cation by ACAT. This cholesteryl ester turnover was discovered by Brown et al. [8] and was termed
‘cholesteryl ester cycle’, which closely related to choles- terol efflux by high-density lipoprotein HDL [9,10].
Probucol [bis3, 5-d-tert-butyl-4-hydroxyphenylthio- propane] is a lipophilic agent with anti-oxidative prop-
erties. It is carried by lipoproteins, mainly in LDL, and is known to prevent oxidative modification of LDL by
its radical-scavenging phenol structure [11]. Oxidative modification of LDL is an early pathogenic event in the
development of atherosclerotic lesions.
Moreover, probucol is also known to lower plasma LDL levels by
a mechanism other than LDL receptor-mediated path- way [12], because probucol lowers LDL cholesterol in
homozygous LDL receptor-deficient patients. There- fore, probucol is expected to inhibit or reduce the
development of atherosclerosis. In fact, subsequent studies showed that probucol reduced atherosclerotic
lesions in Watanabe heritable hyperlipidemic WHHL rabbits
[13,14]. Furthermore,
probucol treatment
caused regression of subcutaneous and tendinous xan- thomas in hypercholesterolemic patients [15]. However,
a large-scale European clinical trial probucol quantita- tive regression Swedish trial, PQRST demonstrated
that probucol treatment did not produce a significant effect on atherosclerotic lesions of human femoral
artery, although it reduced cholesterol levels [16]. More- over, a recent report using apoE − − mice demon-
strated
that probucol
treatment enhanced
the development of atherosclerotic lesions more rapidly
than untreated apoE − − mice. In addition, probucol treatment also accelerated lesion development in apoE
+ − mice fed an atherogenic diet, indicating that the
adverse effect is not dependent on the complete absence of apoE. Interestingly, the plasma lipoprotein profile in
mice lacking both apoE and apoA-I was very similar to probucol-treated apoE − − mice, but did not acceler-
ate atherosclerotic lesions [17]. These reports suggested that probucol might directly affect the development of
atherosclerotic lesions, thereby worsening atherosclero- sis in apoE − − mice. In this regard, Tsujita and
Yokoyama [18] recently reported that probucol selec- tively inhibited apoA-I-mediated cellular lipid efflux
from cultured mouse macrophage-derived foam cells. Thus, it is possible that probucol may also affect
cholesterol metabolism in mouse macrophages.
In the present study, we investigated the effects of probucol on cholesterol metabolism in mouse peri-
toneal resident macrophages, and demonstrated that probucol inhibited HDL-mediated cholesterol efflux
from macrophages, which might be due to the inhibi- tion of binding of HDL to cells and reduction of
HDL-accessible free cholesterol content via activation of ACAT activity. This may be one of the underlying
mechanisms of the harmful effects of probucol on the development of atherosclerosis in apoE − − mice.
2. Materials and methods
2
.
1
. Materials Probucol, [bis3,5-d-tert-butyl-4-hydroxyphenylthio-
propane], and bovine serum albumin Fraction V were purchased from Sigma Chemical St. Louis, MO.
Probucol was dissolved in 99.5 ethanol and then diluted with culture medium. Final concentration of
ethanol was B 0.1 in the culture medium, which did not show any cytotoxic effect, and final concentration
of probucol B 20 mM did not have any cytotoxic effect, determined by MTT assay and lactic dehydrogenase
release. RPMI-1640 was from Gibco BRL Long Is- land, NY. Newborn calf serum NCS was from Hy-
clone
Laboratories [9,10n-
3
H]oleic acid
370 GBqmmol, Na
125
I3.7 GBqml and [1-
14
C]oleoyl CoA 1.85 GBqml were from Amersham Life Science
Buckinghamshire, UK. Other chemicals were the best grade available from commercial sources.
2
.
2
. Lipoproteins and their modifications LDL d = 1.019 – 1.063 gml and HDL d = 1.063 –
1.21 gml were isolated by sequential ultracentrifuga- tion of fresh plasma samples obtained from consented
normolipidemic subjects after overnight fasting. Traces of apoB and E were removed from HDL by a heparin
agarose column [19]. Ac-LDL was prepared by chemi- cal modification of LDL with acetic anhydride as de-
scribed previously [20]. Iodination of lipoproteins with
125
I was performed according to the method of McFar- lane [21]. Protein concentrations were determined by
BCA protein assay reagents Pierce and expressed as mg proteinml. The level of endotoxin associated with
these lipoproteins was B 1.0 pgmg protein, which was measured by a commercially available kit [22]. Under
our experimental conditions, endotoxin at concentra- tions of B 1 ngml did not affect cell viability [23].
2
.
3
. Cells Mouse peritoneal macrophages were collected from
nonstimulated male DDY mice 25 – 30 g with 8 ml of ice-cold PBS, centrifuged at 700 × g for 5 min and
suspended in RPMI-1640 medium containing strepto- mycin 0.1 mgml and penicillin 100 unitsml
medium A together with 3 BSA. The cell suspension was seeded in each well and incubated for 90 min. The
macrophage monolayers thus formed were washed three times with PBS and used in the following experi-
ments. Unless otherwise specified, all cellular experi- ments were performed humidified atmosphere 5 CO
2
in air at 37°C, and culture medium contained 0.1 ethanol as a vehicle control.
2
.
4
. Determination of cellular cholesterol content Macrophage monolayers 3 × 10
6
cells formed in each well 34 mm in diameter were incubated with
medium A containing 10 NCS or 3 BSA together with 20 mgml of Ac-LDL andor 250 mgml of HDL
for 36 h in the presence or absence of 5 mM of probucol simultaneous incubation system. Macrophages were
incubated with 20 mgml of Ac-LDL for 18 h and then incubated with 250 mgml of HDL for 36 h sequential
incubation system. After incubation, cellular lipids were extracted and both total cholesterol and free
cholesterol were quantified using a modified enzymatic fluorometric method [24]. The level of cholesteryl esters
was calculated by subtracting free cholesterol from total cholesterol.
2
.
5
. Cell-association and degradation of lipoproteins Macrophage monolayers 1 × 10
6
cells formed in each well 22 mm in diameter were incubated at 37°C,
for 36 h with 10 mgml of [
125
I]LDL or the indicated concentrations of [
125
I]Ac-LDL with or without 20-fold excess of unlabeled LDL or unlabeled Ac-LDL, respec-
tively, in the presence or absence of 5 mM of probucol. Endocytic degradation of [
125
I]LDL or [
125
I]Ac-LDL was determined by TCA-soluble radioactivity in the
medium after precipitating free iodine with AgNO
3
as described previously [25]. Cells were solubilized with 1.0
ml of 0.1 N NaOH and the cell-associated radioactivity was determined as described previously [25].
2
.
6
. Specific binding of
[
125
I
]
HDL to macrophages Macrophage monolayers 1 × 10
6
cells formed in each well 22 mm in diameter were preincubated for 24
h with medium A containing 3 BSA in the presence or absence of 5 mM of probucol. The monolayers were
washed three times with 1 ml PBS, then incubated at 37°C for 2 h in 1 ml of medium A containing 3 BSA
together with the indicated concentrations of [
125
I]HDL in the absence or presence of excess amount of unla-
beled HDL. Cells were washed and solubilized with 1 ml of 0.1 N NaOH followed by determination of ra-
dioactivity. Specific binding was calculated by subtract- ing non-specific binding from total binding [26].
2
.
7
. Hydrolysis of cholesteryl esters whole cell NCEH
acti6ity Macrophages 1 × 10
6
cells were first converted to form cells by incubation for 18 h with medium A
containing 10 NCS together with 20 mgml of Ac- LDL and 0.1 mM of [
3
H]oleate. After equilibration for 6 h with medium A containing 10 NCS together with
[
3
H]oleate, cells were further incubated for 36 h with medium A containing 10 NCS in the presence or
absence of 5 mM of probucol. Cellular lipids were extracted and resuspended with 180 ml of isopropanol.
Aliquots 30 ml were used for determination of ra- dioactivity of cholesteryl [
3
H]oleate separated by thin layer chromatography TLC [9]. The amount of
cholesteryl [
3
H]oleate was expressed as nmolmg cell protein. The results were expressed as percentage of the
initial amounts of cholesteryl [
3
H]oleate and decreasing rate of cholesteryl [
3
H]oleate determined as NCEH activity.
2
.
8
. Whole cell ACAT acti6ity Macrophage monolayers 1 × 10
6
cells formed in each well 22 mm in diameter were incubated for 24 h
with medium A containing 10 NCS and 20 mgml of Ac-LDL in the presence or absence of 5 mM of probu-
col. Cellular lipids were extracted and resuspended with 180 ml of isopropanol. Aliquots 30 ml were used for
determination of radioactivity of cholesteryl [
3
H]oleate separated by TLC [9]. The radioactivity of cholesteryl
[
3
H]oleate was determined as a whole cell ACAT activ- ity [27]. The amount of cholesteryl [
3
H]oleate was ex- pressed as nmolmg cell protein.
2
.
9
. Preparation of cell homogenate Macrophage monolayers 1 × 10
7
cells formed in each dish 10 cm in diameter were incubated for 36 h
with medium A containing 10 NCS and 20 mgml of Ac-LDL in the presence or absence of 5 mM of probu-
col. Cells were washed with PBS and then lyzed by incubation for 3 min at room temperature with a
hypotonic solution of 1 mM Tris and 1 mM EDTA pH 7.0. The solution was discarded and 400 ml of 50
mM tris – HCl and 1 mM EDTA pH 7.7 was added to each dish. Cells were homogenized by scraping with a
rubber policeman. The cell homogenates thus prepared were finally adjusted to 0.375 mgml with the same
buffer. The homogenate was used for the reconstituted ACAT activity analysis and Western blot analysis.
2
.
10
. Re-constituted ACAT acti6ity Assay for the reconstituted ACAT activity was per-
formed according to the method of Cadigan et al. [28]. This method allowed measurement of ACAT activity
independent of the enzyme’s original lipid environment by adding exogenous cholesterol. A deoxycholate
DOCphosphatidylcholine PC solution was added to cell homogenates to obtain a final concentration of 20
mgml DOC, 4 mgml PC, and 0.3 mgml cell protein. The mixture was incubated for 20 min at 4°C. Aliquots
45 ml of the cell extract were diluted 16-fold vv into cholesterol-PC liposomes prepared by the cholestyra-
mine method [29] cholesterolPC molar ratio = 0.3, and incubated for 10 min at 4°C. After preincubation
for 5 min at 37°C, 180 ml [
14
C]oleoyl CoA-BSA conju- gate 50 mM containing 2.5 mgml fatty acid free BSA
in 0.02 M tris – HCl: 2 × 10
4
dpmnmol was added, followed by incubation for 10 min at 37°C [30,31]. The
reaction was stopped by the addition of CHCl
3
: CH
3
OH 2:1,
v:v [30,31],
and the
radioactive cholesteryl [
14
C]oleate was determined by scintillation spectrophotometer [26].
2
.
11
. Western blot analysis Western blot analysis was performed according to
the method of Cheng et al. [32] using polyclonal rabbit anti-human ACAT-1 antibodies DM10 [33]. DM10
kindly provided by T.Y. Chang is known to cross-re- act with mouse ACAT-1 [34]. Homogenates of cells
were separated by 10 SDS-polyacrylamide gel elec- trophoresis PAGE and transferred to nitrocellulose
membranes at 120 mA for 1 h using a semi-dry blotter Holize Blot ATTO, Tokyo, Japan in buffer system of
25 mM Tris, 190 mM glycine, 20 methanol and 0.01 SDS pH 8.3. The nitrocellulose membranes were then
blocked in 5 Carnation nonfat milk in 20 mM tris – HCl, 150 mM NaCl, and 0.3 Tween-20 pH 7.6 and
reacted with 0.24 mgml of DM10 as a primary anti- body in 1 Carnation milk in the same buffer. The
membranes were washed and then reacted with 0.6 mgml
of goat
anti-rabbit IgG
conjugated with
horseradish peroxidase Bio-Rad Laboratories, Her- cules, CA as a second antibody. The ECL reagent
Amersham visualized
ACAT-1 signals
on the
membranes.
2
.
12
. Statistical analysis Results were expressed as mean 9 SD. Statistical
