Effects of probucol on cholesterol metabolism in mouse peritoneal
macrophages: inhibition of HDL-mediated cholesterol efflux
Toru Takemura *, Masakazu Sakai, Hirofumi Matsuda, Takeshi Matsumura,
Takeshi Biwa, Yoshichika Anami, Takeshi Nishikawa, Takayuki Sasahara,
Motoaki Shichiri
Department of Metabolic Medicine,Kumamoto Uni6ersity School of Medicine,Honjo1-1-1,Kumamoto860-8556,Japan
Received 20 March 1999; received in revised form 16 November 1999; accepted 13 December 1999
Abstract
Macrophage-derived foam cells are known to play an essential role in the development and progression of atherosclerotic lesions. Probucol prevents oxidative modification of low-density lipoprotein (LDL) and lowers plasma contents of LDL and high-density lipoprotein (HDL). A recent report using apoE − / − mice demonstrated that probucol treatment enhanced atherosclerosis in apoE− / − mice more rapidly than that in untreated apoE − / − mice, and a reduction in plasma cholesterol by probucol was not the cause of enhancement of atherosclerotic lesions in probucol-treated apoE − / − mice. Moreover, probucol was reported to inhibit apoA-I mediated cholesterol efflux from mouse macrophages. These reports suggested that probucol might directly affect cholesterol metabolism in mouse macrophages. Thus, we investigated the effects of probucol on cholesterol metabolism in mouse resident peritoneal macrophages. Probucol did not affect degradation of acetylated LDL (Ac-LDL), degradation of LDL and endogenous cholesterol synthesis in mouse macrophages. However, it significantly inhibited HDL-mediated cholesterol efflux. Moreover, probucol partially (30%) inhibited the binding of HDL to mouse macrophages, and significantly activated acyl-coenzyme A:cholesterol acyltransferase (ACAT). Our results suggested that probucol inhibited HDL-mediated cholesterol efflux by inhibiting the binding of HDL to mouse macrophages and reducing HDL-accessible free cholesterol content by ACAT activation, thereby worsening atherosclerotic lesions in apoE − / − mice. However, it remains unclear whether probucol inhibits HDL-mediated cholesterol efflux from human macrophages. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Probucol; Mouse peritoneal macrophage; Acetylated low-density lipoprotein; High-density lipoprotein; Acyl-coenzyme A: cholesterol acyltransferase
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1. Introduction
The presence of a massive cluster of foam cells is a characteristic feature of atherosclerotic lesions [1]. Re-cent immunohistochemical and pathological studies have revealed that foam cells appear in the early stages of atherosclerotic lesions and are mainly blood mono-cyte-derived macrophages [2,3]. These cells play an important role in the development and progression of atherosclerosis via production of various bioactive molecules, such as growth factors and cytokines [1]. Chemically modified low-density lipoproteins (modified
LDLs), such as acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL), are recognized by so-called scavenger receptors [4], and undergo receptor mediated endocyto-sis, followed by intracellular delivery to lysosomes [5]. In these structures, cholesteryl esters in modified LDLs are hydrolyzed to free cholesterol by acid cholesterol esterase (ACEH) in lysosome [6]. Free cholesterol thus formed is then transported across lysosomal membrane into cytoplasm [7] where it is delivered to the endoplas-mic reticulum. Free cholesterol is re-esterified to cholesteryl esters by coenzyme A:cholesterol acyl-transferase (ACAT), leading to accumulation of cholesteryl esters in cytoplasm. Cytoplasmic cholesteryl esters undergo a continual shuttle between hydrolysis by neutral cholesteryl esterase (NCEH) and re-esterifi-* Corresponding author. Tel.: +81-96-3735169; fax: +
81-96-3668397.
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 [bis(3, 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 reducinhibi-tion 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, [bis(3,5-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 B0.1% in the culture medium, which did not show any cytotoxic effect, and final concentration of probucol B20mM 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 [9,10(n)-3H]oleic acid (370 GBq/mmol), Na125I(3.7 GBq
/ml) and [1-14C]oleoyl CoA (1.85 GBq/ml) 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 g/ml) and HDL (d=1.063 – 1.21 g/ml) 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 125I was performed according to the method of
McFar-lane [21]. Protein concentrations were determined by BCA protein assay reagents (Pierce) and expressed as mg protein/ml. The level of endotoxin associated with these lipoproteins was B1.0 pg/mg protein, which was measured by a commercially available kit [22]. Under our experimental conditions, endotoxin at concentra-tions of B1 ng/ml did not affect cell viability [23].
2.3. Cells
2.4. Determination of cellular cholesterol content
Macrophage monolayers (3×106 cells) formed in each well (34 mm in diameter) were incubated with medium A containing 10% NCS or 3% BSA together with 20 mg/ml of Ac-LDL and/or 250 mg/ml of HDL for 36 h in the presence or absence of 5mM of probucol (simultaneous incubation system). Macrophages were incubated with 20mg/ml of Ac-LDL for 18 h and then incubated with 250mg/ml 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 (1×106 cells) formed in each well (22 mm in diameter) were incubated at 37°C, for 36 h with 10 mg/ml of [125I]LDL or the indicated concentrations of [125I]Ac-LDL with or without 20-fold excess of unlabeled LDL or unlabeled Ac-LDL, respec-tively, in the presence or absence of 5mM 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 AgNO3 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 [125I]HDL to macrophages
Macrophage monolayers (1×106 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 5mM 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 bindsubtract-ing from total bindsubtract-ing [26].
2.7. Hydrolysis of cholesteryl esters (whole cell NCEH acti6ity)
Macrophages (1×106 cells) were first converted to form cells by incubation for 18 h with medium A containing 10% NCS together with 20 mg/ml of Ac-LDL and 0.1 mM of [3
H]oleate. After equilibration for 6 h with medium A containing 10% NCS together with [3H]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 [3H]oleate separated by thin layer chromatography (TLC) [9]. The amount of cholesteryl [3H]oleate was expressed as nmol/mg cell protein. The results were expressed as percentage of the initial amounts of cholesteryl [3H]oleate and decreasing rate of cholesteryl [3H]oleate determined as NCEH activity.
2.8. Whole cell ACAT acti6ity
Macrophage monolayers (1×106 cells) formed in each well (22 mm in diameter) were incubated for 24 h with medium A containing 10% NCS and 20 mg/ml of Ac-LDL in the presence or absence of 5mM 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 [3H]oleate separated by TLC [9]. The radioactivity of cholesteryl [3H]oleate was determined as a whole cell ACAT activ-ity [27]. The amount of cholesteryl [3H]oleate was ex-pressed as nmol/mg cell protein.
2.9. Preparation of cell homogenate
Macrophage monolayers (1×107 cells) formed in each dish (10 cm in diameter) were incubated for 36 h with medium A containing 10% NCS and 20 mg/ml of Ac-LDL in the presence or absence of 5mM 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 mg/ml with the same buffer. The homogenate was used for the reconstituted ACAT activity analysis and Western blot analysis.
2.10. Re-constituted ACAT acti6ity
cholestyra-mine method [29] (cholesterol/PC molar ratio=0.3), and incubated for 10 min at 4°C. After preincubation for 5 min at 37°C, 180 ml [14C]oleoyl CoA-BSA conju-gate (50 mM containing 2.5 mg/ml fatty acid free BSA in 0.02 M tris – HCl: 2×104 dpm/nmol) was added, followed by incubation for 10 min at 37°C [30,31]. The reaction was stopped by the addition of CHCl3: CH3OH (2:1, v:v) [30,31], and the radioactive cholesteryl [14C]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 mg/ml 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
mg/ml 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 mean9SD. Statistical analysis was performed using the Student’s t-test. P -values B0.05 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 5mM 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 [125I]Ac-LDL, cell-as-sociation and degradation of [125I]Ac-LDL increased dose-dependently (Fig. 2). Probucol did not affect cell-association of [125I]Ac-LDL to macrophages or endo-cytic degradation of [125I]Ac-LDL by macrophages (Fig. 2). Moreover, synthesis of endogenous cholesterol assessed by the incorporation of [14C]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 mechanism(s) 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 (3×106cells) were incubated at 37°C for 36 h with medium A containing 10% NCS together with 20mg/ml of Ac-LDL in the presence or absence of 5
Fig. 2. Effect of probucol on cell-association to and degradation by macrophages of [125I]Ac-LDL. Mouse resident macrophages (1×106 cells) were incubated at 37°C for 36 h with medium A containing 10% NCS with the indicated concentrations of [125I]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 [125I] LDL in medium A containing 3% BSA. However, probucol did not affect cell-association and degradation of [125I] 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 [125I]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 [125I]HDL in medium A containing 3% BSA, resulted in a dose-de-pendent increase in specific binding of [125I]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 (3×106 cells) were incubated with medium A containing 10% NCS (A and B) or 3% BSA (C, D, E and F) together with 20mg/ml of Ac-LDL and/or 250mg/ml of HDL for 36 h in the presence or absence of 5mM 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
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-tor(s) 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 [3H]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 (1×106 cells) were incubated for 18 h with medium A containing 10% NCS together with 20 mg/ml of Ac-LDL in the presence of 0.1 mM of [3H]oleate. After equilibration for 6 h with medium A containing 10% NCS in the presence of [3H]oleate, cells were chased in medium A containing 10% NCS in the presence () or absence() of 5mM of probucol. At the indicated time itervals, cells were harvested for determination of radioactivity of cholesteryl [3H]oleate. The results are expressed as percentages of the initial amounts of cholesteryl [3H]oleate (A) and the amounts of cholesteryl [3H]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 (1×106cells) were preincubated for 36 h with medium A containing 3% BSA in the presence () or absence () of 5mM of probucol and then incubated at 37°C for 2 h in medium A containing 3% BSA together with the indicated concentrations of [125I]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 PB0.05 compared to control.
4. Discussion
Ac-LDL induced a significant accumulation of cholesteryl esters in mouse macrophages, which was inhibited by simultaneous incubation with HDL (Fig. 3, columns C and E). Since it was reported that HDL does not affect endocytic uptake of Ac-LDL by mouse macrophages, the reduction of Ac-LDL-induced terol accumulation by HDL is explained by its choles-terol efflux [26]. Probucol completely recovered HDL-mediated cholesterol reduction (Fig. 3, columns E and F). Thus, it is possible that probucol inhibits HDL-mediated cholesterol efflux from mouse macrophages. Since the binding of HDL to mouse macrophages was partially (30%) inhibited by probucol (Fig. 4), the inhibition of the binding of HDL, at least in part, is involved in the inhibition of HDL-mediated cholesterol efflux, and there were other inhibitory
Fig. 7. Effect of probucol on re-constituted ACAT activity. Mouse peritoneal macrophages (1×107cells) were incubated for 36 h with medium A containing 10% NCS together with 20mg/ml of Ac-LDL in the presence or absence of 5mM of probucol. Cells were homoge-nized, and then re-constitution ACAT activity was determined as described in Section 2. Data represent the mean of three separate experiments. Error bars represent SD values.
Fig. 6. Effect of probucol on whole cell ACAT activity. Mouse peritoneal macrophages (1×106 cells) were incubated for 24 h with medium A containing 10% NCS together with 20mg/ml of Ac-LDL in the presence or absence of 5 mM of probucol. Cholesteryl [3H]oleate was determined as described in Section 2. Data represent the mean of three separate experiments. Error bar represent SD values.
Fig. 8. Effect of probucol on ACAT-1 protein in macrophages. Mouse peritoneal macrophages (1×107cells) were incubated for 36 h with medium A containing 10% NCS together with 20 mg/ml of Ac-LDL in the presence or absence of 5mM of probucol. Cells were homogenized, and then ACAT-1 protein was determined by western blotting as described in Section 2. The bands corresponding to ACAT-1 are shown. A, medium alone (Control); B, probucol; C, Ac-LDL; D, Ac-LDL and probucol.
of free cholesterol to cholesteryl esters by ACAT, was closely related to cholesterol efflux by HDL [9]. Thus, we examined the effects of probucol on NCEH activity and ACAT activity in mouse macrophages. Our results demonstrated that probucol did not affect NCEH activ-ity (Fig. 5), whereas it significantly enhanced ACAT activity (Fig. 6 and Fig. 7). It is generally accepted that ACAT inhibitor increased free cholesterol accessible to HDL, thereby promoting cholesterol efflux. In fact, it was reported that ACAT inhibitor, 58-035, enhanced HDL-mediated cholesterol efflux from mouse macrophage-derived foam cells [10]. Moreover, in our preliminary experiment, 5 mg/ml of 58-035 completely inhibited probucol-mediated enhancement of choles-terol accumulation induced by Ac-LDL in medium A containing 10% NCS (Takemura et al. unpublished data 1999). Thus, it is possible to speculate that activa-tion of ACAT by probucol may reduce free cholesterol accesible to HDL and then inhibit cholesterol efflux from macrophages, thereby enhancing the accumula-tion of cholesteryl esters inside the mouse macrophages. Tsujita and Yokoyama [18] demonstrated that the lipid-free apoA-I-mediated cholesterol efflux was com-pletely inhibited by probucol, which was explained by the complete inhibition of the binding of apo-A-I to macrophages. They did not show the effect of probucol on the binding of HDL to macrophages, whereas they also demonstrated that probucol completely inhibited HDL-mediated cholesterol efflux [18]. The later result was essentially consistent with our present results, whereas at first glance, the inhibitory effect of probucol on the binding of apoA-I was different from that of HDL. The exact reason remains unclear, but it may be explained by the quite different experimental condi-tions, such as loading of probucol to macrophages, methods of the binding of apoA-I or HDL, and choles-terol acceptors used in the both studies (apoA-I or HDL). Moreover, a following speculation is also possi-ble. If the binding sites in macrophages for free apoA-I would be same to those for apoA-I in HDL, and even if this site would be completely inhibited by probucol, HDL may possess other binding mechanism(s) apart from the apoA-I-mediated binding, because HDL con-sists of apoA-I, apoA-II, apoCs and lipids.
There are two experimental protocols to determine the effect of HDL on cholesterol efflux. One is a conventional protocol, in which macrophages are first incubated with Ac-LDL and converted to foam cells, and then incubated with HDL (sequential incubation). In this system, free cholesterol accessible to HDL is derived from cytoplasmic cholesteryl esters, which is hydrolyzed by NCEH. On the other hand, macrophages are incubated with both Ac-LDL and HDL from the onset of the incubation (simultaneous incubation). In this protocol, free cholesterol is derived from cholesteryl esters in Ac-LDL, which is hydrolyzed
by ACEH in lysosome. One part of lysosome-derived free cholesterol is re-esterified by ACAT, while the other part may be transported to plasma membrane. In this system, HDL-accessible free cholesterol is derived directly from lysosomes in addition to cytoplasmic cholesteryl ester-derived free cholesterol hydrolyzed by NCEH. In the present study, we used the simultaneous incubation system and demonstrated that probucol in-hibited HDL-mediated cholesterol efflux (Fig. 3) and did not affect NCEH activity (Fig. 5). Thus, it is possible to assume that probucol might affect the trans-port of free cholesterol from lysosomes to HDL-accessi-ble free cholesterol pool by activation of ACAT. This conclusion was also supported by the following findings;
1. The enhancing effect of probucol on cholesterol accumulation in sequential incubation system was 5 nmol/mg cell protein, which was significantly weaker than that in simultaneous incubation system (45 nmol/mg cell protein), and
2. ACAT inhibitor, 58-035, completely inhibited the enhancing effect of probucol on cholesterol accumu-lation by Ac-LDL in simultaneous incubation sys-tem (data not shown).
protein life, and translation, would be involved in the regulation of ACAT. The other possibility is the in-volvement of other ACAT spiecies than ACAT-1. Re-cently, Cases et al. [40] demonstrated the presence of other type of ACAT, ACAT-2. Thus, it is possible to assume that ACAT-2 may be activated by probucol or Ac-LDL. Therefore, we should further examine the regulation of ACAT-1 and ACAT-2 by probucol in our future study.
In the present study, we demonstrated that probucol significantly activated ACAT activity (Fig. 6 and Fig. 7), but not affected NCEH activity (Fig. 5). Moreover, probucol partially inhibited the binding of HDL to macrophages (Fig. 4). Since it is well known that ACAT is down-regulated by HDL [41], it is possible to assume that ACAT may be activated by the inhibition of the binding of HDL by probucol. On the other hand, there are conflicting reports demonstrated the interac-tion between the HDL-binding and NCEH activity. Miura et al. demonstrated that HDL could activate NCEH activity in macrophages [42]. However, Oram et al. described in their review that apolipoprotein did not affect NCEH activity [41]. In the present study, NCEH activity was not affected by probucol in the presence of 10% serum which containd 220mg protein of HDL/ml. Moreover, probucol inhibited the binding of HDL to macrophages by 30% (Fig. 4). These results suggested that probucol-mediated inhibition of the binding of HDL might not affect NCEH activity under our exper-imental conditions.
Administration of probucol ameliolates atheroscle-rotic lesions in WHHL rabbits [13,14], but worsenes those in apoE − / − mice [17]. Probucol is known to possess anti-atherogenic properties, such as anti-oxi-dantive effect [11], reduction in plasma LDL cholesterol [12] and alteration of size of HDL particle favourable for cholesterol efflux [43,44]. However, probucol re-duces plasma HDL levels, which might be explained in part by the inhibition of apoA-I-mediated nascent HDL formation [18] and activation of cholesteryl ester transfer protein (CETP) [43 – 45]. The rate of cholesteryl ester cycle was reported to closely relate to HDL-medi-ated cholesterol efflux [9]. Moreover, cholesterol turnover rate was quite slower in rabbit macrophages than that in mouse macrophages [9]. In fact, HDL-me-diated cholesterol efflux from rabbit macrophages was markedly lower than that from mouse macrophages [9]. Thus, even if probucol would completely inhibit HDL-mediated cholesterol efflux in both species, the inhibi-tion of cholesterol efflux by probucol might have a more marked effect on the formation of atherosclerotic lesions in mice than in rabbits.
Another interesting finding observed in this study is that Ac-LDL increased ACAT activity (Fig. 7) and ACAT-1 protein (Fig. 8). Brown et al. [8] previously demonstrated that when mouse macrophages were
in-cubated with Ac-LDL, whole cell ACAT activity was significantly increased, which was partially explained by an increase in free cholesterol, a ACAT substrate. Our results were essentially consistent with their results and explained in part by the mechanism of Ac-LDL-in-duced ACAT activation in mouse macrophages.
In summary, we demonstrated in the present study that probucol inhibited HDL-mediated cholesterol efflux from mouse peritoneal macrophages, probably due to the activation of ACAT and inhibition of HDL binding to mouse macrophages. This conclusion was supported by the following observations:
1. probucol did not affect endogenous cholesterol syn-thesis in mouse macrophages (data not shown), Ac-LDL uptake by mouse macrophages (Fig. 2) and LDL uptake by mouse macrophages (data not shown),
2. probucol completely inhibited HDL-mediated cholesterol efflux from mouse macrophages (Fig. 3), 3. probucol partially inhibited the binding of HDL to
mouse macrophages (Fig. 4),
4. probucol did not affect hydrolysis of cholesteryl esters (Fig. 5), and
5. probucol enhanced ACAT activity in mouse macrophages (Fig. 6 and Fig. 7).
However, it should be noted that it remains unclear whether these mechanisms are also operative in human macrophages, because the present study was performed using mouse macrophages, and because the effect of probucol on human was quite different from those on animals [13 – 18].
Acknowledgements
We gratefully appreciate Prof. Seikoh Horiuchi, Dr Akira Miyazaki and Dr Hideki Hakamata (Department of Biochemistry, Kumamoto University School of Medicine) for helpful discussions, Professor Ta-Yuan Chang (Department of Biochemistry, Dartmouth Medi-cal School) for kindly providing polyclonal rabbit anti-human ACAT-1 antibodies (DM10), Prof. F.G. Issa at Sydney for assistance of preparation of our manuscript and Kenshi Ichinose in our laboratory for helpful tech-nical assistance.
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![Fig. 2. Effect of probucol on cell-association to and degradation bymacrophages of [cells) were incubated at 37°C for 36 h with medium A containing 10%NCS with the indicated concentrations of [ence (125I]Ac-LDL](https://thumb-ap.123doks.com/thumbv2/123dok/3148603.1384469/5.612.30.262.350.496/probucol-association-degradation-bymacrophages-incubated-containing-indicated-concentrations.webp)
![Fig. 4. Effect of probucol on HDL binding to macrophages. Mouserepresent SD values.5containing 3% BSA together with the indicated concentrations of[ c P��) or absence (� �125M of probucol and then incubated at 37°C for 2 h in medium AI]HDL in the absence o](https://thumb-ap.123doks.com/thumbv2/123dok/3148603.1384469/6.612.31.264.337.616/probucol-macrophages-mouserepresent-containing-indicated-concentrations-probucol-incubated.webp)
