EXPERIMENTAL AND MOLECULAR PATHOLOGY 36, 156-163 (1982) The Effect of Bezafibrate and Clofibrate on Cholesterol Ester Metabolism in 3T3 Cells and Smooth Muscle Cells in Tissue Culture KATRINAHUDSON, ALLAN J. DAY, AND ALEXSANDRAMARCEGLIA Department of Physiology, University of Melbourne, ParkviNe, Victoria 3052, Australia Received May 11, 1981, and in revised form September 14, 1981 The effect of bezafibrate and clofibrate on the accumulation and removal of cholesterol ester and on the incorporation, esterification, and removal of *%-labeled cholesterol by both 3T3 cells and pig aortic smooth muscle cells stimulated with cationized low-density lipopro- tein (LDL) has been studied. Bezatibrate at 100 pg/ml reduced the accumulation of choles- terol ester in both cell types. The uptake and esterification of W-labeled cholesterol into cholesterol ester was also reduced by bezatibrate at this concentration. W-labeled choles- terol ester was removed from the cells following its hydrolysis and appeared in the incuba- tion medium as Y-labeled free cholesterol. This process was accelerated by bezafibrate and clotibrate in both cell types. INTRODUCTION The hypolipidemic agent, clofibrate,and its analog, bezafibrate, have been shown to reduce the deposition and to increase the removal of cholesterol ester from cultured 3T3 tibroblasts (Hudson and Day, 1981). In the present work these studies have been continued in order to investigate the effect of these agents on the uptake, esterification, and removal of 14C-labeled cholesterol by both 3T3 cells and aortic smooth muscle cells stimulated by cationized low-density lipoprotein (LDL) in tissue culture. MATERIALS AND METHODS [4-14C]Cholesterol (SO-58 mCi/mmol) was obtained from the Radiochemical Centre, Amersham, and incorporated into serum lipoprotein by incubation at 37°C with fetal calf serum (Medos Pty. Ltd.) for at least 2 hr before its addition to the incubation medium. Bezafibrate was obtained from Boehringer-Mannheim GmbH, West Germany, and clofibrate from Imperial Chemical Industries Ltd., Great Britain. These agents were dissolved in sodium hydroxide and added to Dulbecco’s modified eagle medium (Grand Island Biological Co., New York). The pH was adjusted back to 7.4 with hydrochloric acid. Blood was obtained from the jugular vein of normal pigs or pigs which had been fed for 4-5 months on a diet containing 0.6% cholesterol and 6% beef tallow (Lee et al., 1971)..EDTA, 1 mg/ml blood, was used as anticoagulant. LDL (u’ = 1.006- 1.063) was prepared by ultracentrifugation according to the method de- scribed by Hatch and Lees (1968) and was cationized as described by Basu et al. (1976). 3T3 Balb/c mouse tibroblasts, clone A31 from the American Type Culture Col- lection (Goldberg, 1977) were obtained from St. Vincent’s Hospital, Melbourne. Stock cells were grown in a humidified 5% COZ incubator at 37°C in 75-cm2 flasks (Falcon Plastics, Los Angeles). These cells were passaged twice weekly in a ratio 156 0014-4800/82/020156-08$02.00/O Copyright @ 1982 by Academic Press. Inc. AU rights of reproduction in any form reserved.
Transcript
EXPERIMENTAL AND MOLECULAR PATHOLOGY 36, 156-163 (1982)
The Effect of Bezafibrate and Clofibrate on Cholesterol Ester Metabolism in 3T3 Cells and Smooth Muscle Cells in
Tissue Culture
KATRINAHUDSON, ALLAN J. DAY, AND ALEXSANDRAMARCEGLIA
Department of Physiology, University of Melbourne, ParkviNe, Victoria 3052, Australia
Received May 11, 1981, and in revised form September 14, 1981
The effect of bezafibrate and clofibrate on the accumulation and removal of cholesterol ester and on the incorporation, esterification, and removal of *%-labeled cholesterol by both 3T3 cells and pig aortic smooth muscle cells stimulated with cationized low-density lipopro- tein (LDL) has been studied. Bezatibrate at 100 pg/ml reduced the accumulation of choles- terol ester in both cell types. The uptake and esterification of W-labeled cholesterol into cholesterol ester was also reduced by bezatibrate at this concentration. W-labeled choles- terol ester was removed from the cells following its hydrolysis and appeared in the incuba- tion medium as Y-labeled free cholesterol. This process was accelerated by bezafibrate and clotibrate in both cell types.
INTRODUCTION
The hypolipidemic agent, clofibrate,and its analog, bezafibrate, have been shown to reduce the deposition and to increase the removal of cholesterol ester from cultured 3T3 tibroblasts (Hudson and Day, 1981). In the present work these studies have been continued in order to investigate the effect of these agents on the uptake, esterification, and removal of 14C-labeled cholesterol by both 3T3 cells and aortic smooth muscle cells stimulated by cationized low-density lipoprotein (LDL) in tissue culture.
MATERIALS AND METHODS
[4-14C]Cholesterol (SO-58 mCi/mmol) was obtained from the Radiochemical Centre, Amersham, and incorporated into serum lipoprotein by incubation at 37°C with fetal calf serum (Medos Pty. Ltd.) for at least 2 hr before its addition to the incubation medium.
Bezafibrate was obtained from Boehringer-Mannheim GmbH, West Germany, and clofibrate from Imperial Chemical Industries Ltd., Great Britain. These agents were dissolved in sodium hydroxide and added to Dulbecco’s modified eagle medium (Grand Island Biological Co., New York). The pH was adjusted back to 7.4 with hydrochloric acid.
Blood was obtained from the jugular vein of normal pigs or pigs which had been fed for 4-5 months on a diet containing 0.6% cholesterol and 6% beef tallow (Lee et al., 1971)..EDTA, 1 mg/ml blood, was used as anticoagulant. LDL (u’ = 1.006- 1.063) was prepared by ultracentrifugation according to the method de- scribed by Hatch and Lees (1968) and was cationized as described by Basu et al. (1976).
3T3 Balb/c mouse tibroblasts, clone A31 from the American Type Culture Col- lection (Goldberg, 1977) were obtained from St. Vincent’s Hospital, Melbourne. Stock cells were grown in a humidified 5% COZ incubator at 37°C in 75-cm2 flasks (Falcon Plastics, Los Angeles). These cells were passaged twice weekly in a ratio
156 0014-4800/82/020156-08$02.00/O Copyright @ 1982 by Academic Press. Inc. AU rights of reproduction in any form reserved.
BEZAFIBRATE AND CLOFIBRATE ON CHOLESTEROL METABOLISM 157
of 1:3 to 1:5 and distributed into Falcon petri plates of area 25 cm2 for experi- ments. The incubation medium comprised Dulbecco’s modified eagle medium supplemented with 10% fetal calf serum, 525.6 @g/ml glutamine, 100 units/ml sodium penicillin, and 100 pg/ml streptomycin sulfate (Sigma Chemical Co., St. Louis, MO.).
Pig aortic smooth muscle cells were grown from arterial explants, as described by Ross (1971). Cells began to grow out from the explants after 8 days and grew slowly in multiple overlapping layers. By electron microscopy, the cells contained elongated longitudinally arranged myotilaments approximately 60 A diameter with dense bodies, a centrally located nucleus with prominent nucleoli, well- developed rough endoplasmic reticulum, a basement membrane, and many pinocytotic vesicles. These are characteristics of aortic smooth muscle cells (Ross, 1971). The cells were passaged every 2-3 weeks in a ratio of 1:3 to 1:4 and cells in the seventh passage were used in these experiments.
Experimental Procedure
Confluent 3T3 fibroblasts were incubated in 5 ml incubation medium containing 10% fetal calf serum, 0.44 @Zi 14C-labeled cholesterol, 50 pg/ml cationized hyper- lipidemic pig LDL-cholesterol together with 10 pg/ml (n = 6) or 100 pg/ml (n = 6) bezafibrate or no agent (n = 22). After 48 hr, the plates of cells which had been incubated with 10 and 100 ~&III bezafibrate were taken down for lipid analysis and radio assay (Hudson and Day, 1981) together with a sample (n = 4) from the control group. The remaining 18 plates of cells in the control group were washed five times with Hanks’ solution containing calcium and magnesium, then incu- bated for a further 5 days in normal incubation medium containing 10 &ml (n = 6) or 100 pglml (n = 6) bezafibrate or no agent (n = 6). Two plates of cells were taken down for lipid analysis and radio assay (Hudson and Day, 1981) 1,3, and 5 days after the change from medium containing cationized LDL to normal incubation medium. The medium of the remaining cells (Day 5 groups) was replaced on Day 3.
Pig aortic smooth muscle cells were incubated for 48 hr in 5 ml incubation medium containing 0.38 PCi 14C-labeled cholesterol and 50 pg/ml cationized hyperlipidemic pig LDL-cholesterol together with 10 or 100 &ml bezafibrate or no agent. Lipid content and radioactivity were determined as described previously (Hudson a Id Day, 1981). For the removal experiment, pig aortic smooth muscle cells were incubated in 5 ml incubation medium containing 1.96 &i 14C-labeled cholesterol and 25 pg/ml cationized LDL-cholesterol from a normolipidemic pig. After 48 hr six plates were taken down for lipid analysis and radioassay. The remaining cells were washed and incubated for a further 5 days in normal incuba- tion medium containing 100 pg/rnl bezafibrate (n = 6)) 250 pg/ml clofibrate (n = 6)) or no agent (n = 6).
The cell pellet (of smooth muscle cells) was extracted with chloro- form:methanol, 2:l (Folch et al., 1957). Then 50 Fg of cholestane was added as internal standard, the extract was dried under N2, and the protein was suspended and lipids were dissolved by addition of 5 ml diethyl ether. One milliliter 0.1 M hydrochloric acid was added and the tube buzzed. The top phase was transferred to another tube and the bottom phase containing the protein was washed twice with diethyl ether and the washings were added to the extract. To the protein solution 0.5 ml 4 M sodium hydroxide was added and the tube buzzed and heated for 5 min at 80°C. After neutralization with 0.5 ml 2.6 M hydrochloric acid,
158 HUDSON, DAY, AND MARCEGLIA
aliquots were taken for protein assay (Lowry ef al., 1951). The diethyl ether extract was dried under Nz and taken to 10 ml with chlorofotmmethanol, 2: 1. Aliquots were taken for direct counting, lipid phosphorous assay (Itaya et al., 1966), Iatroscan, neutral lipid thin-layer chromatography, and radioisotope counting as previously described (Hudson and Day, 1981).
RESULTS
Table 1 shows the effect of bezafibrate on the accumulation of free and ester cholesterol and on the uptake and incorporation of 14C-labeled cholesterol in’3T3 fibroblasts stimulated with cationized hyperlipidemic pig LDL. The control cells contained up to 95 hug esterified cholesteroYmg cell protein. This was reduced to 66 pg/rng cell protein by bezafibrate at 100 pg/ml, but not significantly affected by bezalibrate’at 10 /&nl. Free cholesterol content was not affected by bezatibrate at either concentration. The uptake of 14C-labeled cholesterol by the cells was reduced by bezafibrate at 100 pg/ml. The percentage of the 14C-labeled cholesterol incorporated into cholesterol ester was lower in the 100 pg/ml bezafibrate group but did not reach statistical significance.
Table II shows the effect of bezafibrate on the accumulation of free or esterified cholesterol and on the uptake and incorporation of 14C-labeled cholesterol in pig aortic smooth muscle cells stimulated with cationized LDL. The control group contained 32 pg esterified cholesterol/mg cell protein which was reduced (P < 0.05) to 21 pg esterified cholesterol/mg cell protein by 100 &ml bezafibrate. The content of cellular free cholesterol was also reduced (P < 0.05) by 100 p&ml bezafibrate. The lower concentration of bezafibrate, 10 pg/ml, did not signifi- cantly affect the cellular content of esterified cholesterol. As was the case with the 3T3 fibroblasts, the uptake of 14C-labeled cholesterol was reduced by 100 pg/ml bezafibrate. The percentage incorporation of 14C-labeled cholesterol into the cel- lular cholesterol ester fraction was reduced by bezafibrate but not sufficiently to reach statistical significance.
The removal of esterified cholesterol from 3T3 fibroblasts and pig aortic smooth muscle cells stimulated with cationized LDL is shown in Fig. 1. Regression analysis indicated that estetied cholesterol was removed from the 3T3 tibroblasts
TABLE I The Effect of Bezatibrate on Cholesterol Accumulation and Uptake and Incorporation
of W-Labeled Cholesterol in 3T3 Fibroblasts Stimulated with Cationized Hyperlipidemic LDL”
Esterified cholesterol Free cholesterol Total uptake*
Percentage incorporation to cholesterol ester
Control’
95.2 2 5.4 41.8 t- 2.3 390 k 67
14.5 ? 1.6
Bezafibrated Bezaflbrated ( 10 pgiml) (100 Pdn-4
83.2 f 10.9 66.3 f 2.6** 39.9 + 4.6 39.4 k 0.8 203 k 55 170 + 22**
12.9 f 2.4 10.9 r 1.2
o Chemical data are expressed as pg/mg protein. b Dpm/mg protein/1000 dpm of W-labeled cholesterol in the incubation medium. (’ Mean 2 SEM, n = 4. d Mean ? SEM, n = 6. **P < 0.01 by Student t test.
BEZAFIBRATE AND CLOFIBRATE ON CHOLESTEROL METABOLISM 159
TABLE II The Effect of Bezafibrate on Cholesterol Accumulation and Uptake and Incorporation
of “C-Labeled Cholesterol in Pig Aortic Smooth Muscle Cells Stimulated with Cationized Hyperlipidemic LDL”
Control’ Bezatibrate” Bezatibrated
(10 Pi&-a (100 /.&-a
EsteriEed cholesterol 32.3 a 4.4 22.8 f 1.6 20.8 f 0.8* Free cholesterol 19.8 ? 3.9 15.0 ” 1.3 11.5 f 0.8* Total uptake* 195 2 36 125 + 9 101 ” 16*
Percentage incorporation to cholesterol ester 15.8 t 2.9 24.8 f 3.2 9.3 * 2.2
a Chemical data are expressed as pg/mg protein. b Dpm/mg protein0000 dpm of “C-labeled cholesterol in the incubation medium. c Mean -C SEM, n = 3. d Mean f SEM, n = 4. * P < 0.05 by Student t test with Bessel Correction.
in all groups. Analysis of variance of the regression lines indicated that there was no significant difference between the control group and lower concentration of bezafibrate, but a difference at the 1% level of significance between the control group and the high concentration of bezafibrate (Fig. 1). The free cholesterol content of the 3T3 fibroblasts at Day 0 was 41.8 5 2.3 pg/mg protein (mean -e
3T3 FIBROBLASTS
2 E 40 & r PIG AORTIC SMOOTH MUSCLE CELLS
0.
1 1
0 1 3 5 TIME (days)
FIG. 1. The effect of bezatibrate and clofibrate on the removal of esterified cholesterol from 3T3 tibroblasts and pig aortic smooth muscle cells stimulated with cationized LDL. (C-0) Control; (+ . . . +) 10 &ml bezaflbrate; (*- --*) 100 @g/ml bezatibrate; (s---m) 250 &ml clofibrate.
160 HUDSON, DAY, AND MARCEGLIA
SEM, n = 6). Free cholesterol was not significantly removed during the S-day incubation in normal medium nor was it significantly affected by bezatibrate. A removal experiment was attempted on pig aortic smooth muscle cells using the same cationized hyperlipidemic pig LDL at the same concentration that was used in the experiment with 3T3 tibroblasts (50 PgIml). After 48 hr stimulation the cells had filled with vesicles and the medium was changed to normal medium. How- ever, by the following day, many of the cells had lost their plasma membranes and their contents were being dispersed into the incubation medium. A removal ex- periment was therefore set up with pig aortic smooth muscle cells using cationized normolipidemic pig LDL at a concentration of 25 pg/rnl LDL-cholesterol. Es- terified cholesterol was removed from the pig aortic smooth muscle cells but bezafibrate and clotibrate did not affect its removal (Fig. 1). The free cholesterol content of the pig aortic smooth muscle cells at Day 0 was 34.6 + 3.1 pg/mg protein (mean + SEM, n = 6) and its relatively slow removal was not affected by bezafibrate or clofibrate.
The removal of 14C-labeled free cholesterol from the 3T3 cells and its appear- ance in the incubation medium is shown in Fig. 2. Analysis of variance of the regression lines after logarithmic transformation of the data indicated that there was no difference in this removal between the three groups. Most of the label removed from the cells was recovered in the incubation medium for the control group and the cells incubated with the lower concentration of bezafibrate. How- ever in the group of cells incubated with the higher concentration of bezafibrate, more label was found in the free cholesterol fraction of the incubation medium than was removed from the cells.
0 1 3 5 TIME (days)
Fk. 2. The effect of bezatibrate on the removal of W-labeled free cholesterol from 3T3 fibroblasts stimulated with cationized LDL and its appearance in the incubation medium. Solid symbols, label in the cells: open symbols, label in the incubation medium. (60) Control; (* . . . +) 10 &ml bezafibrate; (*- - - *) 100 &ml bezatibrate.
BEZAFIBBATE AND CLOFIBRATE ON CHOLESTEROL METABOLISM 161
As was the case with the 3T3 fibroblasts, 14C-labeled free cholesterol was rapidly removed from the pig aortic smooth muscle cells. Neither bezafibrate nor clofibrate affected this removal. In all groups of smooth muscle cells more label was found in the free cholesterol fraction of the incubation medium than was removed from this fraction of the cells.
The removal of 14C-labeled cholesterol ester from the 3T3 fibroblasts and pig aortic smooth muscle cells is shown in Fig. 3. In the 3T3 fibroblasts, the 14C- labeled cholesterol ester was not significantly removed from the control group or the 10 &nl bezatibrate group. Analysis of covariance (Snedecor and Cochran, 1980) indicated no difference between the control group and the lower concentra- tion of bezafibrate, but a difference at the 1% level of significance between the control group and the higher concentration of bezafibrate. Very little label was recovered in the cholesterol ester fraction of the incubation medium-not more than 800 dpm had accumulated in this fraction during the 5-day incubation period in any group. The increased removal of 14C-labeled cholesterol ester from the 3T3 cells was not matched by an increase in appearance of label in this fraction of the incubation medium. The increased removal of 14C-labeled cholesterol ester from the cells incubated with 100 pg/ml bezafibrate may therefore be accounted for by the increased appearance of 14C-labeled free cholesterol in the incubation medium presumably following cellular cholesterol ester hydrolysis.
14C-labeled cholesterol ester was removed from the pig aortic smooth muscle cells and this removal was increased by both bezafibrate (P < 0.01) and clofibrate
FIG. 3. The effect of bezafibrate and clofibrate on the removal of W-labeled cholesterol ester from 3T3 fibroblasts and pig aortic smooth muscle cells stimulated with cationized LDL. Solid symbols, label in the cells; open symbols, label in the incubation medium. (60) Control; (+ . . . +) 10 pg/rnl bezafibrate; (*---It) 100 &ml bezafibrate; (L.----B) 250 &nl clotibrate.
162 HUDSON, DAY, AND MARCEGLIA
(P < 0.05). In all groups of smooth muscle cells, more label was removed from the cholesterol ester fraction of the cells than was found in this fraction of the incuba- tion medium.
DISCUSSION
In both 3T3 fibroblasts and pig aortic smooth muscle cells 100 pg/ml bezatibrate markedly reduced the accumulation of esterified cholesterol following their incu- bation with cationized LDL. The data with respect to the 3T3 tibroblasts confirms previously reported results (Hudson and Day, 1981). Cholesterol ester accumula- tion under these circumstances results from both increased lipoprotein uptake or to changes in the level of acyl-CoA cholesterol acyltransferase or of cholesterol ester hydrolase.
A decrease in the uptake of lipoproteins from fetal calf serum by bezatibrate was indicated by the decreased uptake of the lipoprotein bound 14C-labeled cho- lesterol. This decreased uptake presumably contributed to the decreased incorpo- ration of 14C-labeled cholesterol into the cellular cholesterol ester fraction by this agent. Previous experiments using labeled fatty acids have indicated a suppression of cellular cholesterol esterification by bezafibrate (Hudson and Day, 1981). In the present experiments decreased cellular esteritication of cholesterol by bezatibrate may also have contributed to the decreased incorporation of 14C-labeled choles- terol into the cholesterol ester fraction but this cannot be asserted with confidence since the percentage incorporation of 14C-labeled cholesterol into cholesterol ester was not statistically significantly reduced.
The increased removal of esterified cholesterol from 3T3 fibroblasts in normal medium by 100 pg/ml bezafibrate confirms previously reported results (Hudson and Day, 1981). The increased removal of 14C-labeled cholesterol ester from both cell types indicates that bezalibrate and clofibrate increased the removal of en- dogenously esterified cholesterol.
The increased removal of 14C-labeled cholesterol ester was not paralleled with an increase of label in this fraction of the incubation medium so that direct re- moval of endogenously esterified cholesterol from the cells by these agents can be excluded. With the pig aortic smooth muscle cells less label had accumulated in the cholesterol ester fraction of the incubation medium than was removed from the cells and more label was recovered in the free cholesterol fraction of the incubation medium than could be accounted for by removal from the cells. This confums that for these cells also the removal of endogenously esterified choles- terol presumably proceeds by cellular hydrolysis and removal of the liberated free cholesterol. In the 3T3 cells the increase of label in the free cholesterol fraction of the incubation medium above that which can be accounted for by removal of this fraction from the cells supports an increase in net hydrolysis of endogenously esteritied cholesterol and removal of the resultant free cholesterol by bezafibrate. The increased removal of 14C-labeled cholesterol ester from cells by bezafibrate and clotibrate together with the fact that little 14C-labeled cholesterol ester appears in the medium indicates that these agents increased the hydrolysis of endoge- nously esterified cholesterol in the cell.
While these agents increased the removal of endogenously esterified choles- terol, an increase in the removal of total cellular esterified cholesterol was ob- served only in the 3T3 cells. One possible explanation for this observation may be given by the proposed deficiency of lysosomal cholesterol ester hydrolase in
BEZAFIBRATE AND CLOFIBRATE ON CHOLESTEROL METABOLISM 163
aortic smooth muscle cells (de Duve, 1974). Cationized LDL is taken up by adsorptive endocytosis into cellular lysosomes where its cholesterol ester is hy- drolyzed and free cholesterol liberated into the cytoplasm where it is esterified, forming lipid droplets (Brown et al., 1976). If the activity of lysosomal cholesterol ester hydrolase is lower in smooth muscle cells than fibroblasts, a smaller propor- tion of the total cellular cholesterol ester would be expected to be present in the lipid droplet compartment of smooth muscle cells compared with the lipid droplet compartment of fibroblasts. Bezafibrate and clofibrate increase the removal of endogenously esterified cholesterol, which is presumably present in the lipid droplet compartment. If there is less cholesterol ester in lipid droplets of smooth muscle cells compared with fibroblasts, an increase in its removal by bezafibrate and clofibrate would have a smaller effect on the total cholesterol ester content of smooth muscle cells compared with that of fibroblasts.
However, the distribution of esterified cholesterol between the lysosomal and lipid droplet compartments in these cells is unknown, as is the comparative lysosomal cholesterol ester hydrolase activity in these two cell types. Further work is currently being undertaken to resolve these possibilities and to determine the mechanism of action of bezatibrate and clofibrate on cholesterol ester hy- drolyzing enzymes.
ACKNOWLEDGMENTS The authors are most grateful to Mr. David Faulkner and Miss Trudi Harris for technical assistance,
and Miss Betty Laby for assistance with the statistical analysis of the data. Support for this work was received from Boehringer-Mannheim GmbH, and a Commonwealth Postgraduate Research Award.
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