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
analysis was performed using the Student’s t-test. P- values B 0.05 denoted the presence of significant statis-
tical differences.
3. Results
3
.
1
. Effect of probucol on Ac-LDL-induced cholesterol accumulation in mouse macrophages
To elucidate the effect of probucol on cholesterol metabolism in mouse macrophages, we first examined
the effect of probucol on Ac-LDL-induced cholesterol- accumulation in macrophages. When mouse resident
peritoneal macrophages were incubated with Ac-LDL in medium A containing 10 NCS, a significant accu-
mulation of cholesteryl esters was observed Fig. 1A. When macrophages were incubated with probucol
alone 5 mM, probucol did not affect cholesterol con- tent in macrophages. In contrast, when cells were incu-
bated with both Ac-LDL and probucol, cholesteryl esters doubled as compared to Ac-LDL alone Fig.
1A. Ac-LDL-induced accumulation of cholesteryl es- ters was enhanced by probucol in a dose-dependent
manner and reached plateau level at 5 mM of probucol Fig. 1B. To elucidate the enhancing mechanism of
probucol
on Ac-LDL-induced
accumulation of
cholesteryl esters in mouse macrophages, we next exam- ined the effects of probucol on Ac-LDL uptake by
macrophages and endogenous cholesterol synthesis in macrophages in medium A containing 10 NCS. When
macrophages were incubated with [
125
I]Ac-LDL, cell-as- sociation and degradation of [
125
I]Ac-LDL increased dose-dependently Fig. 2. Probucol did not affect cell-
association of [
125
I]Ac-LDL to macrophages or endo- cytic degradation of [
125
I]Ac-LDL by macrophages Fig. 2. Moreover, synthesis of endogenous cholesterol
assessed by the incorporation of [
14
C]acetate into sterols was not affected by probucol data not shown. These
findings suggested that probucol significantly enhanced Ac-LDL-induced accumulation of cholesteryl esters in
mouse macrophages in medium A containing 10 NCS by mechanisms other than enhancement of Ac-LDL
uptake or endogenous cholesterol synthesis.
Fig. 1. Effect of probucol on Ac-LDL-induced cholesterol accumula- tion in macrophages. A; Mouse resident macrophages 3 × 10
6
cells were incubated at 37°C for 36 h with medium A containing 10 NCS
together with 20 mgml of Ac-LDL in the presence or absence of 5 mM of probucol. B; Macrophages 3 × 10
6
cells were incubated with 20 mgml of Ac-LDL in the absence or presence of the indicated
concentrations of probucol for 36 h. Cellular lipids were extracted and determination of free cholesterol closed column and cholesteryl
esters open column was performed as described in Section 2. Data represent the mean of three separate experiments. Error bars repre-
sent SD values. In pannel B, data represent an increase in cholesteryl ester content, which was calculated by subtracting cholesteryl ester
content in Ac-LDL-loaded cells from that in cells incubated with Ac-LDL and probucol. ; P B 0.05 as compared with Ac-LDL alone,
; P B 0.01 as compared with Ac-LDL alone.
Fig. 2. Effect of probucol on cell-association to and degradation by macrophages of [
125
I]Ac-LDL. Mouse resident macrophages 1 × 10
6
cells were incubated at 37°C for 36 h with medium A containing 10 NCS with the indicated concentrations of [
125
I]Ac-LDL in the pres- ence
or absencee
of 5 mM of probucol or 20 fold excess
unlabeled Ac-LDL. The cell-associated radioactivity A and TCA- soluble radioactivity in the medium B were determined as described
in Section 2. Specific cell-association and specific degradation were determined by substraction of non-specific association and non-spe-
cific degradation from total-association and total-degradation, respec- tively. Data represent the mean of three separate experiments. Error
bars represent SD values.
probucol-mediated enhancement of Ac-LDL-induced cholesterol accumulation in mouse macrophages. Thus,
to test whether probucol promotes the uptake of LDL in NCS by macrophages, we examined the effect of
probucol on the cell-association and degradation of [
125
I] LDL in medium A containing 3 BSA. However, probucol did not affect cell-association and degradation
of [
125
I] LDL in the presence or abscence of Ac-LDL data not shown. We next examined the effect of
probucol on HDL-mediated cholesterol efflux from mouse macrophages. As shown in Fig. 3 columns C
and D, when cells were incubated with Ac-LDL and probucol in medium A containing 3 BSA, probucol
did not affect cholesterol contents in macrophages. However, incubation of cells with Ac-LDL and HDL
resulted in a significant fall in cholesteryl ester-content as compared to that induced by Ac-LDL alone. These
results suggested that Ac-LDL-induced accumulation of cholesteryl esters was significantly reduced by HDL-
mediated cholesterol efflux Fig. 3, columns C and E. Under these conditions, when cells were incubated to-
gether with probucol, cholesteryl ester-content com- pletely recovered to the level of Ac-LDL-loaded cells
Fig. 3, column E and F. The latter level was similar to probucol-mediated enhancement of cholesteryl ester ac-
cumulation in medium A containing 10 NCS Fig. 3, columns A and B. These results suggested that probu-
col might inhibit HDL-mediated cholesterol efflux from mouse macrophages in simultaneous incubation system.
We next examined the effect of probucol on HDL- mediated cholesterol efflux in medium A containing 3
BSA under sequential incubation system. Cholesteryl esters were accumulated at concentration of 40 nmol
mg cell protein upon incubation with Ac-LDL, which was reduced by HDL to 18 nmol mg cell protein. In
contrast to the result of simultaneous incubation sys- tem, probucol-mediated accumulation of cholesteryl es-
ters in macrophages was only 5 nmol mg cell protein in sequential incubation system.
3
.
3
. Effect of probucol on
[
125
I
]
HDL binding to macrophages
We next examined the effect of probucol on the binding of HDL to mouse macrophages. As shown in
Fig. 4, incubation of macrophages with [
125
I]HDL in medium A containing 3 BSA, resulted in a dose-de-
pendent increase in specific binding of [
125
I]HDL, which was significantly but partially 30 inhibited by probu-
col. Three HDL binding proteins are so far proposed, such as HDL binding protein HBP [35], scavenger
receptor, classB, typeI SR-BI [36] and HDL binding protein 2 HB2 [37]. Among them, we examined the
expression of SR-BI, a well established HDL receptor [36], at mRNA level using RT-PCR. When mouse
peritoneal macrophages were incubated with medium
Fig. 3. Effect of probucol on HDL-mediated cholesterol efflux. Mouse resident macrophages 3 × 10
6
cells were incubated with medium A containing 10 NCS A and B or 3 BSA C, D, E and
F 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. Cellular lipids
were extracted and determination of free cholesterol and cholesteryl esters was performed as described in Section 2. A, Ac-LDL; B,
Ac-LDL and probucol; C, Ac-LDL; D, Ac-LDL and probucol; E, Ac-LDL and HDL; F, Ac-LDL, probucol and HDL. Data represent
the mean of three separate experiments. Error bars represent SD values.
3
.
2
. Effect of probucol on HDL-mediated cholesterol efflux
NCS used in the present study contained 35 mg cholesterolml of LDL 19 mg proteinml and 85 mg
cholesterolml of HDL 220 mg proteinml. It is, there- fore, possible that these lipoproteins are involved in
alone, a significant band of SR-BI mRNA was ob- served. This band was not affected by the presence of 5
mM of probucol data not shown. These results sug- gested that probucol might inhibit the binding of HDL
to mouse macrophages through certain HDL recep- tors other than SR-BI, thereby inhibiting HDL-medi-
ated cholesterol efflux.
3
.
4
. Effect of probucol on cholesteryl ester cycle in macrophages
A previous study demonstrated that the rate of cholesteryl ester turnover in macrophages was a critical
factor for HDL-mediated cholesterol efflux [9]. There- fore, we next examined NCEH activity and ACAT
activity in mouse macrophages. Fig. 5 shows the effect of probucol on decreasing rate of cholesteryl esters
which was determined as NCEH activity. Macrophages were incubated with Ac-LDL in the presence of
[
3
H]oleate for labeling of cellular cholesteryl esters. After equilibration, cells were incubated with probucol
Fig. 5. Effect of probucol on hydrolysis of cholesteryl esters whole cell NCEH activity. Mouse peritoneal macrophages 1 × 10
6
cells were incubated for 18 h with medium A containing 10 NCS
together with 20 mgml of Ac-LDL in the presence of 0.1 mM of [
3
H]oleate. After equilibration for 6 h with medium A containing 10 NCS in the presence of [
3
H]oleate, cells were chased in medium A containing 10 NCS in the presence
or absence
of 5 mM of
probucol. At the indicated time itervals, cells were harvested for determination of radioactivity of cholesteryl [
3
H]oleate. The results are expressed as percentages of the initial amounts of cholesteryl
[
3
H]oleate A and the amounts of cholesteryl [
3
H]oleate decreased by NCEH for 36 h B. Data represent the mean of three separate
experiments. Error bars represent SD values.
Fig. 4. Effect of probucol on HDL binding to macrophages. Mouse resident macrophages 1 × 10
6
cells were preincubated for 36 h with medium A containing 3 BSA in the presence
or absence
of
5 mM of probucol and then incubated at 37°C for 2 h in medium A containing 3 BSA together with the indicated concentrations of
[
125
I]HDL in the absence or presence of excess unlabeled HDL. The specific binding of HDL was determined as described in Section 2.
Data represent the mean of three separate experiments. Error bars represent SD values. c P B 0.05 compared to control.
for indicated time intervals. The rate of decrease in cholesteryl [
3
H]oleate in control cells was 50 within 36 h, a process not affected by probucol Fig. 5. This
result suggested that probucol did not affect hydrolysis of cholesteryl esters in mouse macrophages in medium
A containing 10 NCS. We then evaluated the effect of probucol on whole cell ACAT activity. As shown in
Fig. 6, probucol alone significantly increased whole cell ACAT activity. Moreover, incubation of cells with both
probucol and Ac-LDL resulted in a further increase in whole cell ACAT activity compared to Ac-LDL alone
Fig. 6. To confirm this finding, we next examined the re-constituted ACAT activity. As shown in Fig. 7,
probucol doubled re-constituted ACAT activity, while Ac-LDL increased the activity by 2.3 fold and Ac-LDL
combined with probucol by three fold, compared to non-loaded cells. We also examined the protein content
of ACAT-1 in mouse macrophages by western blotting using polyclonal rabbit anti-human ACAT-1 antibodies
DM10. As shown in Fig. 8, probucol alone signifi- cantly
increased ACAT-1
protein in
mouse macrophages. In contrast to re-constituted ACAT ac-
tivity, ACAT-1 protein was not increased by Ac-LDL combined with probucol compared to Ac-LDL alone.
Finally, we examined mRNA content of ACAT-1 using RT-PCR analysis. The expression of ACAT-1 mRNA
did not increase by probucol or Ac-LDL compared to non-loaded cells data not shown.
4. Discussion
