] [Full Text] [PDF]

				S. Cases, S. Novak, Y.-W. Zheng, H. M. Myers, S. R. Lear, E. Sande, C. B. Welch, A. J. Lusis, T. A. Spencer, B. R. Krause, et al. ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol Acyltransferase. ITS CLONING, EXPRESSION, AND CHARACTERIZATION J. Biol. Chem., October 9, 1998; 273(41): 26755 - 26764. [Abstract] [Full Text] [PDF]

				P. Oelkers, A. Behari, D. Cromley, J. T. Billheimer, and S. L. Sturley Characterization of Two Human Genes Encoding Acyl Coenzyme A:Cholesterol Acyltransferase-related Enzymes J. Biol. Chem., October 9, 1998; 273(41): 26765 - 26771. [Abstract] [Full Text] [PDF]

				A. Miyazaki, N. Sakashita, O. Lee, K. Takahashi, S. Horiuchi, H. Hakamata, P. M. Morganelli, C. C. Y. Chang, and T.-Y. Chang Expression of ACAT-1 Protein in Human Atherosclerotic Lesions and Cultured Human Monocytes-Macrophages Arterioscler. Thromb. Vasc. Biol., October 1, 1998; 18(10): 1568 - 1574. [Abstract] [Full Text] [PDF]

				V. L. Meiner, C. L. Welch, S. Cases, H. M. Myers, E. Sande, A. J. Lusis, and R. V. Farese Jr. Adrenocortical Lipid Depletion Gene (ald) in AKR Mice Is Associated with an Acyl-CoA:Cholesterol Acyltransferase (ACAT) Mutation J. Biol. Chem., January 9, 1998; 273(2): 1064 - 1069. [Abstract] [Full Text] [PDF]

				J. R. Burnett, L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff Inhibition of HMG-CoA Reductase by Atorvastatin Decreases Both VLDL and LDL Apolipoprotein B Production in Miniature Pigs Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2589 - 2600. [Abstract] [Full Text]

				Y. Lange and T. L. Steck Quantitation of the Pool of Cholesterol Associated with Acyl-CoA:Cholesterol Acyltransferase in Human Fibroblasts J. Biol. Chem., May 16, 1997; 272(20): 13103 - 13108. [Abstract] [Full Text] [PDF]

				E. I. Park, E. A. Paisley, H. J. Mangian, D. A. Swartz, M. Wu, P. J. O'Morchoe, S. R. Behr, W. J. Visek, and J. Kaput Lipid Level and Type Alter Stearoyl CoA Desaturase mRNA Abundance Differently in Mice with Distinct Susceptibilities to Diet-Influenced Diseases J. Nutr., April 1, 1997; 127(4): 566 - 573. [Abstract] [Full Text]

				H. Yang, D. Cromley, H. Wang, J. T. Billheimer, and S. L. Sturley Functional Expression of a cDNA to Human Acyl-coenzyme A:Cholesterol Acyltransferase in Yeast. SPECIES-DEPENDENT SUBSTRATE SPECIFICITY AND INHIBITOR SENSITIVITY J. Biol. Chem., February 14, 1997; 272(7): 3980 - 3985. [Abstract] [Full Text] [PDF]

				V. L. Meiner, S. Cases, H. M. Myers, E. R. Sande, S. Bellosta, M. Schambelan, R. E. Pitas, J. McGuire, J. Herz, and R. V. Farese Jr. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals PNAS, November 26, 1996; 93(24): 14041 - 14046. [Abstract] [Full Text] [PDF]

				C. Yu, N. J. Kennedy, C. C.Y. Chang, and J. A. Rothblatt Molecular Cloning and Characterization of Two Isoforms of Saccharomyces cerevisiae Acyl-CoA:Sterol Acyltransferase J. Biol. Chem., September 27, 1996; 271(39): 24157 - 24163. [Abstract] [Full Text] [PDF]

				G. Cao, J. L. Goldstein, and M. S. Brown Complementation of Mutation in Acyl-CoA:Cholesterol Acyltransferase (ACAT) Fails to Restore Sterol Regulation in ACAT-defective Sterol-resistant Hamster Cells J. Biol. Chem., June 14, 1996; 271(24): 14642 - 14648. [Abstract] [Full Text] [PDF]

				H. Yagyu, T. Kitamine, J.-i. Osuga, R.-i. Tozawa, Z. Chen, Y. Kaji, T. Oka, S. Perrey, Y. Tamura, K. Ohashi, et al. Absence of ACAT-1 Attenuates Atherosclerosis but Causes Dry Eye and Cutaneous Xanthomatosis in Mice with Congenital Hyperlipidemia J. Biol. Chem., July 7, 2000; 275(28): 21324 - 21330. [Abstract] [Full Text] [PDF]

				C. C. Y. Chang, N. Sakashita, K. Ornvold, O. Lee, E. T. Chang, R. Dong, S. Lin, C.-Y. G. Lee, S. C. Strom, R. Kashyap, et al. Immunological Quantitation and Localization of ACAT-1 and ACAT-2 in Human Liver and Small Intestine J. Biol. Chem., September 1, 2000; 275(36): 28083 - 28092. [Abstract] [Full Text] [PDF]

				D. K. Spady, M. N. Willard, and R. S. Meidell Role of Acyl-Coenzyme A:Cholesterol Acyltransferase-1 in the Control of Hepatic Very Low Density Lipoprotein Secretion and Low Density Lipoprotein Receptor Expression in the Mouse and Hamster J. Biol. Chem., August 25, 2000; 275(35): 27005 - 27012. [Abstract] [Full Text] [PDF]

				S. Sonda, L.-M. Ting, S. Novak, K. Kim, J. J. Maher, R. V. Farese Jr., and J. D. Ernst Cholesterol Esterification by Host and Parasite Is Essential for Optimal Proliferation of Toxoplasma gondii J. Biol. Chem., September 7, 2001; 276(37): 34434 - 34440. [Abstract] [Full Text] [PDF]

				K. K. Buhman, H. C. Chen, and R. V. Farese Jr. The Enzymes of Neutral Lipid Synthesis J. Biol. Chem., October 26, 2001; 276(44): 40369 - 40372. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Uelmen, P. J. Articles by Chan, L. Search for Related Content PubMed PubMed Citation Articles by Uelmen, P. J. Articles by Chan, L. Social Bookmarking                      What's this?