Seymour Dayton

Characteristics of the Cholesterol-Esterifying Activity in Normal and Atherosclerotic Rabbit Aortas

Esterification of cholesterol with [1-14C]palmityl-CoA by an atherosclerotic cell-free homogenate was approximately 16–50-fold greater than that by a normal cell-free homogenate for a given amount of protein in the homogenate. This difference was due to hyperactivity of the cholesterol-esterifying system in the atherosclerotic cell-free homogenate rather than to depletion of radioactive palmityl-CoA in the reaction mixture containing normal homogenate. Neither an activator of cholesterol esterification in the soluble fraction of the atherosclerotic aortic homogenate nor an inhibitor in the soluble fraction of the normal aortic homogenate could be demonstrated. The pH optimum within the pH range covered for esterification and the apparent Km values were approximately the same in normal and atherosclerotic microsomes, suggesting that the enzymes were probably the same. The results suggested a higher concentration or a higher activity of the enzyme in or on atherosclerotic microsomes. An alternative possibility is that high concentrations of free cholesterol in the atherosclerotic microsomes were responsible for the augmented cholesterol esterification. This possibility seems unlikely, because the observed 2.3-fold increase in the free cholesterol concentration should not produce a 25-fold increase in cholesterol esterification. The rate of cholesterol esterification by atherosclerotic microsomes varied with the substrate: oleyl-CoA > palmityl-CoA > linoleyl-CoA.

Studies of the mechanism of augmented synthesis of cholesteryl ester in atherosclerotic rabbit aortic microsomes.

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 [1-14C]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.

Esterification of cholesterol by homogenates of atherosclerotic and normal aortas

Abstract Cholesterol esterification with palmityl-CoA by cell-free homogenates from atherosclerotic rabbit aortas was 39 times greater than with cell-free homogenates from normal aortas. In a 2 1 2 hr time-trend study using atherosclerotic preparations, the specific activity curve of cholesteryl ester reached an early plateau at a level much lower than that of the fatty acid derived from the β-position of lecithin, with no tendency of the cholesteryl ester specific activity to rise further approaching that of lecithin. A similar type of experiment was done using normal aortic preparations. In contrast to the experiments with atherosclerotic tissue, the specific activity of cholesteryl ester was much higher than that of lecithin at each time interval after 4 minutes of incubation. The data indicate that lecithin is not an intermediate in the observed esterification of cholesterol in our preparations. Esterification of cholestrol occurs upon addition of labeled palmityl-CoA, and terminates after exhaustion of the substrate. Most of the cholesterol-esterifying activity is localized in the microsomes of normal and atherosclerotic aortas. Cholesterol esterification by atherosclerotic and normal microsomes requires ATP and CoA, when free fatty acid is provided as the acyl source. Very little esterification occurs without these factors. These observations together indicate that cholesterol esterification is accomplished by fatty acyl-CoA:cholesterol acyltransferase.

Turnover of Cholesterol in the Artery Walls of Normal Chickens

Studies of cholesterol metabolism in the walls of arteries of normal intact cockerels indicate that plasma cholesterol is the major precursor of the cholesterol in the artery wall, but that local synthesis may contribute a significant fraction. The fractional turnover of cholesterol in the wall of the abdominal aorta is faster than in either the thoracic aorta or the brachiocephalic arteries. However, considered in relation to endothelial surface area, the rate of cholesterol transfer from plasma is smallest in the abdominal aorta. The abdominal aorta also has a lower cholesterol concentration in this species than do the other large arteries.

Influence of a Diet High in Unsaturated Fat upon Composition of Arterial Tissue and Atheromata in Man

Detailed chemical analyses have been carried out on aorta, and on coronary and aortic atheromata, of men who died during a study of prolonged use of diets rich in unsaturated fat and of control subjects. Concentrations of total aortic lipid and of total aortic calcium were not significantly different for the two groups of subjects. Concentrations of cholesterol and cholesterol esters, triglyceride, and phosphatide showed no difference between the two groups in analyses of aorta, uncomplicated aortic atheromata, complicated aortic atheromata, and coronary atheromata. Coronary atheroma lipid contained substantially more triglyceride than did aortic atheroma lipid, whether derived from complicated or uncomplicated plaques.Atheroma triglyceride in experimental subjects contained more linoleic acid than in control subjects, in all types of plaques. Lesser but significant increases in linoleic acid were seen in cholesterol ester and phosphatide of coronary atheromata and complicated aortic atheromata. In the case of uncomplicated aortic atheroma, changes in linoleic acid of cholesterol ester and phosphatide were smaller and not significant. In both groups of subjects, coronary atheroma contained more cholesteryl oleate and less linoleate than did aortic atheroma.Linoleic acid contents of atheroma lipid fractions were positively correlated with the linoleic acid figures for corresponding fractions of the most recent antemortem serum samples. Ratio of linoleic acid in an atheroma lipid fraction to the linoleic acid content of the corresponding serum fraction was, in most cases, constant after the time of the first sample, obtained after 495 days on experimental diet. It is postulated that this ratio constitutes an estimate of the fraction of atheroma fatty acid derived from plasma. In cholesterol ester and triglyceride, the ratio was about 0.65 in experimental subjects, higher in subjects on the control diet. The ratio for phosphatide was about 0.5 in both groups of subjects.

Origin of cholesteryl oleate and other esterified lipids of rabbit atheroma

Rabbit were fed a diet containing [9,10-3H]oleic acid starting at weaning, After 14 weeks, 2% cholesterol was added to the diet to induce atheroma formation, and the experiment was continued for 12 additional weeks. During these 12 weeks serial serum samples were obtained, and at the end of the period the animals were autopsied and aortic atheromata were dissected out. From all serum and atheroma samples, cholesteryl ester, triglyceride, lecithin and free fatty acid were isolated. Oleic acid was obtained from each component and assayed for 3H. The ratio of the atheroma oleic acid specific radioactivity in a given component to the mean serum oleic acid specific radioactivity in that component, designated A/S, was calculated. 1.0-A/S was taken as an estimate of the minimum fractional contribution of local synthesis to the oleate in that component. For cholesteryl ester, the mean value for 1.0-A/S was 0.19 with standard error of the mean ± 0.05; if one outlying value is omitted, the mean is 0.13 with standard error ± 0.02. It is concluded that a measurable and significant fraction of atheroma cholesteryl oleate is derived by local synthesis, and reasons are given for considering the observed value a probable underestimate.

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.

Utilization of Glucose, Octanoate and Palmitate by Normal Rat Aorta, and the Effect of these Acids and of Albumin on Glucose Metabolism

Summary Influence of octanoate and palmitate on the uilization of glucose by rat aortic tissue was examined. Octanoate but not palmitate depressed the oxidation of glucose-U-14C to CO2. No effect was exerted by these fatty acids on glucose uptake, conversion of glucose to lipids, or oxygen uptake. Commercial bovine serum albumin, used as a carrier for these acids, depressed glucose uptake, increased CO2 production from radioactive glucose, and increased oxygen uptake. These effects were abolished with purification of albumin by dialysis and charcoal treatment. The purified albumin did, however, decrease incorporation of glucose radioactivity into tissue lipid. In a short-term experiment (18-min incubation) octanoate was oxidized to water-soluble products and CO2 much more rapidly than was palmitate. These results suggest that oxidation of free fatty acid, at the more rapid rate observed with octanoate, has a sparing effect on the oxidation of glucose to CO2 in aortic tissue.

Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Abstract Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent. Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.

Decline in Rate of Cholesterol Synthesis During Maturation of Chicken Aorta.

Summary In vitro synthesis of labeled cholesterol by aortic tissue of cockerels was found to fall off rapidly with maturation, with either acetate-1-C14 or mevalonate-2-C14 as precursor. Production of C14O2 failed to show a similar relationship, indicating that the declining rate of cholesterol synthesis is not a consequence of decreasing permeability of the tissue to the substrate. In the system employed, mevalonate was only twice as efficient as acetate as a source of sterol carbon, and almost all the sterol radioactivity derived from mevalonate was removed by bromination. Thoracic and abdominal aorta displayed similar rates of cholesterol synthesis.