Cholesterol Ester Metabolism Research Articles

The effect of bezafibrate and clofibrate on cholesterol ester metabolism in rabbit peritoneal macrophages stimulated with acetylated low density lipoproteins

Bezafibrate and clofibrate reduce the quantity of esterified cholesterol, decrease the incorporation of 14C-labeled oleic acid into cholesterol ester and inhibit acyl coenzyme A cholesterol acyltransferase activity in rabbit peritoneal macrophages stimulated with acetylated low density lipoprotein. In all cases, the effect of bezafibrate was more marked than that of clofibrate. The activity of lysosomal cholesterol ester hydrolase in these cells was not affected by these drugs. It is suggested that these drugs exert an anti-atherogenic activity not only by their action on serum lipids, but by influencing cellular cholesterol ester metabolism.

Effects of oleate and compactin on the metabolism and secretion of cholesterol and cholesteryl ester by rat hepatocytes

Incubation of rat hepatocytes with oleate for a period of 1 h gave rise to a decrease in the total (esterified plus unesterified) cholesterol associated with very-low-density lipoprotein (VLDL). This effect was no longer apparent after longer incubation periods. The rate of cholesterol biosynthesis decreased during the first hour of incubation in the presence of oleate. After longer incubation periods, however, more cholesterol was synthesised in the presence of oleate than in its absence. The extracellular presence of oleate gave rise to a 2-fold increase in the concentration of cellular cholesteryl ester. Under these conditions cholesteryl ester contributed a larger proportion of the total cholesterol secreted with the VLDL. The cholesteryl ester associated with VLDL was derived predominantly from cholesteryl ester synthesised intracellularly. Inhibition of cholesterol synthesis with compactin did not significantly alter the rate of secretion of VLDL-cholesterol. Newly synthesised non-esterified cholesterol equilibrated with the bulk of pre-existing cellular cholesterol before secretion with the VLDL. This was true irrespective of the rate of endogenous cholesterol synthesis.

Relationships between circadian cycles of rat adrenal cholesterol ester metabolizing enzymes, cholesterol, ascorbic acid, and corticosteroid secretion

The circadian changes in rat adrenal cholesterol ester (CE) metabolism have been studied and related to the circadian changes in adrenal ascorbic acid and corticosterone levels and plasma corticosterone levels. Significant declines in the ratio of CE/C. cholesterol esterase (CEase) and acyl CoA: cholesterol acyl transferase (ACAT) activities occur during the peak and declining phases of adrenal and plasma corticosteroids, suggesting that when there is a decreased need for steroid precursors the amount of stored CE is decreased, and that the enzymes involved in CE metabolism decline in activity. Adrenal ascorbic acid has a biphasic rhythm with peaks at 0900 and 2200 h. The rhythm of ascorbic acid appears to be inverse to that of ACAT activity, suggesting possible relationships between the two parameters.

Triglyceride and cholesterol ester metabolism of rat 1 cells and Rous Sarcoma Virus-transformed rat 1 cells

The incorporation of 14C-long-chain fatty acids into the neutral lipids of rat 1 cells and B77 Rous Sarcoma Virus-transformed rat 1 cells was compared. Increased labeling of the cholesterol ester and triglyceride fractions was observed in the transformed cells. In temperature shift experiments, using B77 ts src Rous Sarcoma Virus-transformed rat 1 cells, alterations of cholesterol esterification could first be detected 4–6 h after shift to the permissive temperature. Changes in triglyceride metabolism could be shown as early as 4–6 h after activation of the transforming gene function. It is concluded that alterations of neutral lipid metabolism are among the early effects of the transformation process.

Sex difference in aortic cholesterol esterase activity in rats, and changes of the activity following castration and gonadal hormone treatment

Sex differences in aortic cholesterol esterase activity and changes in the activity following castration and gonadal hormone treatment were investigated in rats. Differences in the enzyme activity were apparent after 2.5 months and became most significant after 6 months. The activity in the aorta and the liver was significantly higher in female rats. Prepubertal orchiectomy increased the aortic activity, femiinizing the type of metabolism, whereas postpubertal orchiectomy, and both pre- and postpubertal ovariectomy induced no change in the activity. The administration of testosterone to female rats significantly decreased the aortic activity, masculininzing the type of metabolism. However, the administration of testosterone or of 17β-estradiol to male rats had no effect. These results suggest (1) that there are clear sex differences in aortic cholesterol esterase activity, (2) that prepubertal exposure to andogens plays a critical role in the sexual differentiation in aortic and hepatic cholesterol ester metabolism, and (3) that the administration of testosterone can temporarily masculinize the type of metabolism. These results may partly explain the sexual differences in susceptibility to atherosclerosis.

The effect of bezafibrate and clofibrate on cholesterol ester metabolism in 3T3 cells and smooth muscle cells in tissue culture

The effect of bezafibrate and clofibrate on the accumulation and removal of cholesterol ester and on the incorporation, esterification, and removal of 14C-labeled cholesterol by both 3T3 cells and pig aortic smooth muscle cells stimulated with cationized low-density lipoprotein (LDL) has been studied. Bezafibrate at 100 μg/ml reduced the accumulation of cholesterol ester in both cell types. The uptake and esterification of 14C-labeled cholesterol into cholesterol ester was also reduced by bezafibrate at this concentration. 14C-labeled cholesterol ester was removed from the cells following its hydrolysis and appeared in the incubation medium as 14C-labeled free cholesterol. This process was accelerated by bezafibrate and clofibrate in both cell types.

Lecithin: Cholesterol acyl transfer rate and high density lipoprotein level in coronary artery disease

The lecithin:cholesterol acyl transfer (LCAT) reaction occurs in connection with the high density lipoproteins (HDL) and might have importance for the development of atheromatosis. The LCAT rate and the lipoprotein concentrations in plasma were therefore determined in 82 patients, 40–60 years old, with incapacitating angina pectoris and significant lesions at coronary angiography. Thirty-eight cases were normolipidemic and 22 had type IV hyperlipidemia while the others had different types of hypercholesterolemia (n = 7) or were pharmacologically treated for previously diagnosed hyperlipidemia (n = 11) or diabetes mellitus. The normolipidemic group and the type IV group with coronary artery disease were separately compared to one normolipidemic (n = 44) and one type IV hyperlipidemic (n = 29) control group of healthy subjects of about the same age. The fractional LCAT rate (% · h −1) was lower in patients compared to controls both regarding the normolipidemic and type IV subjects. The molar LCAT rate (μmol · 1 −1 · h −1) did not differ between normolipidemic cases and controls while it was lower in type IV patients with coronary artery disease than in the type IV controls. The HDL total cholesterol (TC) concentration and the HDL-TC/TC ratio were lower in normolipidemic cases than in normolipidemic controls while both cases and controls with type IV hyperlipidemia showed equally low levels of these parameters. In conclusion, both a reduced fractional LCAT rate and a decreased HDL-TC/TC ratio might be indicators of disturbances of the cholesterol ester metabolism and might contribute to the development of coronary atheromatosis.

Purification of acid lipase from rabbit liver

In [l] we demonstrated that acid lipase activity with 4-methylumbelliferyl oleate as substrate represented both acid cholesterol esterase and triacylglycerol lipase activity and that the enzyme might be associated with the lysosomal membrane or hydrophobic macromolecules in rabbit liver. The enzymes seems to be involved in catabolism of lipoproteins and to play an important role in the intracellular metabolism of cholesterol esters and triacylglycerols [2,3]. Purification of the enzyme has been attempted by several groups, but highly purified enzyme has not been obtained because of its instability and hydrophobicity [ 1,4-61. The enzyme was purified 120-fold from rat liver [4] and 2500.fold from human liver [5]. Moreover, lOOO-3000-fold purification of the enzyme from human placenta was reported in [6]. obtained from the Protein Research Foundation (Osaka). The homogenate was centrifuged at 1000 X g for 10 min and the postnuclear supernatant was centrifuged at 3300 X g for 10 min. The resulting supernatant was centrifuged at 14 000 X g for 20 min and the precipitate (lysosome-rich fraction) was suspended in the same medium. The precipitate was washed by recentrifugation at 14 000 X g for 20 min, then homogenized in the same medium containing 0.5% (v/v) digitonin in a volume equivalent to 1/3rd that of the postnuclear supematant. The mixture was stirred at 4°C for 40 min and then centrifuged at 50 000 X g for 30 min and the supematant was collected.

LDLおよびアセチルLDLの大動脈およびマクロファージにおけるコレステロールエステル代謝に及ぼすトコフェロールの影響

To clarify the mechanism of cholesterol ester deposition in the arterial wall, the reconstructed LDL (rLDL) and acetylated LDL (acLDL) cholesterol ester metabolism in the rat peritoneal macrophage and the arterial wall were studied. Reconstructed LDL was prepared by the method of Krieger et al. It is reported that there are acid (pH 4.5) and neutral pH 7.5) cholesterol esterase (CEase) in the arterial wall. rLDL-cholesterol oleate and acLDL-cholesterol oleate were hydrolized by rat arterial homogenate and by rat peritoneal macrophage homogenate only at pH 4.5. Next, microsomal resterification system of cholesterol which was product hydrolized in lysosome was studied. ReLDL[3H]-cholesterol oleate were at first incubated with particulate fractions of arterial wall homogenate or macrophage homogenate at pH 4.5, then were incubated at pH7.5 with the addition of [14C]oleoyl-CoA, and formed amount of [3H]cholesterol-[14C] oleate were measure after separating with thin layer chromatography. The rate of reesterification of cholesterol was enchanced by the addition of supernatant fraction with the arterial wall, but with peritoneal mecrophage, it was decreased by the addition of supernatant fraction. These results suggested that LDL-cholesterol ester metabolism was greatly affected by the factor contained in the supernatant fractions. The hydrolysis of reLDL-cholesterol oleate by both arterial and macrophage homogenate at pH 4.5, was enhanced by the addition of tocopherol. Farther more, tocopheroltreated reLDL cholesterol oleate was much more hydrolized by arterial homogenate or by macrophage homogenate than nontreated reLDL cholesterol oleate, and also tocopherol-treated arterial wall homogenate or macrophage homogenate hydrolized reLDL-cholesterol oleate much more than untreated homogenates. On the other hand, reesterification of once hydrolized reLDL cholesterol by tocopherol-treated arterial wall and macrophage homogenates were decreased comparing to nontreated homogenates.These results suggest that LDL-CE were mainly hydrolized at acid area in the microphage and arterial wall. And once hydrolized cholesterol oleate was resterified in the neutral area. Tocopherol may prevent the deposition of cholesterol ester by facilitating the hydrolysis of LDL-cholesterol ester in lysosome and by reducing the reesterification of once hydrolized cholesterol in microsome.

エタノールの大動脈壁コレステロールエステラーゼ活性に及ぼす影響

The effect of ethanol on cholesterol ester metabolism in rat arterial wall was studied. In rats kept on water containing in 0.5%, 2% and 10% ethanol (0.5%, 2% and 10% EtOH group), cholesterol esterase activity was estimated in homogenate of arterial wall by the method of Brecher, and fatty acid composition in arterial wall was also determined. Each value of EtOH group was statistically compared with the data of control rats kept on plain water (control group).Acid (pH 4.5) cholesterol esterase activity in 10% EtOH group was higher than that in control group. Neutral (pH 7.4) cholesterol esterase activity in all EtOH groups was higher than that in control group. There was no significant difference in fatty acid composition between EtOH group and control group.These results suggest that the change of cholesterol esterase activity in EtOH group might reduce cholesterol ester deposition in arterial wall.

Cholesterol ester metabolism in plasma during estrogen and antiandrogen treatment in men with carcinoma of the prostate.

The male predominance in atherosclerotic disease has been ascribed to differences in lipid and lipoprotein metabolism between men and women. The influences of estrogen and antiandrogen treatment on the cholesterol ester metabolism and lipoproteins in plasma in men were therefore studied. Forty-six men with carcinoma of the prostate were studied before and after 2 and 8 weeks' treatment with either polyestradiol phosphate and ethinyl estradiol or orchidectomy or the testosterone receptor-blocking gestagenic drug cyproterone acetate. The estrogen treatment increased the HDL-TC and the level of TGs in HDL and LDL and reduced the TC and LDL-TC simultaneously with elevations of the fractional and molar cholesterol esterification rates. Orchidectomy caused only slight elevations of the TC level and the molar cholesterol esterification rate. Cyproterone acetate reduced the TC, LDL-TC, and HDL-TC concentrations simultaneously with an increase in the fractional cholesterol esterification rate. The alterations of TC and LDL-TC were positively correlated to the changes in the molar cholesterol esterification rate and negatively correlated to the alteration in the fractional cholesterol esterification rate. High doses of estrogen appeared to raise the TG level and the production of CE in plasma. Both estrogen and cyproterone acetate therapy lowered the LDL-TC level simultaneously with a raised fractional elimination of unesterified cholesterol as CEs from plasma. Although the testosterone production was practically eliminated by all three forms of treatment, there was no change of plasma lipoproteins common to all three groups. Therefore the lower estrogen level rather than the higher testosterone level might cause the lower HDL levels in men compared to women.

Specificities of human and rat brain enzymes of cholesterol ester metabolism toward very long chain fatty acids: implication for biochemical pathogenesis of adrenoleukodystrophy.

Specificities of the cholesterol-esterifying enzyme and the three cholesterol esterases in rat brain with respect to the chain length of fatty acids were examined. For each of the hydrolases, activities toward cholesteryl lignocerate and cerotate were generally less than 1% of that toward cholesteryl oleate. However, both lignoceric and cerotic acids were esterified at rates approximately 10% of that for oleic acid. In postmortem human control and adrenoleukodystrophy brains, the esterifying activity toward cerotic acid was on the average 25% of that toward oleic acid. The abnormal accumulation of cholesterol esters with very long chain fatty acids observed in adrenoleukodystrophy can therefore occur in the absence of deficient activities of the cholesterol esterases, if the free fatty acid pool of the brain contains an abnormal amount of very long chain fatty acids.

Biochemical study of adrenoleukodystrophy (ALD).

Adrenoleukodystrophy (ALD) is an x-linked hereditary neurological disorder characterized by the accumulation of cholesterol ester with long chain fatty acids in the brain and adrenal gland. We examined cholesterol ester metabolism for the postmortem brain tissues of ALD patients, using cholesterol ester with short and long chain fatty acids as the substrate for hydrolyzing enzyme, as well as short and long chain fatty acids for synthesizing enzyme. No enzyme abnormality was found. However, there was a discrepancy between hydrolytic and synthetic activities with short or long chain fatty acids. The findings suggest that the accumulation of cholesterol ester with long chain fatty acids in ALD brain is not due to enzyme abnormalities, but is a secondary phenomenon which comes from abnormal fatty acid metabolism causing a high concentration of long chain fatty acids.

Effect of pantethine on cholesterol ester metabolism in rat arterial wall

The total serum cholesterol level in rats fed on a high cholesterol diet (HCD) for 16 weeks was markedly higher than that in rats fed on a normal diet (ND), but pantethine reduced the increased level in rats fed on HCD ( P < 0.05). Acid cholesterol esterase activity (acid CEase) of arterial wall homogenates from rats fed on HCD was significantly lower than that of rats fed on ND ( P < 0.005). Acid CEase activity in the arterial wall of rats fed on HCD for 8 weeks and then ND for 8 weeks was less than that of rats fed on ND for 16 weeks. Acid CEase activity in the arterial wall was increased in rats fed on pantethine-containing diet. The ratio of cholesterol ester synthesizing activity to neutral cholesterol esterase (neutral CEase) activity was higher in rats fed on HCD than in those fed on ND. The ratio was lower in rats on the pantethine-containing diet than in those on HCD. The relationship between hypercholesterolemia and lipid metabolism in the arterial wall and effects of pantethine are discussed on the basis of these results.

動脈壁の脂質代謝

The following themes were described about the relationship between lipid metabolism and lipid accumulation in arterial wall.1. Introduction of metabolism in arterial wall(1) Lipid metabolism(2) Protein metabolism(3) Glucose metabolism2. Lipoprotein metabolism in arterial wall(1) Lipoprotein and arterial wall(2) Lipoprotein and lipid accumulation3. Cholesterol metabolism in arterial wall(1) Cholesterol ester metabolism in lysosomes(2) Cholesterol ester metabolism in microsomes4. Phospholipid metabolism in arterial wall5. Triglyceride metabolism in arterial wall6. Free fatty acid metabolism in arterial wall7. Lipoperoxide in arterial wall8. Risk factors and lipid metabolism in arterial wall(1) Thrombosis and lipids(2) Hormone and lipid metabolism(3) Hypertension and lipid metabolism9. Regression of atherosclerosis

Study of cultured skin fibroblasts from patients with and without ischemic heart disease Metabolism of low density lipoprotein and cholesterol ester, synthesis of cellular lipids and effect of chloroquine on accumulation of cholesterol ester

The aim of the present study was to determine whether skin fibroblasts derived from patients with ischemic heart disease (IHD), which could not be related to accepted risk factors, would show a metabolic abnormality with respect to lipid or lipoprotein metabolism. Male patients 30–52 years old suffering from IHD were subdivided into two groups: those in whom IHD was not associated with risk factors such as hypertension, hyperlipoproteinemia, diabetes or smoking (group I); and those in whom heavy smoking was the only major risk factor recognized (group II). The controls were patients with angiographically normal coronary arteries (group III). Skin fibroblasts obtained from these patients were cultured and investigated with respect to metabolism of low density lipoprotein (LDL), synthesis of cellular lipids and induction of cholesterol ester accumulation in the presence of chloroquine, an inhibitor of lysosomal hydrolases. After 24 h incubation, the uptake and degradation of LDL protein in cells from patients of group II was significantly higher than in the controls, group III, but not different from those of group I. Hydrolysis of [ 3H] cholesterol linoleate, and incorporation of [ 3H]oleic acid into total lipids and into cholesterol esters was similar in cell cultures of the 3 groups studied. After exposure to chloroquine and LDL, the cells from the different donors accumulated cholesterol ester to a similar extent. Thus, whereas no significant difference was encountered in the lipid and lipoprotein metabolism in cells of patients with IHD without risk factors and controls, some increase in LDL metabolism was seen in cells from patients with IHD and with a history of smoking. It remains to be determined whether this increase was causally related to smoking.

Cholesterol ester metabolism in fat- and cholesterol/fat-fed guinea pigs

Gunea pigs were fed a semisynthetic diet containing 10% (by weight) cottonseed oil with or without 1% cholesterol. In response to cholesterol/fat feeding there was a significant accumulation of cholesteryl ester (CE), particularly in the liver, but also in the kidney, spleen and suprarenal glands. The hepatic acyl-CoA:cholesterol acyltransferase (ACAT) increased 5–10 times when the animals were fed cholesterol fat during 11 weeks while the acid cholesterol esterase (CE-ase) was similar in the two dietary groups. Intestinal lymph showed the highest content of cholesterol (both free and esterified) in guinea pigs fed cholesterol/fat. A low activity of lecithin:cholesterol acyltransferase (LCAT) was present in the intestinal lymph, irrespective of dietary composition. Triglyceride-rich lipoproteins seem to inhibit LCAT activity in the intestinal lymph. Plasma cholesterol levels in animals fed cholesterol/fat increased markedly while LCAT remained unaffected by the diets. Activity of ACAT and CE-ase in kidney and spleen was low compared to liver tissue and the enzyme activities were not affected by the cholesterol/fat feeding.

Participation of phospholipids in arterial metabolism of cholesterol esters.

The increased concentration of cholesterol esters in the arterial wall attracts attention because this could be due to decreased hydrolysis or increased synthesis in atherosclerosis (3, 6). The following enzymes involved in the metabolism of cholesterol esters have been demonstrated in the arterial wall: cholesterol esterase * (5), along with the enzyme-catalyzed acylation of cholesterol by oleic acid without the addition of ATP and CoA (4, 5); lecithin: cholesterol acyltransferase (1) and acyl-CoA: cholesterol acyltransferase (4).

The Effect of Dietary Rape-Seed Oil on Cholesterol-Ester Metabolism and Cholesterol-Ester-Hydrolase Activity in the Rat Adrenal

The effects of stock diet and stock diet supplemented by olive oil and rape seed on rat adrenal cholesterol ester metabolism have been studied. Rats fed rape seed oil failed to gain weight at the same rate as rats fed olive oil. A prominent feature of the rats fed rape seed oil was an accumulation of high concentrations of cholesterol erucate in the adrenal lipid droplets. When these rats were subjected to an ether stress no percentage decrease in the amount of cholesterol erucate was observed. Adrenal cholesterol ester hydrolase activity was higher in rats fed the olive oil and rape seed oil diets than rats fed the stock diet. In rats fed stock or olive oil diets, a ten-minute ether anaesthesia stress resulted in a two-fold increase in activity of adrenal cholesterol ester hydrolase. Cofactor addition of ATP, cyclic AMP and MgCl-2 in vitro resulted in a stimulation of cholesterol ester hydrolase to a similar activity in both quiescent and ether-stressed rats. By contrast rats fed the rape seed oil diet gave no significant stimulation of cholesterol ester hydrolase activity when given an ether stress or when cofactors were added in vitro. Cholesterol erucate was hydrolysed at only 25% to 30% of the rate of cholesterol oleate in vitro in all groups of animals. Oleic acid added in vitro gave an inhibition of cholesterol ester hydrolase activity in rats fed stock diet while erucic acid activated the enzyme. The accumulation of cholesterol erucate in the adrenal when rats are fed rape seed oil could be due to the reduced ability of cholesterol ester hydrolase to hydrolyse this ester.

Effect of exogenous lipids on rat brain cholesterol ester hydrolases

The three cholesterol ester hydrolases in rat brain were activated by varying degrees when a mixture of whole brain lipids was added to the respective assay systems. The lipid mixture could not replace the detergents present in the standard assay systems for the myelin-localized (pH 7.2) and the microsomal (pH 6.0) cholesterol ester hydrolases. However, the lipid mixture not only could replace the detergent in the pH 4.2 hydrolase assay system but also markedly activated the enzyme when the detergent was omitted. Exogenous phosphatidylserine, G M1-ganglioside, stearic acid, and oleic acid all showed distinct and widely varying effects on each of the measured activities of the three cholesterol ester hydrolases. While the mechanism of activation or inhibition of these enzymes by exogenous lipids is not clear at present, these observations suggest potential importance of various lipids in the in vivo metabolic regulation of brain cholesterol ester metabolism.