©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Expression and Cholesterol Regulation of Acylcoenzyme A:Cholesterol Acyltransferase (ACAT) in Mice
MOLECULAR CLONING OF MOUSE ACAT cDNA, CHROMOSOMAL LOCALIZATION, AND REGULATION OF ACAT IN VIVO AND IN VITRO(*)

(Received for publication, July 7, 1995)

Patricia J. Uelmen (1)(§) Kazuhiro Oka (1) Merry Sullivan (1) Catherine C. Y. Chang (2) Ta Yuan Chang (2) Lawrence Chan (1)(¶)

From the  (1)Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030 and the (2)Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the esterification of cholesterol with long chain fatty acids and is believed to play an important part in the development of atherosclerotic lesions. To facilitate the study of ACAT's role in this process, we have used the human ACAT K1 clone previously described (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y.(1993) J. Biol. Chem. 268, 20747-20755) to isolate mouse ACAT cDNA from a liver cDNA library. The 3.7-kilobase cDNA clone isolated contains a 1620-base pair open reading frame which encodes a protein of 540 amino acids. The predicted mouse ACAT protein is 87% identical to the protein product of human ACAT K1 and shares many of the same secondary structural features, including two transmembrane domains, a leucine heptad motif consistent with dimer or multimer formation, and five regions homologous to the ``signature sequences'' found in other enzymes that catalyze acyl adenylation followed by acyl thioester formation and acyl transfer. Using the cDNA as a hybridization probe, we mapped the gene encoding mouse ACAT to chromosome 1 in a region syntenic to human chromosome 1 where the ACAT gene is located. Northern blot analysis and RNase protection assays of mouse tissues revealed that ACAT mRNA is expressed most highly in the adrenal gland, ovary, and preputial gland and is least abundant in skeletal muscle, adipose tissue, heart, and brain. To study the dietary regulation of ACAT mRNA expression in mouse tissues, we fed C57BL/6J mice a high-fat, high-cholesterol (HF/HC) atherogenic diet for 3 weeks and measured ACAT mRNA levels in various tissues by RNase protection. The HF/HC diet had little effect on ACAT mRNA levels in the small intestine, aorta, adrenal, or peritoneal macrophages, whereas hepatic ACAT mRNA levels were doubled in mice fed the atherogenic diet. ACAT activity in liver microsomes was similarly increased in cholesterol-fed mice, suggesting that mouse ACAT is regulated at least in part at the level of mRNA abundance. Additionally, a significant positive correlation was observed between ACAT activity and microsomal free cholesterol levels in chow- and cholesterol-fed mice, supporting the concept of cholesterol availability as a regulator of ACAT. To further investigate the regulation of ACAT activity under controlled conditions, ACAT-deficient Chinese hamster ovary cells were stably transfected with the mouse ACAT cDNA clone driven by a cytomegalovirus promoter. Two transfected Chinese hamster ovary cell lines that expressed the mouse ACAT transgene regained the ability to esterify cholesterol. Cholesterol esterification activity in both of these cell lines was further increased by exposure of these cells to low density lipoprotein. Thus we have demonstrated that mouse ACAT expression in vivo and in vitro is regulated by at least two mechanisms: control of mRNA abundance and post-transcriptional regulation of the enzyme activity, probably by cholesterol availability.


INTRODUCTION

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) (^1)(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

Fatty acid-free bovine serum albumin, bovine serum albumin standard solution, oleoyl-coenzyme A, cholesteryl oleate, oleic acid, 25-hydroxycholesterol, human low density lipoprotein, phenylmethylsulfonyl fluoride, and total cholesterol (#352) and triglyceride INT (#336) assay kits were all obtained from Sigma. Boehringer Mannheim cholesterol assay kit number 139 050 was used for free cholesterol measurements. Restriction enzymes and buffers, culture medium, penicillin/streptomycin, and G418 were obtained from Life Technologies, Inc./BRL. Trypsin/EDTA and glutamine were obtained from Mediatech. Fetal bovine serum was purchased from HyClone; lipoprotein-deficient bovine calf serum was obtained from Biomedical Technologies Inc. (Stoughton, MA). [^14C]Oleoyl-coenzyme A (50-60 mCi/mmol), [alpha-P]dCTP (3000 Ci/mmol), and [alpha-P]UTP (800 Ci/mmol) were obtained from Amersham. [^3H]Cholesteryl oleate (60-100 Ci/mmol) and [^3H]oleic acid (2-10 Ci/mmol) were obtained from DuPont NEN. Reagent grade solvents for RNA and lipid extraction and thin-layer chromatography and all other reagents were obtained from Fisher.

Cloning of Mouse ACAT

The human ACAT cDNA clone K1 previously described (3) was digested with SalI and HindIII to yield a fragment of 1.7 kb containing the entire coding region of human ACAT. This fragment was labeled with [alpha-P]dCTP by random priming (Megaprime labeling kit, Amersham) and used to screen a ZAP mouse liver cDNA library (Stratagene). The nucleotide sequence of this clone was determined on both strands by the dideoxy chain termination method using Sequenase version 2.0 (U. S. Biochemical Corp.).

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

Nylon membranes containing a panel of EcoRI-digested genomic DNA from 94 (C57BL/6J X SPRET/Ei)F(1) X SPRET/Ei backcrossed mice were obtained from the Jackson Laboratory Backcross Panel Service (Bar Harbor, ME)(8) . To probe these blots for ACAT, the mouse ACAT cDNA clone described above was digested with EcoRI and SstII to yield a 1.6-kb fragment containing virtually all of the coding region. This fragment was labeled with [alpha-P]dCTP by random priming and incubated with the blots overnight at 65 °C according to the protocol recommended by the Jackson Laboratory. Bands were visualized by exposure to Kodak X-AR film.

Animals and Diets

The C57BL/6J mice used in these experiments were obtained from the Jackson Laboratory and maintained on a 12-h light/dark cycle and had free access to food and water. Each diet group for each experiment consisted of five age-matched male mice, aged 4-5 weeks at the start of the study, fed either a standard chow diet (Purina) or an atherogenic high-fat, high-cholesterol (HF/HC) diet consisting of a chow diet to which was added 1.25% cholesterol, 0.5% cholic acid, and 11% fat (primarily as cocoa butter; Teklad) for 3 weeks. Mice were fasted for 4 h prior to blood sampling and tissue isolation.

Plasma Lipoprotein Profiles of Chow and Fat-fed Mice

For determination of plasma lipoprotein profiles, mice were anesthetized with an intraperitoneal injection of 0.5 ml of Avertin. 0.5 ml of blood was then removed via the tail vein into tubes containing EDTA in phosphate-buffered saline (pH 7.4; final concentration, 6 mM EDTA). After centrifugation to pellet erythrocytes, the plasma was adjusted to 0.05% NaN(3) and 0.015% phenylmethylsulfonyl fluoride. 10 µl of plasma were assayed for total cholesterol and triglycerides using commercially available total cholesterol and triglyceride INT kits (Sigma).

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(3) 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 isolation of tissue RNA, mice were sacrificed by cervical dislocation and the tissues removed and immediately frozen in liquid nitrogen. RNA was prepared by homogenizing frozen tissue in Ultraspec RNA (Biotecx Inc., Houston TX), 1 ml/100 mg of tissue, using a Janke and Kunkel TP 18-10 homogenizer. The homogenates were extracted with chloroform according to the product protocol, and total RNA was precipitated with isopropyl alcohol, rinsed twice with 70% ethanol, dried briefly, and dissolved in diethylpyrocarbonate-treated distilled water. RNA concentrations were determined by measuring absorbance at 260 nm of samples diluted in distilled water.

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^2 Ultraspec RNA followed by brief tituration. The lysates were processed as above.

Northern Blot Analysis

Total RNA was denatured at 65 °C for 5 min in sample loading buffer (50% formamide, 30 mM MOPS, 7.5 mM sodium acetate, pH 7.0, 1.5 mM EDTA, 11.1% formaldehyde, 0.075 mg/ml ethidium bromide, 0.08% (v/v) glycerol; 2 µl of loading buffer added per µg RNA) and electrophoresed through a 1% agarose, 6% formaldehyde gel using 20 mM MOPS, 5 mM sodium acetate, pH 7.0, 1 mM EDTA as the running buffer. The RNA was transferred to Hybond N nylon (Amersham Life Science) and the blots were blocked for 2 h at 65 °C in MegaBlock II (CEL Associates, Inc., Houston, TX), then incubated overnight at 65 °C in MegaBlock II containing the P-labeled 1.6-kb mouse ACAT probe described above. The membranes were then rinsed twice in 0.5 times SSC buffer (1 times SSC, 15 mM sodium citrate, 150 mM NaCl, pH 7.0) containing 0.1% SDS for 5 min at room temperature, then twice with 0.1 times SSC, 0.1% SDS at 65 °C for 15 min. Bands were visualized by exposure to x-ray film at -80 °C with intensifying screens. In some cases, the blots were then stripped in boiling 0.1% SDS for 10 min, blocked as above, and hybridized with a P-labeled cDNA containing 316 bp of the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (pTRI-GAPDH-Mouse, Ambion) as a control.

RNase Protection Assay to Quantitate ACAT mRNA

A 362-bp EcoRI-BglII fragment of mouse ACAT cDNA was ligated into pBluescript II KS (Stratagene) which had been double-digested with EcoRI and BamHI. The resulting plasmid was digested with EcoRI to generate a linearized cDNA template. A 409-nucleotide antisense RNA probe was synthesized from this template by in vitro transcription using T7 RNA polymerase and [alpha-P]UTP according to the manufacturer's instructions (MAXIscript T7/T3, Ambion). A 318-nucleotide probe for mouse GAPDH was similarly synthesized by polymerase chain reaction amplification of the pTRI-GAPDH-Mouse plasmid to generate a 241-bp EcoRI-BamHI fragment, which was then subcloned into pBluescript II KS; the template was then linearized with BamHI and transcribed using T3 RNA polymerase. The RNase protection assay was performed using an RPA II kit (Ambion) according to the product protocol. Briefly, 70,000 cpm of the ACAT RNA probe and 20,000 cpm of the GAPDH probe were added to 5-20 µg of total RNA from mouse tissues or CHO cells, denatured at 90 °C for 5 min, then allowed to hybridize overnight at 45 °C. The probes were also incubated with 20 µg of yeast tRNA as a negative control. The RNA was then digested with a mixture of RNases A and T1 at 37 °C for 30 min. Protected RNA fragments were precipitated and separated on a 6% polyacrylamide, 8.3 M urea gel. The fragments corresponding to ACAT and GAPDH were detected by overnight exposure to x-ray film. The intensities of the bands were quantitated by exposing the gel to a phosphor screen followed by analysis using a PhosphorImager and the ImageQuant software (Molecular Dynamics).

Liver Microsomal ACAT Assays

Microsomes were isolated from fresh mouse livers by homogenizing approximately 0.5 g of tissue in 3 ml of ice-cold Buffer A (50 mM Tris, pH 7.8, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) with five strokes of a Potter-Elvehjem Teflon pestle homogenizer. The homogenates were centrifuged in a Sorvall RC-3B centrifuge at 10,000 times g for 20 min at 4 °C, the supernatants were removed to fresh tubes, and the centrifugation was repeated. The supernatants were then centrifuged in a Sorvall RCM 100 centrifuge at 100,000 times g for 40 min at 4 °C to pellet microsomal membranes. The pellets were resuspended in 3 ml of fresh ice-cold Buffer A with three strokes of a Teflon pestle homogenizer and the centrifugation was repeated. The resulting pellets were resuspended in 1 ml of ice-cold Buffer A and stored at -80 °C until use. Aliquots of each microsome preparation were assayed for protein by the DC Protein Assay method (Bio-Rad, Inc.) using bovine serum albumin as the standard.

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 times 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 [^14C]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 [^3H]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 ^3H/^14C cpm were quantitated using a Beckman LS 8000 scintillation counter.

CHO Cells Transfected with Mouse ACAT

Mouse ACAT cDNA was digested with HindIII and NsiI to yield a 2.15-kb fragment containing the entire coding region plus approximately 500 bp of the 3`-untranslated region. This fragment was then subcloned into the expression vector pcDNAI (Invitrogen) and used to transfect the ACAT-deficient CHO cell mutant line AC29 previously described(11) . Stable transfectants were selected in 400 µg/ml G418 for 2 weeks and isolated using cloning rings. Five cell lines (mACAT1-mACAT5) were selected from the stable transfectants, purified by recloning once, and maintained in Ham's F-12 supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, 1 mM glutamine, and 400 µg/ml G418. AC29 and 25-RA cells were transfected with the pcDNAI vector alone as controls.

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 times 10^6 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 [^3H]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 [^3H]oleate into cholesteryl [^3H]oleate were measured as above, except that 20,000 dpm of [^14C]cholesteryl oleate was added to each sample prior to lipid extraction as the internal standard.


RESULTS

Cloning and Sequence of Mouse ACAT cDNA

Screening of a mouse liver cDNA library with the human ACAT K1 probe yielded one positive clone out of approximately 1.6 times 10^6 plaques screened. The nucleotide sequence of this clone was determined on both strands and revealed that this clone encodes the mouse equivalent of human ACAT (Fig. 1). The mouse clone has an unusually long 5` leader that contains two short open reading frames. The coding region of the cDNA sequence showed an 83.6% identity with the human ACAT cDNA sequence previously reported(3) . The deduced amino acid sequence of mouse ACAT predicts a 540-residue protein with a calculated molecular mass of 64 kDa. The amino acid sequence is 87% identical and 94% similar to its human counterpart. Several structural motifs are conserved between the human and mouse proteins (Fig. 1). The existence of at least two transmembrane alpha-helical domains is predicted by Kyte-Doolittle hydrophobicity analysis(13) , supporting the experimental observation that ACAT is a membrane-associated protein(1, 3, 14) . A leucine heptad motif found in the N-terminal half of the human ACAT protein is conserved in the mouse protein (Fig. 1a, bold type); this sequence may direct dimer formation(15) . Two potential N-glycosylation sites close to the C-terminal end of the polypeptide were identified (Fig. 1a, brackets). The existence of five domains homologous to the ``signature'' sequences found in firefly luciferase, fatty acid ligase, and other enzymes which catalyze acyl transfer reactions (4) suggested that the product of the human K1 clone possesses acyltransferase activity; these regions are largely conserved in the predicted protein product of the mouse ACAT cDNA clone and are indicated in Fig. 1a by the boxed regions. Regions 1 and 5 are 53 and 47% similar, respectively, to firefly luciferase signature sequence 1; region 3 is 50% similar to firefly luciferase signature sequence 2; and regions 2 and 4 are 50 and 43% similar, respectively, to firefly luciferase signature sequence 3.


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

A distinct restriction fragment length polymorphism was observed in genomic DNA from C57BL/6J and Mus spretus mice following EcoRI digestion (Fig. 2a). The segregation of this restriction fragment length polymorphism within 94 backcross mouse genomic DNAs was compared to those of known loci in order to map mouse ACAT (Fig. 2b). This analysis indicated that the gene encoding mouse ACAT is located on the distal end of chromosome 1, in proximity to a region syntenic with human chromosome 1 (16) and containing the apoA-II and Ath-1 loci(17, 18, 19) (Fig. 2c). Human ACAT is also located on chromosome 1 (1q25) (20) , as is human apoA-II (1q21-22)(21) .


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(1) 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

The distribution of ACAT mRNA in mouse tissues was determined by Northern blot analysis. A major band of approximately 3.9 kb was detected in duodenum, jejunum, spleen, kidney, ovary, testis, preputial, resident peritoneal macrophages, lung, aorta, and adrenal (Fig. 3a). A second species of approximately 9.5 kb was also detected in those tissues which express high levels of ACAT mRNA (ovary, preputial, and adrenal).


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

To determine whether ACAT mRNA levels are regulated by cholesterol feeding, ACAT mRNA in liver, small intestine, and adrenal tissue from chow- and cholesterol-fed C57BL/6J mice was quantitated by an RNase protection assay. Hepatic ACAT mRNA was significantly increased in the cholesterol-fed animals relative to the control mice in two separate experiments (Fig. 4). In contrast, intestinal ACAT mRNA was either unchanged (Experiment 1) or decreased (Experiment 2) in mice fed the atherogenic diet. No change in adrenal ACAT mRNA was detected between chow- and cholesterol-fed mice in either experiment.


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

Plasma total cholesterol in cholesterol-fed mice was elevated up to three times the level in chow-fed mice in each of two separate feeding trials (Table 1). This increase was primarily found in the plasma VLDL and LDL fractions (Fig. 5, top). These particles appeared to be enriched in cholesterol at the expense of triglycerides, as VLDL triglycerides were significantly reduced in mice fed the atherogenic diet (Fig. 5, bottom).




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

To investigate the potential role of ACAT up-regulation in the development of atherosclerosis in susceptible C57BL/6J mice, ACAT mRNA in the aortas and resident peritoneal macrophages of cholesterol- and chow-fed mice was measured by an RNase protection assay. In two separate experiments, the average levels of ACAT mRNA in the aortas of cholesterol-fed mice were 30-60% higher than in chow-fed animals, although the increase did not reach statistical significance (Fig. 7). ACAT mRNA in resident peritoneal macrophages isolated from cholesterol-fed mice increased to a similar degree (Fig. 7).


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

Five G418-resistant cell lines were isolated following transfection of CHO mutant AC29 cells with the mouse ACAT-neo construct and selection with G418. Northern blot analysis of total RNA from the cell lines revealed the presence of a band of the expected molecular weight (2.15 kb) in at least one of these lines (mACAT3, Fig. 8a), and an RNase protection assay showed that this line expressed the transgene-specific mRNA at a high level (Fig. 8b). However, the sensitive RNase protection assay also revealed detectable amounts of mouse ACAT mRNA in three of the other lines (Fig. 8b). The rate of cholesteryl ester synthesis was measured in all five transfected cell lines, and in parental AC29 and 25-RA lines transfected with the neo construct alone, by assaying incorporation of [^3H]oleate into cholesteryl ester by intact cells. These measurements showed that mACAT1 and mACAT3, the two lines that had the highest transgene-specific mRNA levels, had regained the ability to esterify cholesterol; the other three lines with no or barely detectable mRNA and the parental AC29 line were essentially devoid of cholesterol esterification activity (Table 2).


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

The ability of exogenous lipoprotein to regulate ACAT activity and mRNA in the transfected CHO cells was examined by incubating CHO cell lines mACAT1, mACAT3, AC29, and 25-RA in the absence or presence of 20 µg/ml human LDL in the culture medium. The rate of cholesteryl ester synthesis in each cell line under both conditions was assessed by measuring incorporation of [^3H]oleate into cholesteryl esters. Cholesterol esterification in mACAT1 and mACAT3 was increased by 173 and 89%, respectively, following incubation with human LDL (Table 3), demonstrating that the mouse ACAT activity expressed in these cells lines is susceptible to up-regulation by LDL cholesterol. This degree of up-regulation was similar to that of the endogenous ACAT activity in 25-RA cells following incubation with human LDL (increase of 138%, Table 3).




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.

(^1)
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

We thank Lucy Rowe and Mary Barter of the Jackson Laboratory Backcross Panel Service for interpreting the BSS panel data and providing the haplotype and chromosome figures, Oliver Tiebel for the preparation of the mouse GAPDH probes used in these studies, Julie Martinez for excellent technical assistance, and Sally Tobola for expert secretarial assistance.


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