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In the trials on rats it has been found that ethionine injection damages the system of cholesterol esterification in the blood serum. The factors stimulating hepatocyte proliferation prevent partially the toxic action of ethionine.
A group of fourteen healthy young male volunteers was examined to define more exactly the relations between lecithin cholesterol acyltransferase activity (LCAT), fractional cholesterol esterification rate (FER), total cholesterol (TC) and its free and esterified fractions (FC, CE) in skeletal muscles under physiological conditions. The mean values (+/- S.D.) of LCAT activity (95.4 +/- 16.3 mumol .1(-1) per hour), and FER (7.45 +/- 1.54% per hour) corresponded to published data on normolipidaemic healthy men of normal body weight. The mean value of TC in muscles was 332 +/- 83 micrograms per 100 mg of non-collagen protein, of which 14 +/- 7.4 per cent was formed by cholesterol esters. There was positive correlation between TC in muscles and age. Significant positive correlations between FER and the content of esterified cholesterol in muscles, and between FER and the proportion of esterified to total muscle cholesterol were found. These results suggest a close interrelation of cholesterol ester metabolism in the plasma and in slow pool tissues.
Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.
The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.
Pieces of epididymal fat pad from fed and 48-hr-fasted rats were incubated for various periods of time in Krebs-Ringer bicarbonate containing [1-(14)C]glycerol. The radioactive substrate taken up by the tissue increased linearly with time in both groups and was mainly converted to glyceride-glycerol and CO(2). The slopes of the regressions of (14)C-labeled glyceride-glycerol with time were not different between the groups, while those of (14)CO(2) were smaller in the fasted than in the fed animals. Because the radioactive glycerol in the medium is being continuously diluted with the glycerol coming out of the tissue, it is necessary to take account of this factor in calculating the actual amount of glycerol utilized by the tissue. The glycerol produced by the tissues is higher in the fasted than in the fed animals, and in both groups it increases hyperbolically with time. As negligible amounts of the (14)C-labeled glycerol taken up by the tissue recirculates to the medium, the rates of glycerol release (lipolysis), esterification, and oxidation to CO(2) were calculated.
That a necessary requirement for in vivo esterification of 3-hydroxy sterols is the equatorial conformation of the hydroxyl group has been substantiated by the administration of epicoprostanol-3-(3)H (3-OH equatorial) to a man with a complete biliary fistula. Radioactive epicoprostanol was identified in both free and esterified sterols of plasma. The specific activity of biliary sterols was much higher than that of plasma sterols; this indicated a preferential removal of epicoprostanol from circulating sterols. The appearance of tritium in body water demonstrated that a portion of the labeled epicoprostanol underwent oxidation at C-3.
The methyl esterification of the aspartate receptor involved in chemotaxis has been studied in order to clarify the role of receptor modification. Receptors were methyl esterified in an in vitro system using S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Methyl esterified receptors were digested with trypsin and radioactive tryptic peptides were purified using high performance liquid chromatography. Comparing the amino acid composition of the modified peptides with the DNA sequence of the receptor gene, two regions of the polypeptide chain which contain methyl esterified residues were identified. The regions are homologous and contain a strongly conserved 13 amino acid sequence. One region, containing up to three modified residues, is near the middle of the protein; the other, containing one modified residue, is near the carboxyl terminus.
Nine obese women without hyperinsulinemia fasted 22 +/- 2 d under controlled conditions. Blood was drawn weekly and lipids, lipoproteins, and insulin were determined. In five patients an open fat biopsy was performed before and after fasting and the incorporation of free fatty acids (FFA) into total lipids, diglycerides (DG), and triglycerides (TG) was measured. Glycerol release into the medium was also determined. During fasting triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, glucose, and insulin fell considerably. The uptake and esterification of FFA decreased under both basal and stimulated (norepinephrine) conditions (-11 percent to -23 percent). Basal lipolysis increased slightly by 11 percent. Results indicate that fasting-induced changes in adipose tissue metabolism do not account for the decrease in serum triglycerides. The diminished triglyceride synthesis and the enhanced breakdown of triglycerides in adipose tissue do, however, explain the reduction in adipose tissue mass during fasting.
The current studies were undertaken to characterize the localization and regulation of cholesterol synthesis and acyl-CoA:cholesterol acyltransferase activity in rat intestinal crypt and villus cells. Both parameters were determined in groups of animals with widely varying sterol fluxes across the intestinal mucosa. In animals on control diet the rates of cholesterol synthesis, measured by the incorporation of [3H]water per mg of protein, were similar along the villus/crypt axis in the jejunum, whereas in the ileum, villus cells were significantly more active than crypt cells. In both areas, however, the majority of total synthetic activity was found in cells from the crypts and lower villi. In contrast, the highest specific and total acyl-CoA:cholesterol acyltransferase activity was recovered in the villus cells of the jejunum and ileum. Dietary cholesterol did not affect sterol synthesis in any of the cell fractions but increased acyl-CoA:cholesterol acyltransferase activity approximately 2-fold in jejunal cell fractions. Inhibition of cholesterol absorption or sequestration of intestinal bile acids stimulated sterol synthetic activity up to 7-fold, and this occurred mainly in the lower villus and crypt cells in both jejunum and ileum. An increased demand for lipoprotein cholesterol, generated by triglyceride feeding, similarly was associated with enhanced synthetic rates. However, unlike cholesterol feeding, these manipulations did not increase acyl-CoA:cholesterol acyltransferase activity in any of the villus cell fractions. These studies suggest, therefore, that the intracellular pools of cholesterol that regulate the rate of cholesterol synthesis and the rate of cholesterol esterification are functionally distinct.
The metabolism of 9-octadecenoic and 9,12-octadecadienoic acids with different geometrical configurations was compared in isolated perfused rat liver. More ketone bodies were produced when the trans-isomers were infused. In contrast, only the cis-isomer augmented the triacylglycerol secretion almost entirely as very-low-density lipoprotein (VLDL). Although these responses were independent of the difference in the degree of unsaturation in both the cis- and trans-isomers, the trans-monoenic acid compared to the trans-dienic acid was incorporated more readily into perfusate and hepatic lipids. Quantitative information was obtained with radioactive tracer experiments. The hepatic uptakes of 9-[10-14C]octadecenoic acids were comparable in the cis- and trans-isomers. The trans-octadecenoic acid compared to the cis counterpart was oxidized more readily and incorporated more into liver phospholipid but less into perfusate and liver triacylglycerol. These reciprocal responses counterbalanced each other. The lower rates of triacylglycerol synthesis and secretion in the liver perfused with the trans-octadecenoic acid was confirmed using [2- 3H]glycerol as a tracer. The marked difference in the channelling of cis- and trans-fatty acids in the pathways of oxidation and esterification seems to modify the VLDL secretion in perfused rat liver. Present observations indicate a considerable difference in the fate of unsaturated fatty acids with different configurations. trans-Fatty acids are expected to be an efficient energy source in animal tissues and may not be hyperlipidemic.
In a group of 23 newborn infants with necrotizing enterocolitis receiving parenteral nutrition the serum triglyceride levels (347 samples), and the free to esterified cholesterol ratio (188 determinations) were studied. The relationship of both T, and FCH/ECH with bacterial infections were also studied. Mean T level increased significantly with the episodes of infection. In the group of infants with a birth weight less than 1,500 grs. a significantly greater percentage of T levels above 250 mg./dl. than in infants of a greater birth weight is found. FCH/ECH ratio was already abnormal in infants with necrotizing enterocolitis before parenteral nutrition was started. Authors also observed a positive correlation between abnormality of cholesterol esterification and days of parenteral nutrition.
The content of cholesterol and cholesterol ester as well as the levels of acyl coenzyme A:cholesterol acyltransferase activity were determined in the microsomes from Morris hepatomas 7777, 5123D, and 7787. The free cholesterol content, expressed per mg microsomal protein, was significantly increased only in the microsomes from Morris hepatoma 7777 [47.8 +/- 0.4 micrograms (S.D.); p less than 0.001] and hepatoma 7787 (37.6 +/- 6.2 micrograms; p less than 0.01) as compared to normal liver (28.8 +/- 2.4 micrograms). The cholesterol ester content in the microsomes of the three different tumors did not significantly differ from that of normal liver (2.1 +/- 1.2 micrograms cholesterol per mg microsomal protein). The microsomal acyl coenzyme A:cholesterol acyltransferase activity was decreased in Morris hepatoma 7777 (8.6 +/- 2.3 pmol/min/mg protein; p less than 0.01) and in hepatoma 5123D (7.5 +/- 1.7 pmol/min/mg protein; p less than 0.02), and was normal in the hepatoma 7787 (16.5 +/- 7.8 pmol/min/mg protein) as compared to rat liver (16.0 +/- 2.9 pmol/min/mg protein). In a previous study (B. J. H. M. Poorthuis and K. W. A. Wirtz, Biochim. Biophys. Acta, 710: 99-105, 1982), this acyltransferase activity was shown to be stimulated by preincubation of rat liver microsomes with cholesterol-containing vesicles and the nonspecific phospholipid transfer protein. In this paper, a similar 4-fold stimulation of activity was observed for the microsomes of the various hepatomas investigated. The possible role of the nonspecific phospholipid transfer protein in intracellular cholesterol esterification is discussed.
In the present study the effect of 5'methylthioadenosine (MTA), a natural metabolite of S-adenosylmethionine (AdoMet), on the enzymatic methyl esterification of intact erythrocyte membrane proteins has been investigated. The thioether significantly affects the methylation process :50% inhibition being observable at 100 microM MTA. This inhibition is due to the action of MTA on the enzyme protein methylase II. Since MTA is present in micromolar amounts in the cells, the reported effect could be of physiological interest and suggests a new regulatory role of this AdoMet metabolite.
The ratio of unesterified to total serum cholesterol in hypothyreotic rats has been studied. Extreme rat hypothyreosis of identical degree was induced by radiothyroidectomy (group R-HT) and feeding of thyreostatics (groups MTU-HT and MMI-HT). Determination of total serum cholesterol content and the unesterified cholesterol content resulted in following changes (all changes significant for p < 0,001): 1. Higher total cholesterol in hypothyreotic rats 4 and 7 weeks after beginning the experiments as compared to the controls. 2. The ratio of unesterified cholesterol to total cholesterol was increased in all hypothyreotic groups. It is discussed, that the esterification reaction in serum catalyzed by the lecithin-cholesterol-acyltransferase (LCAT) is diminished in the hypothyreotic state.
Direct gas chromatographic method for quantitative determination of plasma neutral lipids was used for the study of cholesterol esterification in vitro. Fully automated computerized method permits the determination of lipid classes (free cholesterol, cholesteryl esters, and triglycerides), as well as their fractions according to the molecular weight, without previous derivatization. All components are determined from one analysis. The method is highly precise and accurate. The coefficient of variation for free and esterified cholesterol is 1.21 and 1.11% in the concentration range 30--430 and 75--545 mg/dl, respectively. The LCAT activity was determined as a decrease in the free cholesterol concentration after one hour's incubation; from the same analysis, the recovery control as an increase in the esterified cholesterol concentration was calculated. The mean difference was 1.06%, maximum difference did not exceed 4%. The LCAT activity in the reference group was 97.2 +/- 28.2 mumol/l/h and was significantly lower than that in hyperlipdemic one. Intravenous administration of heparin had an inhibition effect in subjects with hyperlipidemia of type IV, while only slight changes in the reference group and an increase in hyperlipidemia of type III were observed.
Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors are known to inhibit cholesterol absorption and are under investigation to reduce hypercholesterolemia. These studies examine the effect of an ACAT inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)-dodecanamide (PD128042) on the uptake, metabolism and secretion of cholesterol by the hamster intestinal wall in a short-term model. Preliminary studies in this model indicated that the uptake of 14C-cholesterol and its subsequent esterification 2 hr postoral dosing occurs primarily in the duodenal and jejunal segments of the small intestine and most of the radiolabeled cholesterol and cholesteryl ester in the plasma was associated with chylomicrons. In both single- and multiple-dose studies, PD128042 (50 mg kg-1 day-1) did not inhibit intestinal uptake of [14C]-cholesterol but [14C]-cholesteryl ester formation was inhibited. The free [14C]-cholesterol appearing in plasma was not affected despite a large reduction in [14C]-cholesteryl ester. In contrast, cholestyramine (1 g kg-1 day-1) inhibited the uptake of the radiolabeled free cholesterol and the appearance of cholesteryl ester in the intestine and plasma. The effects of PD128042 on cholesterol and cholesteryl ester mass associated with scraped intestinal mucosa were consistent with the effects observed with the use of the radiolabeled cholesterol. In addition, PD128042 did not affect the uptake of appearance of radiolabeled triglyceride in the intestinal wall after oral gavage of 3H-trioleoylglycerol. Taken together, the data suggest that ACAT inhibition reduces cholesterol absorption by limiting cholesteryl ester incorporation into chylomicrons and has no effect on the intestinal processing of free cholesterol to be secreted into plasma.
We have recently described a novel protein carboxyl methylation system that results in the reversible modification of a 36-kDa polypeptide component of a 178-kDa protein in the cytosol of a variety of eucaryotic cells. This reaction, catalyzed by a cytosolic 40-kDa methyl-transferase, results in the methyl esterification of the alpha-carboxyl group of the C-terminal leucine residue. We have now purified the major methylated 36-kDa polypeptide from bovine brain. N-terminal sequence analysis of a tryptic fragment of this polypeptide revealed identity to the catalytic subunit of protein phosphatase 2A. This enzyme exists in the cell predominantly as a trimeric 151-kDa native species containing the 36-kDa catalytic polypeptide that terminates in a leucine residue. We then fractionated bovine brain cytosolic extracts to separate the major phosphatase isoforms 2A1 and 2A2 and found that both could be methylated by a partially purified preparation of the methyltransferase. A synthetic C-terminal octapeptide based on the sequence of the 36-kDa catalytic subunit is neither a substrate nor an inhibitor of this methyltransferase, suggesting that this enzyme recognizes aspects of the tertiary and/or quaternary structure of the native phosphatase. Because this modification reaction is readily reversible in extracts, it may represent a novel strategy of the cell to modulate the function of this protein phosphatase.
We studied the particle size distribution of plasma high-density lipoproteins (HDL) by gradient gel electrophoresis and by assay of cholesterol esterification rate (FERHDL) in plasma depleted of very low (VLDL) and low-density (LDL) lipoproteins in 32 hypertensive women (53 +/- 10 y old) and in an age-matched group of 21 apparently healthy women. There were no significant differences between the groups with respect to their plasma total, HDL- and LDL-cholesterol. The plasma triglyceride (TG) concentration was significantly higher in the group of hypertensive women, and HDL-free cholesterol was significantly lower in the hypertensive group. There were highly significantly differences in the relative proportions of HDL subclasses between the hypertensive and control women: HDL2b was significantly lower and HDL3b,c was significantly higher in hypertensive patients compared to controls. FERHDL was 12 +/- 4%/h in the control group and 18 +/- 6%/h in patients with hypertension (p < 0.001). There was a strong positive correlation between FERHDL and the relative content of HDL3b,c, and a strong negative correlation between FERHDL and HDL2b in both groups. Comparison of subgroups of healthy and hypertension pre- and postmenopausal women revealed, apart from age, no significant differences between the healthy subjects in any of the above parameters.