Rat Liver Phospholipid Research Articles

Trifluoperazine and other anaesthetics inhibit rat liver CTP: phosphocholine cytidylyltransferase

Chlorpromazine (25 μM) and trifluoperazine (25 μM) inhibited by 5-fold the activity of CTP: phosphocholine cytidylyltransferase, the rate-limiting enzyme for phosphatidylcholine biosynthesis, in rat liver cytosol. Addition of saturating amounts of rat liver phospholipid to the enzyme assay rapidly reversed the drug-mediated inhibition. Three-fold or greater concentrations of these drugs were required to produce a 50% inhibition of the microsomal cytidylyltransferase. Incubation of rat hepatocytes with 20 μM trifluoperazine or chlorpromazine did not inhibit phosphatidylcholine biosynthesis. These results provide additional evidence for the hypothesis that the active form of cytidylyltransferase is on the endoplasmic reticulum and the enzyme in cytosol appears to be latent.

Phospholipid transfer protein-mediated incorporation and subcellular distribution of exogenous phosphatidylcholine and spingomyelin in cultured neuroblastoma cells

Murine neuroblastoma cells (strain N1E-115) were incubated with 2-[ fatty acyl- 14C]acylphosphatidylcholine/sphingomyelin or phosphatidylcholine/[ choline- 3H]sphingomyelin liposomes (1:1, mol/mol; 1.2 μmol total lipid/mg cell protein) in the presence of partially purified rat liver phospholipid transfer protein (2.5 mg/ml), cytochalasin B (50 μM) and 2-deoxyglucose (50 mM) for 10 min. Washed cells were chased for periods of up to 45 min at 37°C with medium containing transfer protein and unlabeled liposomes. Total transfer protein-dependent incorporation of [ 14C]phosphatidylcholine ([ 14C]PC) and [ 3H]sphingomyelin was 136.7 ± 26.5(n = 5) and 23.7 ± 5.4(n = 6) nmol/mg protein per 10 min incubation, respectively, (mean ± S.D.). Incorporation of [ 14C]PC into the mitochondrial membrane fraction was 128-fold greater (nmol/mg protein) than incorporation of [ 3H]sphingomyelin. In contrast, incorporation of [ 3H]sphingomyelin into a fraction enriched in plasma membrane and into microsomes was 1.4- and 2.6-fold greater, respectively, than incorporation of [ 14C]PC. During the chase periods, the specific activities of total cellular phospholipids decreased as intact [ 14C]PC and [ 3H]sphingomyelin accumulated in the culture medium. In the case of cells labeled with [ 14C]PC, the effect was due primarily to a decrease in the amount of labeled phospholipid in the mitochondrial fraction; in the case of cells labeled with [ 3H]sphingomyelin, the decrease in activity was greatest in microsomal and plasma membrane phospholipids. The rate and extent of non-endocytotic incorporation of exogenous phosphatidylcholine into the cell membrane of cultured neuroblastoma cells, and its subsequent subcellular disposition, is different from that of exogenous sphingomyelin. Whereas PC is evidently incorporated into and turned over most rapidly in fraction enriched in mitochondrial membranes, sphingomyelin appears to be preferentially incorporated into microsomal and plasma membrane.

Phospholipid composition of liver in rats fed high levels of 13-cis retinoic acid.

The composition of liver phospholipids was studied in rats fed for 4 weeks diets containing 0, 100 or 300 mg 13-cis retinoic acid per kg diet. There was a significant decrease in phosphatidylcholine content, whereas the levels of phosphatidylethanolamine were slightly increased in liver phospholipids of rats fed 13-cis retinoic acid. The fatty acid composition of total phospholipids, PC, PE, and P1 + PS fractions revealed a general increase in the levels of 18:2 and 20:3 omega 6, whereas the levels of 20:4 omega 6 and C22 fatty acids were reduced in most of the hepatic phospholipids isolated from rats fed 13-cis retinoic acid containing diets. A decrease in the double-bond index of fatty acids was also observed in phospholipids of rats fed 13-cis retinoic acid. The data suggest that high levels of 13-cis retinoic acid may possibly be influencing the activities of microsomal desaturating and chain-elongating enzymes in the liver.

The relative deacylation of different molecular species of endogenous phosphatidylethanolamine in rat liver microsomes by phospholipase activity

The relative deacylation of the 1-palmitoyl and 1-stearoyl homologues of different molecular species of endogenous phosphatidylethanolamine in rat liver microsomes via phospholipase activity was studied. For this purpose, the various molecular species of microsomal phosphatidylethanolamine were labelled specifically in the 1-position by incubation of rat liver microsomes with [ 14C]palmitoyl-CoA or [ 14C] stearoyl-CoA plus 2- acyl-sn- glycero-3- phosphorylethanolamine containing 18:1, 18:2, 20:4, 22:6 , etc. followed by resuspension of the microsomal pellet. The loss of radioactivity from the total 1-[ 14C]palmitoyl and 1-[ 14C]stearoyl homologues of phosphatidylethanolamine amounted to 31 and 29%, respectively, when these microsomal preparations were incubated for 1 h in 50 mM Tris-HCl buffer (pH 8.5) containing 10 mM Ca 2+. Regardless of whether palmitate or stearate resided in the 1-position, the susceptibility of the phosphatidylethanolamines to deacylation was not influenced significantly by the nature of the unsaturated fatty acid in the 2-position, as judged by selectivity indices for the relative disappearance of radioactivity from the individual classes (monoenoic, dienoic, trienoic, tetraenoic, pentaenoic and hexaenoic). A moderate discrimination against 1-stearoyl 2-saturated species was indicated. The findings indicate that fatty acid selectivity in the microsomal deacylation of phosphatidylethanolamine cannot account for the unique fatty acid and molecular species composition of this phospholipid in rat liver.

Changes in phospholipid methyltransferases and membrane microviscosity during induction of rat liver microsomal cytochrome P-450 by phenobarbital and 3-methylcholanthrene.

Rat liver microsomes contained two methyltransferases which converted phosphatidylethanolamine (PE) to phosphatidylcholine (PC). The first methyltransferase converted PE to phosphatidyl-N-methylethanolamine (PME) and the second methyltransferase converted PME to PC. Previous work has shown that increased PME synthesis decreases membrane microviscosity. Therefore, changes in the rat liver microsomal cytochrome P-450, phospholipid methyltransferases and membrane microviscosity after induction by phenobarbital and 3-methylcholanthrene were studied. Phenobarbital and 3-methylcholanthrene increased cytochrome P-450 levels 2- to 3-fold. At low SAM concentration, the proportion of PME among the total phospholipids formed increased significantly, and at a high SAM concentration, the proportion of PC among the total phospholipids formed decreased significantly in microsomes of treated rats. Treatment of rats with phenobarbital and 3-methylcholanthrene also decreased microviscosities of the microsomal membranes and liposomes which were prepared from phospholipids extracted from the microsomes. In synthetic liposomes containing PE, PME and PC, microviscosity decreased when the proportion of PME was increased or the proportion of PC was decreased. These results suggest that the membrane fluidity increases with phenobarbital and 3-methylcholanthrene treatment, and changes in phospholipid methyltransferases may contribute to the process of enzyme induction. During induction with phenobarbital, all three factors known to increase membrane fluidity (linoleic acid content, the formation of phosphatidyl-N-methylethanolamine, and decreases in the cholesterol/phospholipid ratio) contribute to the decrease in microviscosity. During induction with 3-methylcholanthrene, alterations in phospholipid methylation is possibly the primary cause of the decrease in membrane microviscosity.

Effects of Linolenic Acid Deficiency on the Fatty Acid Patterns in Plasma and Liver Cholesteryl Esters, Triglycerides and Phospholipids in Female Rats

These experiments were performed to measure the effects of linolenate deficiency upon neutral lipids of plasma and liver, and to search for a metabolic interaction between dietary choline and linolenic acid. Rats were fed for two generations on a linolenic acid-deficient diet containing methyl linoleate as the only source of lipid. Control rats were supplemented with methyl linolenate. Second-generation linolenate-deficient rats and control rats were fed low-methionine, choline-deficient diets for 2 weeks. Half the animals in each group were given choline-supplemented diets. Plasma and liver total cholesterol, esterified cholesterol, triglyceride and major phospholipid classes, and the fatty acids of these classes were measured. Linolenic acid deficiency reduced the concentrations of plasma triglycerides in both choline-deficient and choline-supplemented rats. Evidence for a metabolic interaction between choline and linolenic acid was not obtained because the rats responded very weakly to the choline deficiency. Linolenate deficiency reduced the proportions of n-3 fatty acids, particularly 22:6n-3, in all the lipids analyzed.

Effects of Vitamin B-12 Deprivation on Phospholipid Fatty Acid Patterns in Liver and Brain of Rats Fed High and Low Levels of Linoleate in Low Methionine Diets

Experiments were conducted to determine how 20 weeks of deprivation of vitamin B-12 (B-12) would influence polyunsaturated fatty acid (PUFA) patterns in cerebral and liver phospholipids of female rats. All experimental diets included 24% soybean protein and either 3.5 of 0.35% of linoleate (18:2). Deprivation of B-12 and restricted intakes of 18:2 significantly inhibited growth of the rats. Rats depleted of B-12 had 6.7 times more methylmalonic acid in their liver, and significantly more odd-numbered acids (15:0 + 17:0) were present in phosphatidyl choline (PC) of their liver and cerebrum. Deprivation of B-12 promoted 63.3 to 97.3% increases of 18:2 in PC and phosphatidyl ethanolamine (PE) of the liver. Rats deprived of B-12 had significantly less arachidonate (20:4 omega 6) and 22:5 omega 6 in their liver PC, as well as smaller amounts of 20:4 omega 6 and 22:6 omega 3 in their cerebral PC. Some of the observed changes appear to be due to a significant interaction between the effects of B-12 and linoleate. Changes in PUFA patterns of cerebral and hepatic PC suggest that B-12 deprivation may interfere with the conversion of 18:2 omega 6 leads to 20:4 omega 6 leads to 22:5 omega 6. Some of the observed changes may also be related to an inhibited ability to convert PE to PC.

Effect of clofibrate on the phospholipid biosynthesis in rat liver.

The effect of clofibrate on rat liver phospholipid biosynthesis was studied using 32P as a precursor. Phospholipid classes, levels and specific radioactivity were evaluated. Significant increases in levels of phosphatidylethanolamine and phosphatidylcholine were found and could account for the observed increase in total phospholipids. Specific activity of phosphatidylserine increased and that of phosphatidylethanolamine decreased. This fact suggests that clofibrate seems to alter the systems engaged in the transformation occurring within the different classes of phospholipids but not the de novo biosynthesis.

Role of lipid peroxidation and?-tocopherol in conductivity of artificial membranes made from liver phospholipids of rats with burns

Antibodies on sepharose immunosorbents containing A-polysaccharide-sepharose or synthetic beta-N-acetylglucosamine, were isolated from the sera of rabbits immunized with streptococci, group A, by means of affinity chromatography. Antibodies obtained from some sera with both immunosorbents reacted with streptococcus, group A and L polysaccharides. Partial identity of these polysaccharides was revealed by the immunodiffusion test. Absorption of antibodies with polysaccharides, group A and L, showed their different specificities. These antibodies could apparently be directed against the end parts of molecules of streptococcus, group A polysaccharide.

Phospholipid content and activity of pure uridine diphosphate-glucuronyltransferase from rat liver.

Rat liver phospholipids were radioactively labeled in vivo before purification of UDP-glucuronyltransferase to homogeneity. The pure enzyme contained very little phospholipid (approx. 0.7 mol of phospholipid/mol of protein). The solubilization detergent Lubrol 12A9 appeared to act as a phospholipid substitute, capable of supporting UDP-glucuronyltransferase activity. Phospholipase C did not inhibit the pure enzyme activity and pure UDP-glucuronyltransferase was stimulated by 40--100% by the addition of phospholipid dispersions.

Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [ 32P]orthophosphate or [Me- 14C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of 32Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [ 14C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [ 14C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both 32Pi and 14C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in 32Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of 14C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.

Fatty Acid and Glucose Metabolism in Selenium Deficient Rats and Lambs

Fatty acid analyses were done on tissues of lambs from ewes fed purified diets, and injected with selenium and/or vitamin E in a 2 X 2 factorial treatment. The concentrations of arachidonic acid averaged 9.3% of the total fatty acid content in semitendinosus muscle from lambs given vitamin E and selenium, but averaged 19.4% in this muscle from lambs given vitamin E without selenium. Arachidonic acid comprised 28.3% and 33.7%, respectively, of the total fatty acids in livers from selenium supplemented and deficient rats. Twice as much radioactivity from [1-(14)C]acetate was recovered in rat liver phospholipid arachidonate in selenium deficient rats as in selenium supplemented ones, indicating a greater turnover rate of this fatty acid in deficient rats. Fifty-five percent of dosed 14C-glucose (either [1-(14)C] or [6-(14)C]glucose) was recovered within 3 hours as 14CO2 from selenium deficient rats. This recovery dropped to one-half this value when these rats were fed a diet containing 0.1 ppm selenium for only 7 days. This increased glucose metabolism is suggestive of a greater metabolic rate in selenium deficient animals, which may be responsible for the differences observed in tissue fatty acid composition.

Intracellular phospholipid transfer and exchange

The intracellular transfer of phospholipids in rat liver was studied. The factors affecting the transport and the role of phospholipid transfer proteins in this process were investigated. The procedure was based upon the labelling of microsomal phospholipids with either 14C or 32P, and incubation with unlabelled mitochondria in an in vitro system. The re-isolated mitochondria became labelled demonstrating an exchange of phospholipid between the two membranes. The transfer was stimulated by the addition of high-speed supernatant, was unaffected by the addition of ATP, but did not occur at 0°C. Phospholipid transfer between liposomes and mitochondria was shown to occur, suggesting that the participation of natural membranes is not required for transfer to take place. The conditions required were found to be similar, and the high-speed supernatant promoted transfer to the same extent as when labelled microsomes acted as the donor. The transfer activity of the high-speed supernatant was abolished by treatment with sulphydryl-blocking agents. Beef supernatant was adjusted to pH 5.1 and the activity remained in the supernatant. The pH 5.1 supernatant was applied to a Sephadex G-75 column and two active fractions were collected. The higher molecular weight fraction contained several proteins and stimulated the transfer of phosphatidylcholine and phosphatidylethanolamine. The lower molecular weight active fraction contained a protein which specifically stimulated the transfer of phosphatidylcholine.

Effect of starvation on the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.

A new rat liver phospholipid exchange protein

The soluble fraction from several mammalian tissue homogenates is known to stimulate phospholipid exchange between cell membrane fractions or artificial vesicles. All phospholipid exchange proteins purified to data exhibit an acidic isoelectric point. Using an assay that measures the transfer of [ 32P]phosphatidylcholine from liposomes to beef heart mitochondria, we report the presence of a new phospholipid exchange protein with a basic isoelectric point (8.4) in rat liver cytosol. A purification procedure, consisting of pH adjustment to 5.1, gel filtrations on Sephadex G-75 and DE 52 cellulose, isoelectric focusing between a pH of 5 and 10, and gel filtration on Sephadex G-50, yielded a fraction with high phosphatidylcholine exchange activity per mg of protein. This fraction exhibits a major band and two minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the major band (18 700) is close to that for basic exchange protein fraction obtained by gel filtration (17 000). The distribution of basic and acidic exchange proteins differs markedly in various tissues and animal species. About 50 and 35% of phosphatidylcholine exchange activity from rat liver and rat intestine respectively are due to basic phospholipid exchange proteins. In contrast, no basic exchange protein was found in beef heart and only a small amount in beef liver. In the latter organ, less than 10% of phosphatidylcholine exchange activity was due to a basic phospholipid exchange protein fraction.

Hepatoma, host liver, and normal rat liver phospholipids as affected by diet

Individual phospholipid classes derived from hepatoma, host liver, and normal liver of rats maintained on chow and fat free diets were examined in detail and the sphingomyelin and phosphoglyceride structures compared. The concentration of hepatoma spingomyelin was higher while phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol were only one-fourth to one-half normal liver concentrations, irrespective of diet. Hepatoma phosphatidylcholine, phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol contained higher percentages of 18:1 and, except phosphatidylinositol, much lower percentages of most polyunsaturated fatty acids than liver. The 1-position of host liver phosphatidylcholine and phosphatidylethanolamine, normal liver phosphatidylcholine and phosphatidylethanolamine, and hepatoma phosphatidylcholine from animals on both diets had the same approximate fatty acid composition, but the percentage of 16:0 in hepatoma phosphatidylethanolamine was reduced dramatically. The low percentage of 16:0 at the 1-position of both phosphatidylethanolamine and triglycerides suggests that the 1-position fatty acids of these two classes may have a similar origin. The fat free diet reduced the percentage of 18:2 in liver diphosphatidylglycerol 3-fold and the decrease was offset by increased percentages of 16:1 and 18:1; whereas the very low percentage of 18:2 in hepatoma diphosphatidylglycerol was offset by increased percentages of 18:0 and 16:0. Liver phosphatidylinositol and phosphatidylcholine from the animals fed the fat free diet contained the highest percentage of 20:3, which replaced 20:4. Hepatoma sphingomyelin contained a much higher concentration of 24:0 and 24:1 than liver. The hepatoma sphingomyelin also contained a C-24 dienoic acid, which was not detected in host and normal liver. Host liver contained a higher percentage of 22:6 than normal liver. The diglycerides derived from host liver PC contained a significantly higher percentage of carbon number 38 than normal liver. Diglycerides derived from hepatoma phosphatidylcholine and phosphatidylethanolamine exhibited a 1-random-2-random distribution of fatty acids, whereas diglycerides from liver phosphatidylcholine and phosphatidylethanolamine showed pairing of specific fatty acids.

Blood and liver phospholipids of albino rats at various stages of development of experimental pancreatitis

A marked decrease in the content of neutral phospholipids in the plasma and blood cells of albino rats with experimental pancreatitis was observed, especially on the 3rd–7th day of the disease. With the development of pancreatitis no polyglycero-phospholipids — an important faction of acid phospholipids participating in the formation of oxidative enzyme systems — could be found in the liver. The phenomena described are regarded as essential pathogenetic factors leading to the development of fatty infiltration of the liver in pancreatitis.

Blood serum and liver phospholipids of rats with chronic choline—protein deficiency

Serum and liver phospholipids were studied in rats at different stages of pathological processes induced by a diet with a high fat content, but deficient in protein and choline. Such cirrhosogenic diet caused fatty infiltration, fibrosis and nodular liver cirrhosis in rats, depending on the period of its administration. This diet resulted in a considerable decrease of the phospholipid content in the liver and the serum of experimental rats. Addition of choline to the cirrhosogenic diet prevented formation of fatty infilitration in rats, but failed to protect completely from a fall of phospholipids in the liver.

Drugs affecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amphetamine, cinchocaine, chlorpromazine, demethylimipramine, mepyramine and some of their derivatives.

The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered.

Influence of long-term exercise and training on heart and liver phospholipids in rats

1. 1. Comparative studies were made in heart and liver phospholipids between sedentary controls and rats subjected to a long-term programme of exercise and training. 2. 2. Statistically significant changes occurred between experimental animals and controls. 3. 3. An increase in phosphatidylcholine and phosphatidylethanolamine was observed both in heart and liver of experimental animals. 4. 4. A decrease was found in heart sphingomyelin and in liver phosphatidylserine. 5. 5. High concentrations of diphosphatidylglycerol were found in the heart compared to the liver.