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Atherosclerosis, 28 (1977) 441-452 @ Elsevier/North-Holland Scientific Publishers, Ltd.
STUDIES OF THE MECHANISM OF AUGMENTED SYNTHESIS OF CHOLESTERYL ESTER IN ATHEROSCLEROTIC RABBIT AORTIC MICROSOMES
SAM HASHIMOTO and SEYMOUR DAYTON Research Service and Medical Service, VA Wadsworth Hospital Center, Los Angeles, Calif. 90073; Department of Medicine, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.) (Received 20 May, 1977) (Revised, received 2 September, 1977) (Accepted 14 September, 1977)
Summary A study was undertaken to test the hypothesis that an abnormally high concentration of acyl-CoA:cholesterol acyltransferase in atherosclerotic microsomes is partly responsible for augmented esterification of cholesterol. We approached the problem indirectly by measuring the incorporation of radioactivity into cholesteryl ester from [ l-14C]palmityl-CoA in normal microsomes after enrichment of their concentration of microsomal free cholesterol to levels characteristic of atherosclerotic microsomes. Elevation of free cholesterol content induced increased cholesterol esterification approximately linearly over the range studied. The cholesterol-esterifying activity of atherosclerotic microsomes was not greater than that of normal microsomes having the same concentration of cholesterol. The results suggest that, with acyl-CoA constant, augmented cholesterol esterification in atherosclerotic microsomes is an effect of high microsomal cholesterol concentrations and not due to an increase in the concentration of the enzyme.
Introduction It is well established that cholesteryl ester synthesis in the artery is augmented during atherogenesis [l-4]. This phenomenon occurs in experimental animals as early as 3-10 days after starting an atherogenic diet [ 1,5], indicating that it is an early biochemical lesion. The major pathway responsible for accelerated cholesterol esterification is catalyzed by acyl-CoA:cholesterol acyltransferase ( ACCAT) [ 41.
448
The mechanisms of augmented cholesterol esterification have not been fully clarified. Increased cholesterolesterifying activity in atherosclerotic microsomes is attributed in part to high concentrations of unesterified cholesterol [6] and perhaps of acyl-CoA [ 71. There is also the suggestion that augmented synthesis of cholesteryl ester is due either to an abnormally high concentration of the enzyme, or to augmented accessibility of cholesterol substrate to the cholesterol esterifying enzyme, in atherosclerotic microsomes [ 81. We report here further studies on the mechanisms of cholesterol esterification in atherosclerotic microsomes. We undertook to learn whether enrichment of normal arterial microsomes with cholesterol can stimulate esterification of cholesterol, and we tested indirectly the possibility that a high concentration of the enzyme may be responsible for augmented esterification of cholesterol in atherosclerotic microsomes. The latter was approached by comparing the cholesterol-esterifying activity of atherosclerotic microsomes and of normal microsomes enriched to the same concentration of microsomal cholesterol, with acylCoA concentration controlled at an optimal level. Methods Male New Zealand rabbits, weighing approximately 2.5 kg, were fed a Purina Chow pellet diet, supplemented with 10% olive oil and 1% cholesterol, for 3-6 months. Control rabbits were fed a pellet diet with no supplements. Animals were killed by injections of sodium pentobarbital. The thoracic aorta was washed in ice-cold 0.9% NaCl and stripped of adventitia. Microsomes were isolated from the intima + media layers by the procedure described elsewhere [ 61. The microsomal protein concentration was adjusted to 2 mg/ml. Total lipoproteins from hyperlipidemic rabbit serum of cholesterol-fed rabbits were isolated by the procedure of Have1 et al. [9]. Serum was adjusted to a density of 1.21 with a concentrated salt solution containing 153 g NaCl and 354 g KBr per liter. The sample was centrifuged at 100,000 X g for 20 h. The floating layer was removed, redispersed in a salt solution (density (d) = 1.21) and centrifuged. The washed lipoprotein solution was dialyzed against 0.15 M NaCl-1 mM EDTA overnight. The lipoprotein solution was heated at 60°C to destroy (if present) lecithin :cholesterol acyltransferase. Normal microsomes were enriched with cholesterol during a 6 h incubation at 37°C with varying amounts of lipoproteins (d < 1.21) from hyperlipidemic serum. For a control, an aliquot of the same microsomal preparation was carried through the procedure in the absence of lipoproteins. The lipoprotein solution had total and free cholesterol concentrations of 555 and 236 mg/dl, respectively. The mass of lipoprotein used for loading contained at least 4 times as much free cholesterol as did the microsomes. After incubation, the microsomes were washed twice by resuspension and centrifugation. The microsomes were redispersed in 0.1 M Tris-10 mM mercaptoethanol buffer (pH 7.4). Atherosclerotic microsomes were treated in the same manner as normal microsomes except that the incubation with lipoproteins was omitted. Cholesterol-esterifying activity of microsomes was measured by the incorporation of radioactivity into cholesteryl ester from [ 1-14C]palmityl-CoA during 0.5 h incubation at 37°C. The assay system consisted of 0.5 ml 0.1 M Tris-
449
10 mM mercaptoethanol buffer (pH 7.4) containing albumin (1 mg/ml), 8 nmoles of [l-‘4C]palmityl-CoA (spec. act. 58 &3/pmole), and microsomes (approx. 100 pg protein). Palmityl-CoA was present at a saturating concentration, with added acyl-CoA greatly in excess of estimated endogenous acyl-CoA [4]. The reaction was stopped with 20 ml chloroform-methanol (2 : 1). The procedure for the isolation and the chemical and radioactive assay of microsomal cholesterol and cholesteryl ester is described elsewhere [ 81. Results Normal aortic microsomes became enriched with cholesterol during incubation with mixed lipoproteins (d < 1.21) from hyperlipidemic rabbit serum. The data from 4 experiments are shown in Fig. 1. The free cholesterol concentration per mg microsomal protein was increased as much as 5-fold from a normal level (open symbols) of about 140 pg/mg protein. The cholesterol-esterifying activity, as measured by the incorporation of radioactivity into cholesterol ester from [1-14C]palmityl-CoA, was increased (r = 0.88, P < 0.01) as the free cholesterol concentration of normal microsomes was increased. When the regression line is extrapolated to the x intercept, the microsomal free cholesterol concentration is approximately 60 pg/mg microsomal protein. The 4 experiments with atherosclerotic microsomes all yielded results (Fig.1) comparable to the results with cholesterol-enriched normal microsomes. That is, at a given cholesterol concentration, the cholesterol-esterifying activity of the atherosclerotic microsomes was in no instance higher than the activity in
loo
XK)
I 300
I 400
FREE CHOLESTEROL MNXOSOMAL
I so0
(w)
PROTEIN (ma)
Fig. 1. Influence of microsomal free cholesterol concentration on the cholesterol-esterifying activity of rabbit aortic microsomes. Cholesterol-esterifying activity was measured by the incorporation of radioactivity into cholesteryl ester from [l- 14C]pabnityl-CoA during 0.5 h incubation at 37’C. Normal microsomes were enriched with free cholesterol by weincubation with total lipoproteins (d < 1.21) from hyperlipidemic rabbit serum. Four experiments were done with normal and atherosclerotic microsomes. Experiments with normal microsomes were designated by symbols (0. 0, a, 0); open symbol designates a control; closed symbol designates normal microsomes treated with lipoproteins. Values for atherosclerotic microsomes are indicated by X’s. The solid line designates the regression line for normal microsomes; interrupted lines designate the standard error of estimate.
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normal microsomes enriched to comparable cholesterol concentration, as estimated from the regression line. As a control, we measured ACCAT activity of normal microsomes before and after a 4 h incubation at 37°C in the absence of lipoproteins, in quadruplicate. Incorporation of radioactivity into cholesteryl ester (mean 2 SE) was 1342 f 137; after incubation, 1225 ? 192. The results show that incubation alone does not increase microsomal cholesterol-esterifying activity. We checked the lipoproteins for possible contamination by ACCAT. Incorporation of radioactivity into cholesteryl ester from [ l-‘4C]palmityl-CoA was 178, 162, and 171 counts/min (cpm) as compared to the buffer control, 150, 186, and 200 cpm. The results show that lipoproteins contained no ACCAT activity. Discussion In many previous efforts (unpublished) we have failed to load normal microsomes appreciably with cholesterol by exposure in vitro to high cholesterol concentrations presented in many physical forms, including normal human lipoproteins which had been concentrated by ultracentrifugation. In the present experiment, enrichment was successful when mixed plasma lipoproteins of cholesterol-fed rabbits were used. Since the lipoproteins of density
Atherosclerosis, 28 (1977) 441-452 @ Elsevier/North-Holland Scientific Publishers, Ltd.
STUDIES OF THE MECHANISM OF AUGMENTED SYNTHESIS OF CHOLESTERYL ESTER IN ATHEROSCLEROTIC RABBIT AORTIC MICROSOMES
SAM HASHIMOTO and SEYMOUR DAYTON Research Service and Medical Service, VA Wadsworth Hospital Center, Los Angeles, Calif. 90073; Department of Medicine, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.) (Received 20 May, 1977) (Revised, received 2 September, 1977) (Accepted 14 September, 1977)
Summary A study was undertaken to test the hypothesis that an abnormally high concentration of acyl-CoA:cholesterol acyltransferase in atherosclerotic microsomes is partly responsible for augmented esterification of cholesterol. We approached the problem indirectly by measuring the incorporation of radioactivity into cholesteryl ester from [ l-14C]palmityl-CoA in normal microsomes after enrichment of their concentration of microsomal free cholesterol to levels characteristic of atherosclerotic microsomes. Elevation of free cholesterol content induced increased cholesterol esterification approximately linearly over the range studied. The cholesterol-esterifying activity of atherosclerotic microsomes was not greater than that of normal microsomes having the same concentration of cholesterol. The results suggest that, with acyl-CoA constant, augmented cholesterol esterification in atherosclerotic microsomes is an effect of high microsomal cholesterol concentrations and not due to an increase in the concentration of the enzyme.
Introduction It is well established that cholesteryl ester synthesis in the artery is augmented during atherogenesis [l-4]. This phenomenon occurs in experimental animals as early as 3-10 days after starting an atherogenic diet [ 1,5], indicating that it is an early biochemical lesion. The major pathway responsible for accelerated cholesterol esterification is catalyzed by acyl-CoA:cholesterol acyltransferase ( ACCAT) [ 41.
448
The mechanisms of augmented cholesterol esterification have not been fully clarified. Increased cholesterolesterifying activity in atherosclerotic microsomes is attributed in part to high concentrations of unesterified cholesterol [6] and perhaps of acyl-CoA [ 71. There is also the suggestion that augmented synthesis of cholesteryl ester is due either to an abnormally high concentration of the enzyme, or to augmented accessibility of cholesterol substrate to the cholesterol esterifying enzyme, in atherosclerotic microsomes [ 81. We report here further studies on the mechanisms of cholesterol esterification in atherosclerotic microsomes. We undertook to learn whether enrichment of normal arterial microsomes with cholesterol can stimulate esterification of cholesterol, and we tested indirectly the possibility that a high concentration of the enzyme may be responsible for augmented esterification of cholesterol in atherosclerotic microsomes. The latter was approached by comparing the cholesterol-esterifying activity of atherosclerotic microsomes and of normal microsomes enriched to the same concentration of microsomal cholesterol, with acylCoA concentration controlled at an optimal level. Methods Male New Zealand rabbits, weighing approximately 2.5 kg, were fed a Purina Chow pellet diet, supplemented with 10% olive oil and 1% cholesterol, for 3-6 months. Control rabbits were fed a pellet diet with no supplements. Animals were killed by injections of sodium pentobarbital. The thoracic aorta was washed in ice-cold 0.9% NaCl and stripped of adventitia. Microsomes were isolated from the intima + media layers by the procedure described elsewhere [ 61. The microsomal protein concentration was adjusted to 2 mg/ml. Total lipoproteins from hyperlipidemic rabbit serum of cholesterol-fed rabbits were isolated by the procedure of Have1 et al. [9]. Serum was adjusted to a density of 1.21 with a concentrated salt solution containing 153 g NaCl and 354 g KBr per liter. The sample was centrifuged at 100,000 X g for 20 h. The floating layer was removed, redispersed in a salt solution (density (d) = 1.21) and centrifuged. The washed lipoprotein solution was dialyzed against 0.15 M NaCl-1 mM EDTA overnight. The lipoprotein solution was heated at 60°C to destroy (if present) lecithin :cholesterol acyltransferase. Normal microsomes were enriched with cholesterol during a 6 h incubation at 37°C with varying amounts of lipoproteins (d < 1.21) from hyperlipidemic serum. For a control, an aliquot of the same microsomal preparation was carried through the procedure in the absence of lipoproteins. The lipoprotein solution had total and free cholesterol concentrations of 555 and 236 mg/dl, respectively. The mass of lipoprotein used for loading contained at least 4 times as much free cholesterol as did the microsomes. After incubation, the microsomes were washed twice by resuspension and centrifugation. The microsomes were redispersed in 0.1 M Tris-10 mM mercaptoethanol buffer (pH 7.4). Atherosclerotic microsomes were treated in the same manner as normal microsomes except that the incubation with lipoproteins was omitted. Cholesterol-esterifying activity of microsomes was measured by the incorporation of radioactivity into cholesteryl ester from [ 1-14C]palmityl-CoA during 0.5 h incubation at 37°C. The assay system consisted of 0.5 ml 0.1 M Tris-
449
10 mM mercaptoethanol buffer (pH 7.4) containing albumin (1 mg/ml), 8 nmoles of [l-‘4C]palmityl-CoA (spec. act. 58 &3/pmole), and microsomes (approx. 100 pg protein). Palmityl-CoA was present at a saturating concentration, with added acyl-CoA greatly in excess of estimated endogenous acyl-CoA [4]. The reaction was stopped with 20 ml chloroform-methanol (2 : 1). The procedure for the isolation and the chemical and radioactive assay of microsomal cholesterol and cholesteryl ester is described elsewhere [ 81. Results Normal aortic microsomes became enriched with cholesterol during incubation with mixed lipoproteins (d < 1.21) from hyperlipidemic rabbit serum. The data from 4 experiments are shown in Fig. 1. The free cholesterol concentration per mg microsomal protein was increased as much as 5-fold from a normal level (open symbols) of about 140 pg/mg protein. The cholesterol-esterifying activity, as measured by the incorporation of radioactivity into cholesterol ester from [1-14C]palmityl-CoA, was increased (r = 0.88, P < 0.01) as the free cholesterol concentration of normal microsomes was increased. When the regression line is extrapolated to the x intercept, the microsomal free cholesterol concentration is approximately 60 pg/mg microsomal protein. The 4 experiments with atherosclerotic microsomes all yielded results (Fig.1) comparable to the results with cholesterol-enriched normal microsomes. That is, at a given cholesterol concentration, the cholesterol-esterifying activity of the atherosclerotic microsomes was in no instance higher than the activity in
loo
XK)
I 300
I 400
FREE CHOLESTEROL MNXOSOMAL
I so0
(w)
PROTEIN (ma)
Fig. 1. Influence of microsomal free cholesterol concentration on the cholesterol-esterifying activity of rabbit aortic microsomes. Cholesterol-esterifying activity was measured by the incorporation of radioactivity into cholesteryl ester from [l- 14C]pabnityl-CoA during 0.5 h incubation at 37’C. Normal microsomes were enriched with free cholesterol by weincubation with total lipoproteins (d < 1.21) from hyperlipidemic rabbit serum. Four experiments were done with normal and atherosclerotic microsomes. Experiments with normal microsomes were designated by symbols (0. 0, a, 0); open symbol designates a control; closed symbol designates normal microsomes treated with lipoproteins. Values for atherosclerotic microsomes are indicated by X’s. The solid line designates the regression line for normal microsomes; interrupted lines designate the standard error of estimate.
450
normal microsomes enriched to comparable cholesterol concentration, as estimated from the regression line. As a control, we measured ACCAT activity of normal microsomes before and after a 4 h incubation at 37°C in the absence of lipoproteins, in quadruplicate. Incorporation of radioactivity into cholesteryl ester (mean 2 SE) was 1342 f 137; after incubation, 1225 ? 192. The results show that incubation alone does not increase microsomal cholesterol-esterifying activity. We checked the lipoproteins for possible contamination by ACCAT. Incorporation of radioactivity into cholesteryl ester from [ l-‘4C]palmityl-CoA was 178, 162, and 171 counts/min (cpm) as compared to the buffer control, 150, 186, and 200 cpm. The results show that lipoproteins contained no ACCAT activity. Discussion In many previous efforts (unpublished) we have failed to load normal microsomes appreciably with cholesterol by exposure in vitro to high cholesterol concentrations presented in many physical forms, including normal human lipoproteins which had been concentrated by ultracentrifugation. In the present experiment, enrichment was successful when mixed plasma lipoproteins of cholesterol-fed rabbits were used. Since the lipoproteins of density
