©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Acyl-Coenzyme A:Cholesterol Acyltransferase by Cholesterol or by Oxysterol in a Cell-free System (*)

(Received for publication, August 19, 1994; and in revised form, September 30, 1994)

Dong Cheng Catherine C. Y. Chang Xian-ming Qu Ta-Yuan Chang (§)

From the 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) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.


INTRODUCTION

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) (^1)is an intracellular enzyme that catalyzes the conjugation of long chain fatty acids and cholesterol to form cholesteryl esters. ACAT is believed to play important roles in lipoprotein assembly, in dietary cholesterol absorption, and in cholesterol homeostasis (for reviews see Suckling and Stange, 1985; Chang et al., 1994b). Under pathological conditions, accumulation of ACAT reaction products as foamy lipid droplets in the cytoplasm of macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaques (for reviews see Brown and Goldstein, 1983; Ross, 1986). Cholesterol ester synthesis and ACAT activity in the arteries of experimental animals are increased upon feeding of cholesterol to induce experimental atherosclerosis (for early references see Chang and Doolittle, 1983). Experiments performed in cultured human fibroblasts and CHO cells have shown that the rates of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium (Goldstein et al., 1974; Brown et al., 1975; Doolittle and Chang, 1982a). Results from studies employing mammalian cell mutants defective in intracellular cholesterol trafficking (Kruth et al., 1986; Liscum et al., 1989; Cadigan et al., 1990; Dahl et al., 1992) suggest that the release of LDL-derived free cholesterol from the lysosomal compartment may be required for ACAT activation. Other evidence suggests that most of the newly liberated LDL-bound cholesterol released from the lysosome may not move directly to ACAT, but first move to the plasma membrane (Tabas et al., 1988; Lange et al., 1993; for a review, see Liscum and Faust, 1994).

The process of activation of ACAT by LDL or by 25-hydroxycholesterol is insensitive to the protein synthesis inhibitor cycloheximide (Goldstein and Brown, 1977; Doolittle and Chang, 1982b), implying that the elevation of ACAT activity is not due to a change in enzyme content. A reconstituted vesicle assay for measuring ACAT activity in vitro (Cadigan and Chang, 1988; Doolittle and Chang, 1982a) has been developed. This method allowed ACAT activity to be measured independently of the enzyme's original lipid environment. Cell extracts prepared from CHO cells or human fibroblasts incubated in sterol-containing medium or in sterol-depleted medium had ACAT activities that vary up to 20-fold. The differences in the activity were essentially abolished in the reconstituted vesicle assay, supporting the notion that modulating the amount of ACAT enzyme may not be an important mechanism for regulating ACAT activity by sterols in mammalian cells. However, the molecular mechanism(s) responsible for sterol regulation of ACAT remains to be elucidated.

ACAT is an integral membrane protein located in the endoplasmic reticulum (ER). This protein exists only in small quantities and little progress has been made to purify the protein in an active form from crude cell homogenates (Doolittle and Chang, 1982a). Recently, our laboratory reported the cloning of human ACAT cDNA K1 by a somatic cell genetic and molecular biological approach (Chang et al., 1993, reviewed in Chang et al., 1994b). This gene has been localized to human chromosome 1 q25 (Chang et al., 1994a). Transfecting of the coding region of cDNA K1 into an ACAT-deficient mutant CHO cell line resulted in the expression of ACAT activity. Heat inactivation studies indicated that the expressed ACAT activities were of human origin. This result, along with protein sequence homology analysis, indicated that the open reading frame (ORF) of cDNA K1 encodes a polypeptide essential for ACAT catalysis (Chang et al., 1993).

In the current study, we report the expression of ACAT human cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity (see ``Result''). Infecting these cells with recombinant virus containing ACAT cDNA K1 caused these cells to express high levels of ACAT protein and high levels of ACAT activity assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. We also observed that the cellular cholesterol content of Sf9 cells grown in medium with 10% fetal bovine serum (FBS) is much lower (by approximately 10-20-fold) than the values typically found in various mammalian cells grown under the same condition. The combination of high level ACAT protein expression and the low level cellular cholesterol content in the infected cells has provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this cell-free system, we have shown that cholesterol itself can serve as an ACAT activator, in addition to its role as an ACAT substrate.


EXPERIMENTAL PROCEDURES

Materials

Sf9 insect cells, Grace's medium, wild-type baculovirus, recombinant baculovirus containing beta-galactosidase gene, cationic liposomes, linearized Autographa California nuclear polyhedrosis virus (AcMNPV) DNA, and the transfer vector pBlueBacIII were from Invitrogen. Methionine-free Grace's medium was from Life Technologies, Inc. as were the restriction endonucleases EcoRI, HindIII, and PstI. [S]Methionine (>1000 Ci/mmol) and the ECL Western blotting detection reagent were from Amersham Corp.. Prestained protein molecular weight marker and the horseradish peroxidase-conjugated enzyme immunoassay grade affinity-purified goat anti-rabbit IgG were from Bio-Rad. The antiserum against the synthetic peptide (EKNNHRAKDL) was raised in rabbit by Research Genetics. The ImmunoPure Ag/Ab immobilization kit was from Pierce. Immobilon-P membrane was from Millipore. Autofluor was from National Diagnostics. 7-Ketocholesterol, 6-ketocholestanol, beta-sitosterol, and 7alpha-hydroxycholesterol were from Steraloids, Inc.; cholesterol and 25-hydroxycholesterol were from Sigma; cholate and deoxycholate (both as sodium salt) were from Calbiochem-Novabiochem Corporation. 25-[26,27-^3H]Hydroxycholesterol was from DuPont NEN. O-Phthaldialdehyde was from Sigma. ACAT inhibitors CI 976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide), octimibate (8-[1,4,5-triphenyl-1H-imidazole-2-yl)-oxy]octanoic acid) and Dup 128 (N`-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pentyl]-N-heptylurea) were generous gifts from Drs. Roger Newton and Brian Krause at Parke-Davis Pharmaceutical Division of Warner-Lambert Co. Other reagents were from the same source as described previously (Chang et al., 1986, 1993; Cadigan et al., 1989).

Methods

Standard molecular biology techniques (Sambrook et al., 1989) and Sf9 cell tissue culture methods (O'Reilly et al., 1992) were used. Sf9 cells were grown as monolayers at 27 °C in Grace's medium + 10% FBS. The medium contained 10 µg/ml of gentamycin as antibiotics.

Construction of an ACAT cDNA-containing Baculovirus Transfer Vector

A 1.7-kb fragment of cDNA K1, from nucleotide 1282 to 3050, encompassing the entire predicted protein coding region (nucleotides 1397-3046) as well as 115 additional base pairs as part of the 5`-untranslated element upstream from the presumptive ATG initiation codon (Chang et al., 1993) was produced by digesting the 4.0-kb cDNA K1 insert in pBluescript with EcoRI and HindIII. This fragment was gel-purified (using a GeneClean II kit from Bio 101 Inc.) and ligated with a EcoRI-HindIII linearized pBluescript (SK-) plasmid to construct pBluescript-K1 1.7 kb. To construct the transfer vector pBlueBacIII-K1 1.7 kb, pBluescript-K1 1.7 kb was digested with PstI and HindIII to release the 1.7-kb fragment containing the entire predicted ACAT ORF. This fragment was gel purified and ligated with a PstI-HindIII linearized pBlueBacIII plasmid.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting Analysis

Electrophoresis was done essentially according to the method by Laemmli(1970), using 10% polyacrylamide for the resolving gel and 5% polyacrylamide for the stacking gel. Protein samples were prepared by lysing the cells directly in 8 M urea, 2% SDS after the cells were washed several times in phosphate-buffered saline (PBS) at 4 °C.

An antiserum was raised in rabbit against a synthetic decapeptide (EKNNHRAKDL) located at amino acid residues 103-112 of the predicted human ACAT protein sequence (Chang et al., 1993). The specific IgGs of this antiserum were purified by affinity column chromatography using the peptide EKNNHRAKDL as the ligand. The affinity column was synthesized and utilized according to the instructions of Pierce ImmunoPure Ag/Ab Immobilization Kit. Western blots were prepared essentially according to the method of Towbin et al.(1979) and briefly described as follows: after the samples were separated by SDS-PAGE on 10% acrylamide slab gels, the proteins were transferred to Immobilon-P (Millipore, Bedford, MA) at 300 mA for 4 h. The transfer buffer consisted of 25 mM Tris, 190 mM glycine, 20% methanol, and 0.005% SDS. Proteins were immunoblotted as follows: the blots were blocked in 5% Carnation nonfat milk and probed with 2.5 µg/ml of the affinity-purified specific antibody in 1% Carnation milk. The horseradish peroxidase-conjugated enzyme immunoassay grade affinity-purified goat anti-rabbit IgG (from Bio-Rad) at 1/3000 dilution in 1% milk was used as the secondary antibody to probe the immunoabsorbed proteins on the blot. The buffer used for blotting and washing was 20 mM Tris-HCl, 150 mM NaCl, and 0.3% Tween-20 at pH 7.6. The ECL Western blotting detection reagent was used to for detection.

Metabolic Labeling of Insect Cells

Sf9 cells were seeded in prewarmed fresh Grace's medium + 10% FBS in 6-well dishes at confluence. Cells were incubated for at least 30 min at 27 °C to allow the cells to attach to the dishes. The cells were infected with baculovirus at appropriate multiplicity of infection, usually 5-10 pfu/cell. One h before labeling, the medium was removed, and the cells were washed with prewarmed PBS at 27 °C without disturbing the monolayer. The washed cells were incubated with 2 ml/well of prewarmed methionine-free Grace's medium + 10% FBS for 1 h to deplete the endogenous methionine. This medium was replaced with prewarmed 0.5 ml/well of methionine-free medium containing 25 µCi of [S]methionine, and the cells were incubated at 27 °C for 1 h. The tissue culture fluid was removed, and the monolayer was rinsed with ice-cold PBS three times. Washed cells were collected by low speed centrifugation at 4 °C, and lysed with 200 µl of 8 M urea, 2% SDS/well. Twenty µl of the cell lysates were loaded per lane for SDS-PAGE analysis. After electrophoresis, gels were fixed with 5% glacial acetic acid, 5% isopropyl alcohol, and 90% water for 30 min. The gels were incubated in Autofluor (from National Diagnostics) for 1 h and then dried and autoradiographed using x-ray film (Fuji Medical x-ray Film) at -80 °C for 1-3 h.

ACAT Assays

Reconstituted Vesicle Assay (Assay A)

This was carried out according to the procedure as described previously (Cadigan and Chang, 1988). Unless otherwise stated, the cholesterol/phosphatidylcholine molar ratio of the vesicles used was kept at 0.2. To prepare the cell homogenates, the infected Sf9 cells were harvested by detaching cells from the dishes and collected by low speed centrifugation (1000 revolutions/min in a Beckman TJ-6 centrifuge for 5 min) and washed with cold PBS. The washed cells were exposed to hypotonic shock buffer at room temperature for 2 min as described previously (Chang et al., 1981). The swollen cells were further Dounce homogenized at 4 °C with 40 strokes to assure complete cell lysis. The fresh whole cell homogenates, with protein concentration ranging from 3 to 5 mg/ml, were used immediately for solubilization and reconstitution into vesicles. The specific ACAT inhibitors, CI 976, octimibate, and Dup 128, were dissolved in dimethyl sulfoxide (Me(2)SO) to prepare 20 mM stock solutions and stored at 4 °C. Each ACAT inhibitor was delivered to the reconstituted vesicle from serially diluted Me(2)SO solutions; the Me(2)SO concentration in each assay tube was kept at 2.5%. Control experiments showed that Me(2)SO at this concentration caused less than 10% inhibition on ACAT activities assayed in reconstituted vesicle form.

Cell-free Assay Using Endogenous Cholesterol as the Substrate (Assay B)

The infected Sf9 cells were harvested, collected, and washed with cold PBS as described above. The washed cells were exposed to hypotonic shock buffer as described above but at 4 °C for 3 min. The swollen cells were lysed by Dounce homogenization with 40 strokes at 4 °C. Complete cell lysis was assured by examining the broken cell homogenates under the microscope. Sixty µl of cell lysates with cellular protein concentration ranging from 3 to 5 mg/ml were used for each assay reaction. The ACAT reaction was initiated by adding 40 µl of assay mixture containing 0.25 mM [^3H]oleyl-CoA (0.1 µCi), 12.5 mg/ml fatty acid-free bovine serum albumin, and 100 mM Tris-HCl at pH 7.8 (Doolittle and Chang, 1982b). Assays were carried out at 37 °C for 5-10 min. We found that the ACAT activity gradually deviated from linearity (decreasing) after 10 min. Termination of reaction was by addition of chloroform/methanol (2:1). Lipid extraction and thin layer chromatography (TLC) analyses were performed as described previously (Chang et al., 1986).

Cell-free Assay Using [^3H]25-Hydroxycholesterol as the Substrate (Assay C)

Cell extracts were prepared in the same way as was described above. Sixty µl of cell extracts were used for each assay reaction, after the cell extracts were incubated with 1 µl of ethanol containing 0.25 pmol (0.05 µCi) of [^3H]25-hydroxycholesterol, with or without varying amounts of cholesterol at 37 °C for 30 min. The final concentration of ethanol in the incubation mixture was 2%. The ACAT reaction was initiated by adding 40 µl of assay mixture containing 0.25 mM oleyl-CoA, 12.5 mg/ml fatty acid-free bovine serum albumin, 100 mM Tris-HCl at pH 7.8. After incubation at 37 °C for 5 min, or for various indicated assay times, chloroform/methanol (2:1) was added to stop the reaction. Lipids were extracted as described previously (Chang et al., 1986). Three different solvent systems were used for TLC analyses on Whatman LK5D silica gel plates to verify the identities of 25-hydroxycholesterol and 25-hydroxycholesteryl-3beta-oleate: I, petroleum ether/ether/acetic acid (90:10:1); II, benzene/ethyl acetate (3:1); and III, benzene. The corresponding R values for 25-hydroxycholesteryl-3beta-oleate were: 0.39, 0.84, and 0.22; R values for 25-hydroxycholesterol were: 0.13, 0.45, and 0.13, respectively. The percent esterification of 25-hydroxycholesterol was determined by the H counts/min found in the ester band divided by the sum of the H counts/min found in the 25-hydroxycholesterol band and the H counts/min found in the ester band. The ACAT activity was expressed as picomoles of ester formed per milligram of protein per time period.

Determination of Cholesterol Esterification Rate in Intact Sf9 Cells

[^3H]Oleate pulse assays using intact Sf9 cells were carried out according to the procedure as described previously (Chang et al., 1986) for the analysis of cholesterol esterification rate in intact mammalian cells. The pulse medium consisted of 4 ml of Grace's medium + 10% FBS containing 5 times 10M oleate (4 µCi) per 25-cm^2 flask; pulse periods ranged from 5 to 40 min in various experiments. The pulse assays were performed at 37 °C. Under this condition, we found that the cholesterol esterification rates in ACAT-expressing Sf9 cells were linear up to 40 min.

Determination of Cholesterol Synthesis Rate and Cholesterol Content in Sf9 Cells

The method for [^14C]acetate pulse to determine the cholesterol biosynthesis rate in intact cells was carried out according to the procedure described previously (Chang et al., 1986). To determine cholesterol content in Sf9 cells, after lipid extraction of Sf9 cell lysates with chloroform/methanol (2:1) and separation of lipids by TLC on Whatman LK5D plate (solvent system: dichloromethane/ethyl acetate (97:3); R: 0.3), cholesterol was determined according to the procedure described by Rudel and Morris (1973).


RESULTS

Functional Expression of Human ACAT K1 in Sf9 Cells

The recombinant baculovirus transfer vector pBlueBacIII-K1 1.7 kb contains the entire predicted human ACAT K1 protein-coding sequence directly downstream from the baculovirus polyhedrin protein promotor (see ``Experimental Procedures'' for the construction of this vector). The orientation and identity of the ACAT K1 cDNA insert were confirmed by DNA sequencing at the insert-vector junction. This vector was used to perform cotransfection experiments according to standard procedures (O'Reilly et al., 1992). Two days after cotransfection of Sf9 cells with the transfer vector and the linearized AcMNPV baculovirus DNA, the tissue culture fluid was collected as the primary viral stock. This viral stock was used to infect Sf9 cells to identify plaques arising from recombinant ACAT cDNA containing viruses. The recombinant viruses were detected by visually screening for the absence of polyhedrin-containing occlusion particles and by screening for blue plaques stained with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) digestion product by beta-galactosidase on plaque assay plates (O'Reilly et al., 1992). Three plaques were isolated and purified. Pilot experiments showed that the ACAT activity expressed in Sf9 cells infected with these three viruses were the same. Therefore, only one was chosen for further characterization.

The [S]methionine metabolic labeling technique was utilized to analyze the newly synthesized proteins after the Sf9 cells were infected with the recombinant virus. It is known that baculovirus infection results in shut-off of host gene expression and host protein synthesis such that, by 24-36 h post-infection, gene expression is primarily viral-specific (O'Reilly et al., 1992). As shown in Fig. 1A, at 48-h post-infection, two prominent metabolically labeled protein bands, with apparent sizes at 50 and 56 kDa, were detected after SDS-PAGE (lane 4). In contrast, cells infected with the control recombinant virus containing beta-galactosidase (lane 8) or with the wild-type virus (lane 12) produced the expected 110-kDa beta-galactosidase protein band or the 32-kDa viral polyhedrin protein band, respectively. We next examined proteins of the infected Sf9 cells by Western blot analysis. An affinity purified, anti-peptide antibody raised in rabbit against the synthetic decapeptide EKNNHRAKDL, located at amino acid 103-112 of the predicted human ACAT protein sequence, was employed to probe the Western blot. This antibody specifically recognized proteins produced in Sf9 cells infected with the ACAT-recombinant virus (lanes 2-5, Fig. 1B). In contrast, no signal was detectable by this antibody in cell extracts prepared from Sf9 cells infected with the control recombinant virus or with the wild-type virus (lanes 6 and 7). Before the 36 h post-infection time point, the antibody mainly recognized the 50 kDa protein band only. However, after 48 h, an additional 56 kDa band, as seen in Fig. 1A, lane 4, became apparent. The same results were obtained irrespective of whether the following methods were used to prepare the protein samples: 1) lysis buffer (8 M urea and 2% SDS) with or without 1% beta-mercaptoethanol, with or without various protease inhibitors; 2) lysis buffer with different SDS concentrations (from 1 to 5%); 3) boiling urea-SDS buffer to solubilize the cells, or long periods (30-60 min) of low heating (50 °C) to treat protein samples prior to electrophoresis; 4) treatment of intact cells with the N-glycosylation inhibitor tunicamycin at 20 µg/ml in the growth medium for 6 h before cell lysis. In addition, the same results were seen using three independently isolated and purified recombinant viral particles. Another unusual, and perhaps related observation is that, the apparent size of the 56-kDa protein and the 50-kDa protein in SDS-PAGE deviate significantly from the estimated size of the hypothetical ACAT protein (63.8 kDa) deduced from the ORF analysis of human ACAT cDNA K1 1.7 kb (Chang et al., 1993). We noticed from the protein sequence analysis that the hypothetical ACAT protein K1 is a hydrophobic, membrane-spanning protein with a calculated isoelectric point (PI) of 9.78. In the literature, there is ample precedent for a discrepancy in molecular weight estimation among membrane proteins (for example, Kartner et al., 1991, and earlier references cited therein). Perhaps the combination of the hydrophobic and basic nature of the ACAT protein causes a higher amount of SDS associated with it and makes the ACAT protein move faster in SDS-PAGE. The reason(s) for the appearance of the 50/56 kDa doublet protein bands at 48 h post-infection time remains obscure. It is possible that, due to the abnormal protein structure, the 56-kDa protein may represent a minor portion of the ACAT protein that maintains a certain non-random configuration under various denaturing conditions. This hypothesis can explain the existence of the 100 and 200 kDa bands seen in the immunoblots, which may be the dimeric and tetrameric forms of the ACAT protein; these aggregates can not be completely dissociated into monomers even under the harsh conditions described above. However, other possibilities cannot be ruled out. More experiments are needed to clarify these observations.


Figure 1: Functional expression of ACAT cDNA K1 in Sf9 cells. A, time course of protein synthesis in baculovirus-infected Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing ACAT cDNA K1 1.7 kb (lanes 1-4) or beta-galactosidase (lanes 5-8) or with wild-type baculovirus (lanes 9-12). The cells were metabolically labeled with [S]methionine and were lysed at 12 (lanes 1, 5, and 9), 24 (lanes 2, 6, and 10), 36 (lanes 3, 7, and 11), or 48 (lanes 4, 8, and 12) h post-infection as described under ``Experimental Procedures.'' Each lane was loaded with 50 µg of protein and was analyzed by 10% SDS-PAGE. The gel was fixed and autoradiographed at -80 °C for 1 h. B, immunoblot analysis of proteins from Sf9 cells uninfected or infected with recombinant virus containing ACAT cDNA K1 or control viruses. Cell lysates of uninfected Sf9 cells (lane 1), Sf9 cells infected with ACAT-recombinant virus (lanes 2-5), with beta-galactosidase-recombinant virus (lane 6), or with wild-type virus (lane 7) were prepared after 12 h (lane 2), 24 h (lane 3), 36 h (lane 4), or 48 h (lanes 5-7) of infection. Infections were performed in a staggered manner such that at the end, cells were harvested at the same time. Approximately 50 µg of protein was loaded in each lane. After 10% SDS-PAGE, proteins were transferred onto a filter, and Western blot analysis was performed as described under ``Experimental Procedures.'' C, analysis of ACAT activity in Sf9 cells infected with recombinant viruses at various points post infection. Sf9 cells were infected with ACAT-recombinant virus (circle), beta-galactosidase-recombinant virus (box), or wild-type virus (up triangle). At each time point post-infection, the cell lysates were prepared, and the ACAT activities were measured by the reconstituted vesicle assay as described under ``Experimental Procedures.'' Similar results as reported in A-C were obtained in three independent experiments. The inset of C gives the result of a separate experiment, which showed dependence of ACAT activity on cholesterol concentration present in the reconstituted vesicles. ACAT activities were measured by the reconstituted vesicle assay; vesicles containing 10 mg/ml egg phosphatidylcholine (PC) and various indicated concentrations of cholesterol, prepared according to the procedure described previously (Ventimiglia et al., 1986). The values are the averages of duplicate assays from the same detergent-treated extracts and varied within 10% of the mean.



To test whether these virally produced proteins possess ACAT enzyme activity, we measured ACAT activity in Sf9 cells in vitro at various post-infection time points (Fig. 1C). To ensure that the measured ACAT activity would be independent of cellular lipid composition, we employed a solubilization and reconstitution into vesicle procedure (Cadigan and Chang, 1988). Uninfected Sf9 cells or cells infected with the control recombinant virus or with the wild-type virus did not exhibit any ACAT activity throughout the time course. In contrast, Sf9 cells infected with the ACAT recombinant virus began to express increasing amount of ACAT activity with time. At 48 h post-infection, the specific activity of ACAT was at least 10-fold higher than those detected in various human cell lines, including human A431 cells and human fibroblast cells (Cadigan and Chang, 1988). In the reconstituted vesicle assay, the ACAT enzyme residing in the vesicle utilizes cholesterol present in the same vesicle as its substrate. This assay avoids the step of cholesterol transfer between vesicles. Thus, a sensitive activity-cholesterol concentration curve could be demonstrated. In the inset of Fig. 1C, the shape of the ACAT activity-cholesterol concentration curve was shown to be sigmoidal (instead of hyperbolic), suggesting cooperative interaction between cholesterol and the ACAT protein during catalysis. A similar observation was made when extracts of human fibroblasts, or CHO cells, or pig liver microsome were used as the ACAT enzyme source (Doolittle and Chang, 1982a, 1982b; Cadigan and Chang, 1988).

This laboratory previously reported that the ACAT activity in CHO cells is more thermolabile than the ACAT activity in human cells (Cadigan et al., 1989; Chang et al., 1993). This result may reflect the structural difference between the CHO cell ACAT protein and the human cell ACAT protein. In order to examine the heat stability of ACAT activity expressed in the Sf9 cells, a heat inactivation experiment was carried out. As shown in Fig. 2, ACAT activity expressed in Sf9 insect cells exhibits heat stability comparable to that of human ACAT; its heat stability is significantly greater than that of hamster ACAT.


Figure 2: Heat inactivation of ACAT activities in CHO 25-RA cells (circle), T2-8 cells (CHO cell line transfected with human ACAT) (bullet), human A431 cells (+), and Sf9 cells infected with the human ACAT-recombinant virus (up triangle). Sf9 cells infected with ACAT recombinant virus at 5 pfu/cell were harvested at 48 h post-infection time point. 25-RA and T2-8 cells were grown in F-12 medium + 10% fetal bovine serum. A431 cells were grown in Dulbecco's modified Eagle's medium + 10% fetal bovine serum. They were harvested, and the cell extracts were prepared at 2 mg protein/ml, treated with 1% deoxycholate, and reconstituted into vesicles according the previously described method (Cadigan and Chang, 1988). The reconstituted vesicles were incubated at 45 °C for various indicated times, then placed on ice until assayed for enzyme activity. The control activity (without heat treatment) for 25-RA, T2-8, A431, and infected Sf9 cells were 97.9, 41.8, 18.9, 479.8 pmol/min/mg protein, respectively. The values are the averages of duplicate assays from the same detergent-treated extracts and varied within 10% of the mean.



Three active site-directed ACAT inhibitors (CI 976, octimibate, and Dup 128), each with distinct structural features, were employed to probe the sensitivity of active site(s) of ACAT expressed in Sf9 cells. The ACAT inhibition curves (Fig. 3) indicate that insect cell-expressed ACAT activity shows similar sensitivity to inhibition as the CHO cell ACAT activity and the human cell ACAT activity. For all the ACAT activities examined, the IC values for CI 976, octimibate, and Dup 128 were approximately 5, 50, and 0.5 µM, respectively. These values are similar to those reported by Harte et al.(1993) using rat liver ACAT as the enzyme source.


Figure 3: Dose-response curves of ACAT inhibitors on ACAT activities from various sources. Sf9 cells infected with ACAT recombinant virus at 5 pfu/cell for 48 h were used. Cell extracts were prepared from infected Sf9 cells (times), 25-RA cells (box), T2-8 cells (bullet), A431 cells (), or wild-type CHO cells (). The reconstituted cell lysates, prepared according to the previously described method (Cadigan and Chang, 1988), were incubated with CI 976 (A), octimibate (B), or Dup 128 (C) at various final concentrations for 20 min on ice. Me(2)SO (DMSO) was used as the solvent of the ACAT inhibitors. Final concentration of Me(2)SO in all the assay tubes was 2.5% (v/v). The ACAT assays were performed as described (Cadigan and Chang, 1988). The curves were drawn according to the values obtained from Sf9 cell-expressed ACAT activity (times). The control ACAT activities (2.5% Me(2)SO treated) for 25-RA, T2-8, A431, wild-type CHO, and infected Sf9 cells were 93.6, 51.0, 19.3, 67.8, and 550.9 for pmol/min/mg protein, respectively. The values are the averages of duplicate assays from the same detergent extracts and varied within 10% of the mean.



Activation of Cholesterol Esterification Rate by Adding Sterols to Growth Medium of Insect Sf9 Cells

We determined that the cellular cholesterol content of Sf9 cells grown in 10% FBS is 1.8 µg/mg protein, which is less than 10% of the values found in various mammalian cells grown under similar condition (Brown et al., 1975; Cadigan et al., 1988). This is probably due to the following features of Sf9 cells: unlike the observation reported in mammalian cells (Chang et al., 1986), we found that the basal cholesterol esterification rate in ACAT-expressing Sf9 cells did not change in response to the removal or the readdition of lipoproteins in the growth medium within a 2-h period (result not shown), suggesting that Sf9 cells do not possess efficient mechanism(s) for taking up and utilizing lipoprotein-bound cholesterol from the growth medium; we also found that Sf9 cells do not possess a measurable capability to biosynthesize cholesterol (by performing [^14C]acetate pulse in intact cells; result not shown).

To find out whether the human ACAT enzyme expressed in Sf9 cells can be activated by sterols added in growth medium, we used the [^3H]oleate pulse method to measure the cholesterol esterification rate in intact cells. Sf9 cells infected with the ACAT-expressing virus grown in 10% FBS medium exhibited basal esterification rate, at approximately 0.03-0.1 pmol/min/mg, which was at least 300-fold less than the values found in mammalian cells grown under similar condition (30-100 pmol/min/mg; Goldstein et al., 1974; Chang et al., 1986). Adding 25-hydroxycholesterol to the growth medium caused a very large increase in cholesterol esterification rate; the fold increase varied between 20-60-fold in several different experiments. The activation by 25-hydroxycholesterol was both time- (Fig. 4A) and concentration-dependent (Fig. 4B). At a concentration of 5 µg/ml, the time required to cause maximal activation was approximately 60 min. The concentration to cause half-maximal activation was approximately 1-2 µg/ml when the cells were treated with 25-hydroxycholesterol for 60 min. In control experiments, we found that in uninfected or mock infected Sf9 cells, treated or untreated with 25-hydroxycholesterol, the cholesterol esterification rate was undetectable (result not shown). To examine if sterols other than 25-hydroxycholesterol possess a similar capacity to activate cholesterol esterification, we tested 7-ketocholesterol, 6-ketocholestanol, 7alpha-hydroxycholesterol, beta-sitosterol, cholate, deoxycholate, and cholesterol. The result (Fig. 5) showed that none of these sterols were nearly as effective as 25-hydroxycholesterol in activating cholesterol esterification in intact cells.


Figure 4: Activation of cholesterol esterification rate by 25-hydroxycholesterol in intact Sf9 cells expressing human ACAT. A, time dependence. B, concentration dependence. Sf9 cells grown in 25-cm^2 flasks were infected with ACAT recombinant virus at 5 pfu/cell. After infection for 48 h, cells were incubated with 5 µg/ml of 25-hydroxycholesterol at 37 °C for various time as indicated (A) or were incubated with various indicated concentrations of 25-hydroxycholesterol at 37 °C for 60 min (B). Cholesterol esterification rate as measured by the [^3H]oleate pulse assay in intact cells was performed according to the procedure described under ``Experimental Procedures.'' The final concentrations of ethanol in the media were kept at 0.1% in all the flasks. The values are the averages of duplicate flasks and varied within 10% of the mean. Similar results were reproduced in three other independent experiments.




Figure 5: Sterol specificity in activating the cholesterol esterification rate in intact Sf9 cells expressing ACAT. The experiment was performed as described in Fig. 4. The symbols were: bullet, 25-hydroxycholesterol; circle, cholesterol; , 7-ketocholesterol; box, 6-ketocholestanol; box, beta-sitosterol; , 7alpha-hydroxycholesterol; , cholate; up triangle, deoxycholate. The values are the averages of duplicate flasks and varied within 10% of the mean.



To examine if the activation by 25-hydroxycholesterol involves an increase in ACAT protein content in cells, we used immunoblotting to monitor the ACAT protein levels and found that neither the content nor the eletrophoretic mobility of ACAT protein underwent detectable changes, after the intact cells were treated with 25-hydroxycholesterol with concentrations ranging from 0.1 to 10 µg/ml in the medium (Fig. 6).


Figure 6: ACAT protein electrophoretic mobility and content after treating intact cells with varying concentrations of 25-hydroxycholesterol. The experiment was conducted as described in Fig. 4B. A parallel set of cell culture were used for infection and incubations, and whole-cell protein lysates were prepared by lysing the cells in 8 M urea, 2% SDS. Western blot analysis was performed as described under ``Experimental Procedures.'' Lane 1 represents lysate from the infected cells without any treatment. Lanes 2-7 represent lysates from infected cells treated with 0, 0.1, 0.5, 1, 5, or 10 µg/ml 25-hydroxycholesterol, respectively. 25-Hydroxycholesterol was delivered from ethanol stock solution. The final concentrations of ethanol in media during the incubation for lanes 2-7 were 0.1%.



Increase of ACAT Activity by Adding Oxysterol or Cholesterol to Cell Extracts

We found that, similar to the situations demonstrated in intact mammalian cells, ACAT activities assayed using endogenous cholesterol as its substrate in the extracts prepared from cells incubated with 25-hydroxycholesterol in the growth medium were highly elevated (Table 1, compare line 1 in columns 3 and 4). In a parallel experiment, we added 25-hydroxycholesterol to extracts prepared from cells unexposed to 25-hydroxycholesterol, then measured ACAT activities. The results (Table 1, lines 2 and 3) showed that 25-hydroxycholesterol added to the cell extracts caused significant activation of ACAT activity. In five other experiments, a 3.5-7-fold increase in ACAT activity by adding 25-hydroxycholesterol in vitro was seen.



To characterize the in vitro ACAT activation system, we next tested the effect of incubation temperature on the degree of activation. The result presented in lines 2 and 3 of Table 1showed that incubating 25-hydroxycholesterol with extracts at two different temperatures (4 or 37 °C) did not affect the degree of activation (6.7- versus 7.2-fold); we did observe that incubating cell extracts at 37 °C for 30 min caused approximately a 2-fold increase in basal ACAT activity (from 8.6 pmol/min/mg to 16.5 pmol/min/mg; similar observations were made in two separate experiments). In another experiment, we tested the possible effect of incubation time on the degree of activation and found that 25-hydroxycholesterol added at 10 µg/ml to the cell extracts at 37 °C reached its maximal activating effect within 10 min (result not shown). For the purpose of consistency, we chose a 30-min sterol incubation time at 37 °C to perform the remainder of the experiments reported in this article.

Using the in vitro condition described above, we examined the effect of sterol concentration and found that the concentration of 25-hydroxycholesterol needed to cause half-maximal activation of ACAT activity was 1-2 µg/ml (Fig. 7), a value similar to the value found to cause half-maximal activation of cholesterol esterification rate in intact cells (Fig. 4B). We next compared the effect of various sterols (at 10 or 100 µg/ml) on ACAT activity and found that two oxysterols, 7-ketocholesterol and 6-ketocholestanol, added at high concentration (100 µg/ml), caused modest increase in ACAT activity, while 7alpha-hydroxycholesterol caused significant inhibition in ACAT activity. beta-Sitosterol, cholate, or deoxycholate did not cause detectable change in ACAT activity. Among the various sterols tested, other than 25-hydroxycholesterol, the only sterol that caused significant increase in ACAT activity was cholesterol (Fig. 8). Since cholesterol is the substrate for the ACAT enzyme, we could not conclude from this result whether the stimulation of ACAT by cholesterol was due to an increase in substrate availability, or was due to a specific activating effect in addition to the substrate effect.


Figure 7: Activation of ACAT activity in vitro by adding varying amounts of 25-hydroxycholesterol to the cell extracts. Sf9 cells were infected with ACAT-recombinant virus for 48 h at 5 pfu/cell. Cell extracts were prepared as described under ``Experimental Procedures.'' The cell extracts were incubated with various indicated concentrations of 25-hydroxycholesterol at 37 °C for 30 min. After incubation, extracts were assayed immediately for ACAT activity. The final ethanol concentration present in each assay tube was 1%. The values are the averages of duplicate assays from the same cell extracts and varied within 5% of the mean.




Figure 8: Sterol specificity in activating ACAT activity in vitro. Conditions for virus infection, cell extract preparation, and incubation with different sterols and ACAT assay in vitro were the same as described in Fig. 7, except cholate and deoxycholate were delivered from stock solutions in water at 1/100 volume of the cell extract. The final concentration of ethanol in each tube was adjusted to 1%. The control value indicated by the arrow is the value obtained after the cell extract was incubated with 1% ethanol alone. Solid bars represent the values obtained after cell extracts were incubated with 10 µg/ml of sterol; hatched bars represent the values obtained after the cell extracts were incubated with 100 µg/ml of sterol. The values are the averages of the duplicate assays from the same cell extracts and varied within 5% of the mean.



We tested the stimulation effect of 25-hydroxycholesterol on ACAT in vitro in the presence of varying amounts of cholesterol. Cell extracts from cells unexposed to 25-hydroxycholesterol were preincubated with increasing amounts of cholesterol, then incubated with or without 10 µg/ml 25-hydroxycholesterol, and assayed for ACAT activity. The result (Fig. 9A, replotted in Fig. 9B) showed that as the cholesterol concentration added to the cell extracts increased, the fold activation of ACAT activity by 25-hydroxycholesterol decreased (from 3.5- to 1.5-fold).


Figure 9: A, cholesterol dose-response curve of ACAT activity in vitro, with or without preincubating the cell extracts with 25-hydroxycholesterol. Conditions for virus infection, cell extract preparation, and ACAT assay were as described in Fig. 7. Cell extracts were preincubated with (box) or without () 10 µg/ml 25-hydroxycholesterol at 37 °C for 30 min. Then various indicated amounts of cholesterol were added, and an additional incubation at 37 °C for 30 min was performed before the extracts were assayed for ACAT activity. Final concentration of ethanol in each tube was at 2%. B, fold activation of ACAT activity by 25-hydroxycholesterol diminishes as cholesterol concentration in the cell extracts increases. This is a replot of A. Fold activation of ACAT activity is defined as ACAT activity with 25-hydroxycholesterol preincubation divided by ACAT activity without 25-hydroxycholesterol preincubation at a given cholesterol concentration in cell extracts. Similar results were reproduced in two other independent experiments.



Cholesterol Added in Vitro Serves as an Activator of ACAT

The result presented in Fig. 9B indicated that cholesterol added in the cell extract diminished the ability of 25-hydroxycholesterol to serve as an ACAT activator, suggesting that these two sterols may be competing against each other for the activating effect, i.e. this result implies that cholesterol may itself serve as an ACAT activator. To investigate this possibility, we took advantage of the following observation: by adding [^3H]25-hydroxycholesterol and nonradioactive oleyl coenzyme A to the cell extracts prepared from ACAT-expressing cells, a typical enzymatic conversion to form [^3H]25-hydroxycholesteryl-3beta-oleate was demonstrated (Fig. 10A). The reaction rate at 37 °C was linear for at least 5 min. Additional results showed that the reaction product was absent if oleyl coenzyme A was omitted from the incubation mixture or if the specific ACAT inhibitor CI 976 was added to the cell extract at 10 µM prior to adding oleyl coenzyme A or if extracts prepared from uninfected cells were used as the enzyme source. Together, these results showed that the ACAT enzyme expressed in Sf9 cells was able to use 25-hydroxycholesterol as an alternative substrate and converted the sterol to 25-hydroxycholesteryl-3beta-oleate.


Figure 10: Cholesterol activation of ACAT activity in vitro using 25-hydroxycholesterol as the substrate. Conditions for Sf9 cell infection, preparation of cell extracts were as described in Fig. 7. [^3H]25-Hydroxycholesterol (2.5 pmol, 0.05 µCi) in 1 µl of ethanol was delivered to each 60 µl of cell extracts kept at 0 °C. After a brief vortex, either 10 µg/ml final concentration of cholesterol was added (bullet) or vehicle ethanol (circle) was added (A); various amounts of cholesterol as indicated were added (B). Final concentration of ethanol in each tube was adjusted to 2%. The mixtures were incubated at 37 °C 30 min. Esterification of 25-hydroxycholesterol was initiated by adding 40 µl of assay mixture containing 12.5 mg/ml fatty acid-free bovine serum albumin, 0.25 mM oleyl-CoA, 100 mM Tris-HCl, pH 7.8. The assay time was as indicated in A and was 5 min in B. Lipid extraction procedure and TLC analysis was as described under ``Experimental Procedures.'' The values are the averages of duplicate assays from the same cell extracts and varied within 5% of the mean. The same results were seen in two independent experiments.



Using [^3H]25-hydroxycholesterol as the substrate, we found that cholesterol added at 10 µg/ml activated the 25-hydroxycholesteryl ester formation by approximately 3-fold (Fig. 10A). If cholesterol was not an activator, but served only as a substrate for ACAT, one would have seen that cholesterol competed with the [^3H]25-hydroxycholesterol and reduced [^3H]25-hydroxycholesteryl ester formation. The result shown in Fig. 10, therefore, is a demonstration that cholesterol also serves as an activator for ACAT. The activation by cholesterol was concentration-dependent; approximately 1-2 µg/ml of cholesterol was required to cause half-maximal activation (Fig. 10B).


DISCUSSION

In this study, we have shown that expression of human ACAT cDNA K1 1.7 kb in Sf9 insect cells caused these cells to produce ACAT activity. Sf9 cells do not contain endogenous ACAT-like activity. Infecting these cells with the ACAT gene-containing recombinant baculovirus for 48 h caused these cells to express ACAT, with specific activities at least 10 times higher than those found in various human cell lines. Time course experiments showed that the expression of ACAT activities correlated well with the appearance and accumulation of new polypeptides, which were detected by both the [S]methionine metabolic labeling and Western blotting. The heat stability characteristics of the Sf9 cell-expressed ACAT activity were very similar to that of the human enzyme. Additionally, the inhibition constants of three structurally different ACAT inhibitors against the Sf9 cell-expressed ACAT activity were very similar to those against the ACAT activities produced by various mammalian cell lines. These ACAT inhibitors contain different functional groups (^2)and are believed to be targeting at the ACAT active site(s). Our results thus strongly suggest that the active site(s) of Sf9 cell-expressed ACAT is very similar to the ACAT enzyme present in human and hamster cell lines. Taken together, these data demonstrate that the cDNA K1 1.7 kb encodes an essential catalytic component of the ACAT enzyme. At present, we cannot unambiguously prove that the protein encoded by cDNA K1 alone constitutes the entire ACAT holoenzyme. This question can only be appropriately addressed by examining the biological activities of the protein purified to homogeneity. High level expression of the ACAT K1 protein reported here will facilitate this investigation.

Using the intact Sf9 cells expressing ACAT to study ACAT regulation by sterols, we showed that the cholesterol esterification rate and the ACAT activity (using endogenous cholesterol as its substrate) in these cells was highly elevated by adding 25-hydroxycholesterol to the culture medium. Cholesterol added to the medium in the same manner failed to elicit the same response. The dependence in sterol concentration and the rapidity of the activation process (Fig. 4, A and B) are very similar to the earlier observations made in various mammalian cells (Goldstein et al., 1974; Doolittle and Chang, 1982a). Since the DNA elements of the ACAT gene other than the protein coding region were not included in constructing the recombinant virus, we can conclude that the ACAT activation process described in Fig. 4is strictly a post-translational event. Western blotting analysis showed that the increase in ACAT activity in oxysterol-treated cells did not involve any increase in the levels of the ACAT protein. However, our results do not exclude the possibility of transcriptional or translational mechanism in sterol-dependent regulation of ACAT in mammalian cells. The high specific activity of ACAT found in the extracts of ACAT-expressing Sf9 cells (higher than 1 nmol/min/mg; Fig. 1C) was measured by using in vitro assay A, utilizing optimal amounts of cholesterol present in reconstituted vesicle as the substrate. If in vitro assay B, utilizing endogenous cholesterol present in the Sf9 cell extract as the substrate was used, the specific activity of ACAT of these cells (5-20 pmol/min/mg) was actually lower than those found in various mammalian cells grown and assayed under the similar condition (30-50 pmol/min/mg in human fibroblasts or in CHO cells; Cadigan and Chang, 1988). We also noted that the cholesterol esterification rate in these cells grown in medium containing 10% FBS was much lower (0.03-0.1 pmol/min/mg, Fig. 4) than the rates found in various mammalian cells grown under similar conditions (versus 50-100 pmol/min/mg; Goldstein et al., 1974; Chang et al., 1986). Taken together, these results suggest that the ACAT enzyme residing within the cell interior rarely encounters cholesterol; when extracts were prepared from these cells, the ACAT activity remains largely dormant (based on the results using assay B). Using the cell extracts prepared from ACAT-expressing Sf9 cells as the enzyme source, we were able to demonstrate significant activation of ACAT by 25-hydroxycholesterol in vitro (Fig. 7). The capacity of 25-hydroxycholesterol to act as an ACAT activator was shown to be gradually diminished by adding increasing amounts of cholesterol to the cell extracts (Fig. 9). This result could explain why in earlier studies, when extracts of mammalian tissues or mammalian cells were used as the ACAT enzyme source, it was difficult to show a large increase in ACAT activity (no more than 2-fold) by adding oxysterol in vitro (Lichtenstein and Brecher, 1980; Erickson et al., 1980). During the cell homogenization step, the relatively high cholesterol content endogenously present in mammalian tissues might have adhered onto the ACAT protein and largely masked the effect of 25-hydroxycholesterol.

Previously, we and others (for early references, see Chang and Doolittle, 1983) had shown significant stimulation of ACAT catalyzed reaction by adding cholesterol in vitro. However, since cholesterol is a substrate of ACAT, one could not determine from these studies whether the stimulatory effect of cholesterol was due to its effect as a substrate, or as an activator, or as both. Using the in vitro system described here, and by exploiting the enzymatic characteristics of ACAT, which can use 25-hydroxycholesterol as an alternative substrate, we have now demonstrated that cholesterol itself served as an ACAT activator (Fig. 10). The unique features of the in vitro system described here (i.e. high enzyme expression in a low cholesterol environment) may be generally applicable for demonstrating specific effects of cholesterol on other enzymes or proteins involved in cholesterol or other metabolic pathways (Jingami et al., 1987; Skalnik et al., 1988; Wang et al., 1994).

It has long been speculated that inside the mammalian cells the true activator of ACAT may be an oxysterol, such as 25-hydroxycholesterol, rather than cholesterol itself. This hypothesis was based on the finding that in intact cells certain oxygenated sterols, when added from ethanol stock solutions to the culture medium, greatly stimulated cholesterol esterification rate and increased ACAT activity; when cholesterol was tested, it failed to elicit the same response (reproduced in the current study (Fig. 5)). To explain the negative result when cholesterol was tested as the stimulating agent in this type of experiment, an alternative interpretation is that the highly compartmentalized distribution of cholesterol in intact cells (DeGrella and Simoni, 1982; Lange and Ramos, 1983; reviewed by Dawidowicz, 1987) has caused the spatial separation of the incoming cholesterol from ACAT located at the cell interior. Once broken cell homogenates are used cholesterol added to the cell extracts is readily available to ACAT. If cholesterol itself can activate ACAT, then under carefully chosen conditions its activating effect should be readily demonstrable. The result of our current work has provided direct evidence in support of the latter interpretation. While not negating the possibility that oxysterol(s) may be physiological regulator(s) of ACAT under certain situations, our current work provides the experimental basis to hypothesize that, inside the mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.

The molecular mechanism by which cholesterol activates ACAT in vitro is not clear at present. In principle, cholesterol could activate ACAT activity by one of at least three mechanisms: first, it is possible that binding of cholesterol by ACAT causes a ligand induced structural/configurational change in the ACAT protein, which converts the enzyme from an inactive form to an active form (Monod et al., 1963; Koshl and and Neet, 1968). The ligand-induced configurational change may occur through interactions among multiple subunits in the ACAT holoenzyme, since the molecular mass of the functional ACAT enzyme has been reported to be approximately 220 kDa (Erickson et al., 1994), which is about four times the value as estimated by SDS-PAGE. This model explains the recurring finding that, the ACAT activity-cholesterol concentration curve is sigmoidal, once ACAT is reconstituted in vesicles with a defined lipid composition (inset of Fig. 1C; Doolittle and Chang 1982a, 1982b; Cadigan and Chang, 1988). If this model turns out to be correct, it would be interesting to find out, within the same ACAT protein, whether the cholesterol-binding site that causes activation to occur is the same as or distinct from the catalytic site for cholesterol esterification. It would also be important to determine, if 25-hydroxycholesterol shares the same site(s) with cholesterol. Since ACAT is believed to span the phospholipid bilayer (Chang et al., 1993), it is possible that the interaction between sterol and ACAT protein may involve phospholipid as another lipid participant. The second possibility is that there may be a cholesterol-induced specific covalent modification of ACAT protein, which in turn activates the enzyme. It is difficult to rationalize why such a mechanism would exist in the extracts of Sf9 cells which normally do not contain endogenous ACAT-like activity. However, the ACAT protein expressed in these cells may contain intrinsic activity for covalently modifying and activating itself upon encountering cholesterol. This model can be rigorously tested once the ACAT protein is purified to homogeneity. The third possibility is that cholesterol activates the ACAT catalysis not by acting on the ACAT protein, but by facilitating the translocation/delivery of substrate to the ACAT catalytic site(s). While this remains as a viable mechanism to explain some of the activation effects of oxysterols on ACAT activity seen in intact mammalian cells, it is not compatible with the result presented in Fig. 10, in which 25-hydroxycholesterol at 0.1 µg/ml was used as the substrate to measure ACAT activity. At this concentration, 25-hydroxycholesterol is 100% soluble in water (Sinensky(1981) reported that the solubility of 25-hydroxycholesterol in water is approximately 0.4 µg/ml). It is therefore difficult to envision a specific mechanism by which cholesterol added to the extracts could facilitate the delivery of a water-soluble substrate to ACAT. In any event, utilizing the cell-free system reported in the current study, further biochemical analyses will shed light on the molecular mechanism(s) by which cholesterol/oxysterol activates the ACAT activity.

During the preparation of this article, Becker et al.(1994) reported that expression of the full-length cDNA encoding the human macrophage carboxylesterase enzyme in wild-type CHO cells led to an approximately 3-fold increase in cellular ACAT activity. This gene was previously cloned and sequenced by Munger et al.(1991) who showed that it encodes the major serine esterase contained within and secreted by alveolar macrophages. This enzyme is also present in abundance in liver microsomes (Ozols, 1989). Shown by various investigators, this enzyme is capable of catalyzing the hydrolysis of esters, amides, and thioesters and is believed to function as a detoxication enzyme (Heymann, 1980). In vitro experiments have shown that long chain fatty-acyl CoAs are poor substrates for this enzyme (Kroetz et al., 1993). Ozols provided evidence(1989) that this enzyme resides within the lumen of the ER. This result suggests that physiologically this enzyme would not be able to use long chain fatty-acyl CoAs as its normal substrates, since the ER membranes are not permeable to long chain fatty-acyl CoAs (for a review, see Coleman and Bell, 1983). Since the human carboxylesterase gene has been expressed in Sf9 cells (Kroetz et al., 1993) it would be interesting to see if it constitutes ACAT-like enzyme activity with catalytic and regulatory properties consistent with what would be expected from the literature.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL36709. 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.

§
To whom correspondence should be addressed. Tel.: 603-650-1622; Fax: 603-650-1483.

(^2)
CI 976 is a derivative of fatty acid amide (Krause et al., 1989); octimibate is a derivative of triphenylimidazole (Lautenschlager et al., 1986); Dup 128 is a derivative of trisubstituted urea (Billheimer et al., 1989).

(^1)
The abbreviations used are: ACAT, acyl-coenzyme A:cholesterol acyltransferase; AcMNPV, Autographa California nuclear polyhedrosis virus; CHO, Chinese hamster ovary; Me(2)SO, dimethyl sulfoxide; ER, endoplasmic reticulum; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; TLC, thin layer chromatography; LDL, low density lipoprotein; FBS, fetal bovine serum; kb, kilobase(s); PBS, phosphate-buffered saline; pfu, plaque-forming unit(s).


ACKNOWLEDGEMENTS

We thank Drs. Gus Lienhard, Larry Chan, Roger Newton, Bo-liang Li, Zining Wu, and Brian Lavan for valuable discussions during the course of this work. We also thank Drs. Roger Newton and Brian Krause for providing ACAT inhibitors.


REFERENCES

  1. Becker, A., Bottcher, A., Lackner, K. J., Fehringer, P., Notka, F., Aslanidis, C., and Schmitz, G. (1994) Arterioscler. Thromb. 14, 1346-1355 [Abstract]
  2. Billheimer, J. T., Gillies, P. J., Wexler, R. R., Higley, C. A., and Maduskuie, T. P., Jr. (1990) European Patent 0372445
  3. Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223-261 [CrossRef][Medline] [Order article via Infotrieve]
  4. Brown, M. S., Dana, S. E., and Goldstein, J. L. (1975) J. Biol. Chem. 250, 4925-4027
  5. Cadigan, K. M., and Chang, T. Y. (1988) J. Lipid Res. 29, 1683-1692 [Abstract]
  6. Cadigan, K. M., Heider, J. G., and Chang, T. Y. (1988) J. Biol. Chem. 263, 274-282 [Abstract/Free Full Text]
  7. Cadigan, K. M., Chang, C. C. Y., and Chang, T. Y. (1989) J. Cell Biol. 108, 2201-2210 [Abstract]
  8. Chang, C. C. Y., and Chang, T. Y. (1986) Biochemistry 25, 1700-1706 [Medline] [Order article via Infotrieve]
  9. 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]
  10. Chang, C. C. Y., Noll, W., Nutile-McMenemy, N., Lindsay, E. A., Baldini, A., Chang, W., and Chang T. Y. (1994a) Somatic Cell Mol. Genet. 20, 71-74 [Medline] [Order article via Infotrieve]
  11. Chang, T. Y., and Doolittle, G. M. (1983) Enzymes 16, 523-539
  12. Chang, T. Y., Limanek, J. S., and Chang C. C. Y. (1981) Anal. Biochem. 116, 298- 302
  13. Chang, T. Y., Chang, C. C. Y., and Cadigan, K. M. (1994b) Trends Cardiovas. Med. 4, 223-230 [CrossRef]
  14. Coleman, R. A., and Bell, R. M. (1983) Enzymes 16, 605-625
  15. Dahl, N. K., Reed, K. L., Daunais, M. A, Faust, J. R, Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896 [Abstract/Free Full Text]
  16. Dawidowicz, E. A. (1987) Annu. Rev. Biochem. 56, 43-62 [CrossRef][Medline] [Order article via Infotrieve]
  17. DeGrella, R. F., and Simoni, R. D. (1982) J. Biol. Chem. 257, 14256-14262 [Abstract/Free Full Text]
  18. Doolittle, G. M., and Chang, T. Y. (1982a) Biochim. Biophys. Acta 713, 529-537 [Medline] [Order article via Infotrieve]
  19. Doolittle, G. M., and Chang, T. Y. (1982b) Biochemistry 21, 674-679 [Medline] [Order article via Infotrieve]
  20. Erickson, S. K., Shrewsbury, M. A., Brooks, C., and Meyer, D. J. (1980) J. Lipid Res. 21, 930-941 [Abstract]
  21. Erickson, S. K., Lear, S. R., and McCreery, M. J. (1994) J. Lipid Res. 35, 763-769 [Abstract]
  22. Goldstein, J. L. and Brown, M. S. (1977) Annu. Rev. Biochem.. 46, 897-930 [CrossRef][Medline] [Order article via Infotrieve]
  23. Goldstein, J. L., Dana, S. E., and Brown, M. S. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4288-4292 [Abstract]
  24. Harte, R. A., Jackson, B., Suckling, K. E., and Yeaman, S. J. (1993) Biochem. Soc. Trans. 21, 3255
  25. Heymann, E. (1980) in Enzyme Basis of Detoxication (Jakoby, W. B., ed) Vol. II, pp. 291-323, Academic Press, New York
  26. Jingami, H., Brown, M. S., Goldstein, J. L., Anderson, R. G. W., and Lusckey, K. L. (1987) J. Cell Biol. 104, 1693-1704 [Abstract]
  27. Kartner, N., Hanrahan, J. W., Jensen, T. J., Naismith, A. L., Sun, S., Ackerly, C. A., Reyes, E. F., Tsui, L. -C., Rommens, J. M., Bear, C. E., and Riordan, J. R. (1991) Cell 64, 681-691 [Medline] [Order article via Infotrieve]
  28. Koshland, D. E., Jr., and K. E. Neet (1968) Annu. Rev. Biochem. 37, 359-410 [CrossRef][Medline] [Order article via Infotrieve]
  29. Krause, B. R., Hoefle, M. L., Holmes, A., Roth, B. D., Kieft, K. A., Uhlendorf, P. D., Standfield, R. L., Sekerke, C. S., Bocan, T. M. A., and Newton, R. S. (1989) 10th International Symposium on Drugs Affecting Lipid Metabolism, November 8th-11th, 1989, Houston, TX, (Abstr. 422)
  30. Kroetz, D. L., McBride, O. W, and Gonzalez, F. J. (1993) Biochemistry 32, 11606-11617 [Medline] [Order article via Infotrieve]
  31. Kruth, H. S., Comly, M. E., Butler, J. D., Vanier, M. T., Fink, J. K., Wenger, D. A., Patel, S., and Pentchev, P. G. (1986) J. Biol. Chem. 261, 16769-16744 [Abstract/Free Full Text]
  32. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  33. Lange, Y., and Ramos, B. V. (1983) J. Biol. Chem. 258, 15130-15134 [Abstract/Free Full Text]
  34. Lange, Y., Strebel, F., and Steck, T. L. (1993) J. Biol. Chem. 268, 13838-13843 [Abstract/Free Full Text]
  35. Lautenschlager, H. H., Prop, G., and Niemann, R. (1986) Drugs Future 11, 26
  36. Lichtenstein, A. H., and Brecher, P. (1980) J. Biol. Chem. 255, 9098-9104 [Free Full Text]
  37. Liscum, L., and Faust, J. R. (1994) Curr. Opin. Lipid. 5, 221-226
  38. Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625-1636 [Abstract]
  39. Monod, J., Changeux, J.-P., and Jacob, F. (1963) J. Mol. Biol. 6, 306-329
  40. Munger, J. S., Shi, G. Mark, E. A., Chin, D. T., Gerard, C., and Chapman, H. A. (1991) J. Biol. Chem. 266, 18832-18838 [Abstract/Free Full Text]
  41. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Company, NY
  42. Ozols, J. (1989) J. Biol. Chem. 264, 12533-12545 [Abstract/Free Full Text]
  43. Ross, R. (1986) New. Engl. J. Med. 314, 488-500 [Medline] [Order article via Infotrieve]
  44. Rudel, L. L., and Morris, M. D. (1973) J. Lipid Res. 14, 364-366 [Abstract/Free Full Text]
  45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  46. Sinensky, M. (1981) Arch. Biochem. Biophys. 209, 321-324 [Medline] [Order article via Infotrieve]
  47. Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841 [Abstract/Free Full Text]
  48. Suckling, K. E., and Stange, E. F. (1985) J. Lipid Res. 26, 647-671 [Medline] [Order article via Infotrieve]
  49. Summers, M. D., and Smith, G. E. (1987) Tex. Agric. Exp. St. Bull. 1555, 10-39
  50. Tabas, I., Rosoff, W. J., and Boykow, G. C. (1988) J. Biol. Chem. 263, 1266-1272 [Abstract/Free Full Text]
  51. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acd. Sci. U. S. A. 76, 4350-4354 [Abstract]
  52. Ventimiglia, J. B., Levesque, M. C., and Chang, T. Y. (1986) Anal. Biochem. 157, 323-330
  53. Wang, X., Sato, R., Brown, M. S., Hua, X., Goldstein, J. L. (1994) Cell 77, 53-62 [Medline] [Order article via Infotrieve]

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