Microsomal Phosphatidylcholine Research Articles

Clofibric Acid Increases the Formation of Oleic Acid in Endoplasmic Reticulum of the Liver of Rats

The effects of 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) on the formation of oleic acid (18:1) from stearic acid (18:0) and utilization of the 18:1 formed for phosphatidylcholine (PC) formation in endoplasmic reticulum in the liver of rats were studied in vivo. [¹⁴C]18:0 was intravenously injected into control Wistar male rats and rats that had been fed on a diet containing 0.5% (w/w) clofibric acid for 7 days; and the distribution of radiolabeled fatty acids among subcellular organelles, microsomes, peroxisomes, and mitochondria, was estimated on the basis of correction utilizing the yields from homogenates of marker enzymes for these organelles. The radioactivity was mostly localized in microsomes and the radiolabeled fatty acids present in microsomes were significantly increased by the treatment of rats with clofibric acid. The formation of radiolabeled 18:1 in microsomes markedly increased and incorporations of the formed [¹⁴C]18:1 into PC and phosphatidylethanolamine in microsomes were augmented in response to clofibric acid. The [¹⁴C]18:1 incorporated into PC was mostly located at the C-2 position, but not the C-1 position, of PC, and the radioactivity in 18:1 at the C-2 position of PC was strikingly increased by clofibric acid. These results obtained from the in vivo experiments directly link the findings that clofibric acid treatment induces microsomal stearoyl-CoA desaturase and 1-acylglycerophosphocholine acyltransferase in the liver and the findings that the treatment with the drug elevated absolute mass and mass proportion of 18:1 at the C-2 position, but not the C-1 position, of PC in the liver together.

Flip-Flop of Fluorescently Labeled Phospholipids in Proteoliposomes Reconstituted with Saccharomyces cerevisiae Microsomal Proteins

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.

Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats.

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.

Effects of dietary eritadenine on delta6-desaturase activity and fatty acid profiles of several lipids in rats fed different fats.

Effects of dietary eritadenine on liver microsomal delta6-desaturase activity and the fatty acid profile of phosphatidylcholine, cholesteryl esters, and triglycerides of liver microsomes or plasma were investigated in rats fed different fats (palm oil, olive oil, and safflower oil). The activity of delta6-desaturase was influenced by both dietary fat types and eritadenine. In rats fed control diets, delta6-desaturase activity was higher in the order of the palm oil, olive oil, and safflower oil groups. In rats fed eritadenine-supplemented diets, the enzyme activity was markedly decreased to a constant level irrespective of dietary fat type. The 20:4n-6/18:2n-6 ratio of phosphatidylcholine and cholesteryl esters, as compared with triglycerides, was highly sensitive to eritadenine. The results suggest that the activity of delta6-desaturase is regulated by dietary fats and eritadenine independently, and that the effect of eritadenine is stronger than that of dietary fats.

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys 55 present in one of the β-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys 55 be involved in the binding of the PC molecule.

Molecular properties of the oleoyl-phosphatidylcholine desaturase from Arachis hypogaea L.

Deficiencies in the activity of the microsomal oleoyl phosphatidylcholine (oleoyl-PC) desaturase from peanuts are the basis of the high oleate oil. Mutation of aspartate-150 to asparagine and the attendant decrease in activity, together with the loss in expression of the higher activity transcript, was the molecular basis of the high oleate trait. The ability of oleoyl-PC desaturase to desaturate palmitoleate, oleate and 10(Z) nonadecenoate to methylene-interrupted products was not consistent with description of this activity as a Delta(12) or omega-6 desaturase. Electrospray MS was used to examine the intact phospholipid products of desaturation by the oleoyl-PC desaturase. PC and phosphatidylinositol containing unsaturated moieties could be desaturated. The enzyme can act on either sn-1 or sn-2 moieties. Phosphatidylethanolamine was a poor substrate.

Dietary cholesterol induces changes in molecular species of hepatic microsomal phosphatidylcholine.

After 21 days on a diet containing 1 g% cholesterol and 0.5 g% cholic acid, rats had an increased content of cholesterol in liver microsomal lipids. In liver, both cholesterol content and delta9 desaturase activity increased, whereas delta6 and delta5 desaturase activities decreased. These changes correlated with increases in oleic, palmitoleic, and linoleic acids and decreases in arachidonic and docosahexenoic acids in total microsomal lipids. Similar fatty acid changes were found in phosphatidylcholine (PC), the principal lipid of the microsomal membrane. In PC the predominant molecular fatty acid species (67% of the total) in the control rats were 18:0/20:4, 16:0/20:4, and 16:0/18:2; and they mainly determined the contribution of PC to the biophysical and biochemical properties of the phospholipid bilayer. The cholesterol diet decreased specifically the 18:0/20:4 species, and to a lesser extent, 16:0/20:4 and 18:0/22:6. The 18:1-containing species, especially 18:1/18:2 and less so 16:0/18:1 and 18:1/20:4, were increased. A new 18:1/18:1 species appeared. The independent effects of the presence of cholesterol and change of the fatty acid composition of the phospholipid bilayer of liver microsomes on the packing were studied by fluorescence methods using 6-lauroyl-2,4-dimethylaminonaphthalene, 1,6-diphenyl-1,3,5-hexatriene and 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which test different parameters and depths of the bilayer. Data showed that the increase of cholesterol in the membrane, and not the change of the fatty acid composition of phospholipids, was the main determinant of the increased bulk packing of the bilayer. The increase of fluid oleic- and linoleic-containing species almost compensated for the drop in 20:4- and 22:6-containing molecules. But the most important effect was that the general drop in essential n-6 and n-3 polyunsaturated fatty acids meant that this endogenous source for the needs of the animal decreased.

The Assembly of Very Low Density Lipoproteins in Rat Hepatoma McA-RH7777 Cells Is Inhibited by Phospholipase A2Antagonists

In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.

Regulation of guinea pig hepatic acyl-coa:cholesterol acyltransferase activity by dietary fat saturation and cholesterol

We measured the interactive effects of dietary cholesterol and fat on the regulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity and its relationship to hepatic microsomal lipid composition in guinea pigs fed 15 g/100 g (w/w) fat diets (corn oil, olive oil, or lard) with 0.01, 0.08, 0.17, or 0.33 g/100 g (w/w) added cholesterol. Guinea pigs exhibited a dose dependent increase in hepatic microsomal ACAT activity, with increasing levels of cholesterol intake ( P < 0.001) in all dietary fat groups. Animals fed monounsaturated olive oil had the highest hepatic ACAT activity with the exception of the 0.33 g/100 g cholesterol diet ( P < 0.001). There were no differences in ACAT activity with intake of polyunsaturated corn oil or saturated lard. Dietary cholesterol resulted in increased microsomal free cholesterol (FC) concentrations in a dose dependent manner but had no effects on microsomal phosphatidylcholine (PC) concentrations. Guinea pigs fed olive oil generally had the highest microsomal FC/PC molar ratios, and hepatic ACAT activities correlated significantly with this parameter. After modification of the lipid compositions of the microsomes from guinea pigs fed the 12 test diets with FC/PC liposome treatment, microsomal ACAT activities remained significantly related to the microsomal FC/PC molar ratios, and dietary fat type did not affect this correlation. Our findings do not support the hypothesis that the stimulation of hepatic ACAT activity with cholesterol intake is enhanced by polyunsaturated fat intake. The data demonstrate that although dietary fat type and cholesterol amount have differential effects on hepatic ACAT activity, substrate availability, expressed as microsomal FC/PC molar ratio, is a major regulator of hepatic microsomal ACAT activity.

The effect of ethanol and acetaldehyde on microsomal and mitochondrial membrane fatty acid profiles in cultured rat astroglia.

It has been shown that free radical damage may be involved in ethanol-induced cytotoxicity in cultured neural cells. Since changes in oxidative metabolism and the resulting lipid peroxidation readily modify biological membranes and alter cell functions we studied the effect of ethanol and its metabolite acetaldehyde on rat astroglial fatty acids profiles in the most common lipid classes of mitochondrial and microsomal membranes, i.e. phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Rat astroglial cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To examine acetaldehyde effects we used a 4-day co-culture model consisting of astroglial cells and alcohol dehydrogenase-transfected Chinese hamster ovary (CHO) cells. Acetaldehyde produced by these cells reached 172 mu M and 265 mu M, respectively, for ethanol concentrations of 10 and 20 m M. Mitochondrial and microsomal membranes were prepared by differential centrifugation, phosphatidylcholine and phosphatidylethanolamine were separated using thin layer chromatography and fatty acid quantitation was performed by GLC. Neither ethanol nor acetaldehyde changed the mitochondrial phosphatidylcholine or phosphatidylethanolamine profiles of total saturated, mono-unsaturated or polyunsaturated fatty acids. However, some significant alterations in particular fatty acids appeared especially after acetaldehyde but also after the highest ethanol dose. In microsomal phosphatidylcholine monounsaturated fatty acids were significantly increased after both, ethanol and acetaldehyde exposure. Among polyunsaturated fatty acids, arachidonic acid was found to be especially affected by both ethanol and acetaldehyde. Similar decreases were observed in adrenic, docosapentaenoic and docosahexaenoic acids in the groups treated with ethanol. In microsomal phosphatidylethanolamine, ethanol and acetaldehyde decreased monounsaturated and some polyunsaturated fatty acids. These data support the role of peroxidative processes in cultured rat astroglia exposed to ethanol and point to the role of acetaldehyde in this mechanism.

Molecular species of phosphoglycerides in liver microsomes of rats fed a fat-free diet.

The influence of a fat-free diet on the molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) of rat liver microsomes was studied by using reversed-phase high-pressure liquid chromatography. In the three phosphoglyceride classes analyzed, the fat-free diet produced a large decrease in the 18:0/20:4n-6 species but less important changes were found in the 16:0/20:4n-6 species. In PC, the most abundant phosphoglyceride class of rat liver microsomes, the fall in the 18:0/20:4n-6 species was counterbalanced mainly by an enhancement in the 16:0/18:1n-9 species although it was not evident in PE. In PI, the decrease in the 18:0/20:4n-6 species was counterbalanced by an increase in the 18:0/20:3n-9 species. Fluorescence polarization measurements of 1,7-diphenyl-1,3,5-hexatriene in liposomes of 16:0/18:1n-9, 18:0/18:1n-9-, 16:0/20:4n-6-, and 18:0/20:4n-6-PC indicated that the change in the saturated fatty acid in the sn-1 position accompanying the replacement of 20:4n-6 by 18:1n-9 could be very important for a homeoviscous compensation, maintaining the membrane physical properties without large alterations in spite of the essential fatty acid deficiency due to the fat-free diet.

Methionine Content of Dietary Proteins Affects the Molecular Species Composition of Plasma Phosphatidylcholine in Rats Fed a Cholesterol-Free Diet

The effects of dietary protein types and methionine supplementation on phospholipid metabolism were investigated to clarify the mechanism of the hypocholesterolemic action of soybean protein in rats fed a cholesterol-free diet. The effect of switching from a casein diet to a soybean protein diet was also investigated. Rats were fed casein, soybean protein or soybean protein + methionine diet for 14 d. Compared with casein diet, feeding of soybean protein diet led to significantly higher proportions of linoleic acid and linoleic acid–containing molecular species, especially 16:0–18:2, in plasma and liver microsomal phosphatidylcholine (PC). In addition, significantly lower plasma cholesterol concentration, hepatic S-adenosylmethionine concentration and liver microsomal PC:phosphatidylethanolamine ratio resulted. These alterations caused by the soybean protein diet were significantly suppressed by supplementing methionine to the level of the casein diet (3.4 g/kg diet). The proportion of the sum of certain plasma PC molecular species, which contain 18:1 or 18:2 in the sn-2 position, increased in response to the switch from the casein diet to the soybean protein diet at a rate similar to the decrease in plasma cholesterol concentration; there was a significant correlation between the two variables (r = −0.992, P < 0.001). These results indicate that about 40% of the hypocholesterolemic action of soybean protein is due to the low methionine content of the protein and might be associated with alterations of the plasma phospholipid molecular species profile.

Thresholds and kinetics of fatty acid replacement in different cellular compartments in rat liver as a function of dietary [formula omitted] fatty acid content

The kinetics of fatty acid (FA) replacement in different membrane compartments in the rat liver were investigated using diets with varying n-6 n-3 FA ratios. Rats at different stages of growth, i.e. after weaning and at 150 g body weight, were either fed a modified AIN 76A diet containing sunflower oil as fat source or the same diet containing sunflower oil and fish oil to achieve n-6 n-3 FA ratios of 12:1 and 6:1 (diets A and B, respectively). In the adult rats, fed diet A for 8 weeks, C18:2n-6 increased significantly at week 2 in the phosphatidylcholine (PC) fraction of the plasma membranes, microsomes and plasma but not in phosphatidylethanolamine (PE). C20:3n-6 increased significantly at week 2 in the plasma membrane and microsomal PC, but only increased in PE of both compartments by week 8. C20:4n-6 and the n-3 FAs significantly decreased and increased, respectively, at week 2 in PC and PE of both membrane compartments and plasma PC. The experimental diets led to a change in the plasma membrane fluidity but not in the microsomes. The FA changes in the weaned rats followed a similar pattern as in the adult rats although the changes were greater, depending on the phospholipid fraction and specific FA. The decrease in C20:4n-6 was significantly greater in the microsomal PC and PE and plasma PC but not in the plasma membrane PC and PE. The n-3 FAs increased significantly above the adult levels in the plasma membrane PC and PE respectively but not in the microsomal phospholipid fractions. A plateau for maximal n-3 and n-6 FA incorporation was achieved in the adult rats fed diet A in the microsomes after 2 weeks with no further alterations occurring with diet B. In the plasma PC and plasma membranes most of the n-3 FAs achieved a threshold incorporation after 2 weeks on diet A, except for C22:6n-3 in the plasma membranal PE and certain n-6 FAs in the plasma membrane PC and PE. The present data shows that differences exist in the kinetics of FA incorporation and replacement depending on the specific phospholipid fraction, membrane compartment, age and to a certain extent the dietary n-6 n-3 FA ratio.

Methylamine decreases trafficking and packaging of newly synthesized phosphatidylcholine in lamellar bodies in alveolar type II cells.

Lung lamellar bodies, the storage organelles for lung surfactant phosphatidylcholine (PC), maintain an acidic pH that can be increased with weak bases. This study investigates the effect of a weak base, methylamine, on the pH in lamellar bodies and on the trafficking and packaging of newly synthesized PC in lamellar bodies. Methylamine increased the pH of isolated lung lamellar bodies and of lamellar bodies in intact cells. Metabolic labelling of isolated type II cells with [methyl-3H]choline showed that although methylamine (2.5-10 mM) did not alter the labelling of cellular or microsomal PC and disaturated PC, it decreased the labelling of the PC and disaturated PC in lamellar bodies. The packaging of PC in lamellar bodies (the specific activities ratio between the PC in lamellar bodies and the microsomal PC) also decreased in a time- and concentration-dependent manner. The cellular synthesis of PC or its packaging into lamellar bodies was unaltered by brefeldin A, suggesting that the Golgi was not involved in PC packaging. Although methylamine also increased surfactant secretion, the inhibition of PC packaging in lamellar bodies seems unrelated to the secretagogue effect, (1) on the basis of metabolic consequences of increased secretion and (2) because ATP, another secretagogue, did not inhibit PC packaging. Methylamine seems to inhibit PC packaging by inhibiting trafficking of PC to lipid-rich light subcellular fractions. Together our results suggest that the trafficking of surfactant PC into lamellar bodies might be sensitive to changes in the pH of lamellar bodies.

Dietary eritadenine-induced alteration of molecular species composition of phospholipids in rats.

The effect of dietary eritadenine, a hypocholesterolemic compound found in the mushroom Lentinus edodes, on the fatty acid and molecular species profiles of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver microsomes was investigated in relation to the hypocholesterolemic action of the compound in rats. Animals were fed the control or eritadenine-supplemented (50 mg/kg diet) diet for 14 d. Eritadenine supplementation significantly decreased the plasma concentrations of cholesterol and phospholipids, but not triglycerides. The PC/PE ratio of liver microsomes, but not plasma, was also markedly decreased by eritadenine. Eritadenine supplementation was found to increase the proportion of 18:2n-6 and, inversely, to decrease the proportion of 20:4n-6 and 22:5n-6 in plasma PC and liver microsomal PC and PE, indicating that eritadenine depressed the metabolism of linoleic acid. The effect of eritadenine on the profile of n-3 fatty acids was dissimilar in PC and PE. These changes in fatty acid composition were selectively reflected in the molecular species composition of both PC and PE; the extent of increase in 16:0-18:2 molecular species or decrease in 18:0-20:4 molecular species was apparently greater than that of other molecular species containing 18:2 or 20:4 in the sn-2 position. These results suggest that, in addition to the decrease in liver microsomal PC/PE ratio, the alteration of plasma PC molecular species composition might also participate in the hypocholesterolemic action of eritadenine.

Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation ( ca 10% of administered) for 10 μg microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2–0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1- 14C]oleoyl-CoA concentration is higher than 0.2–0.3 nmol/50 μl. The labeled acyl group is mainly inserted in position 2 of the glyceroposphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 μg proteins. It is evaluated that, in the presence of 10 μg proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Amino acid composition of dietary proteins affects plasma cholesterol concentration through alteration of hepatic phospholipid metabolism in rats fed a cholesterol-free diet

This study was designed to investigate the relationship between the amino acid composition of dietary proteins and the plasma cholesterol concentration and to examine whether the alteration of hepatic phospholipid metabolism participates in the effect of dietary proteins in rats fed a cholesterol-free diet. There was a significant positive correlation between the plasma total cholesterol concentration and the plasma concentration of methionine and valine and the hepatic concentration of valine and alanine in rats fed seven types of proteins. In contrast, the plasma cholesterol concentration exhibited a significant negative correlation with the hepatic-free ethanolamine concentration. As far as methionine and ethanolamine, their positive and negative correlation with the plasma cholesterol concentration was consistent with their known hyper- and hypocholesterolemic effects, respectively. The phospholipid profile as represented by the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in liver microsomes, but not in other tissues tested, was largely influenced by the type of dietary proteins; the PC PE ratio exhibited a significant positive correlation with the plasma total cholesterol concentration. There was a significant correlation between the methionine content of dietary proteins and several biochemical variables including plasma cholesterol and liver microsomal PC PE ratio. A significantly lower concentration of hepatic S-adenosylmethionine was observed with soybean protein diet than with casein diet suggesting a decrease in the PC biosynthesis via the PE N-methylation pathway in rats fed soybean protein. From these results, it is suggested that the plasma cholesterol concentration might be influenced by the methionine content of dietary proteins at least in part through an alteration of hepatic phospholipid metabolism.

Choline resistance and phosphatidylcholine biosynthesis in tobacco cell suspensions or calli

Addition of choline to the culture medium of tobacco cells or calli of a wild cell line promoted an increase in the level of phosphatidylcholine (PC) as well as a rise in the phosphatidylcholine: phosphatidylethanolamine (PC/PE) molar ratio. Both of these events are inconsistent with cell or tissue survival. The in vitro activities of the enzymes involved in the synthesis of PC by the choline diphosphate (CDP) pathway, were examined in tobacco cells sensitive to (ChS line) or resistant to choline (ChR line), grown in suspension in a liquid medium or as calli on a solid medium. During the whole growth cycle, ChR cells or calli showed no changes in the enzymatic activities related to the CDP pathway, in contrast to ChS cells or calli which exhibited large changes. Alterations in the levels of these enzymatic activities mostly occurred at the onset of the subculture of ChS calli or cells transferred on an enriched choline medium. During the lag phase of ChS calli development, an abrupt drop in the in vitro activity of phosphocholine cytidylyltransferase (CT) occurs, which may be linked to a high level of the microsomal PC. Moreover during this phase an increase in the phosphotransferase activity (CPT) occurred in ChS calli, by contrast with a transient decrease of this activity found in suspension-cultured ChS cells. In choline-treated ChS cultures, an extended elevation of cholinekinase (CK) activity promoted a large entry of exogenous choline into ChS cells. By comparison with the behaviour of choline-sensitive cells, the choline-resistance might be correlated with low and stable levels of CK and CT activities associated with a high level of CPT activity both in tobacco cells or calli.

Hypocholesterolemic Action of Eritadenine Is Mediated by a Modification of Hepatic Phospholipid Metabolism in Rats

The hypocholesterolemic action of eritadenine, a compound found in the mushroom Lentinus edodes, was investigated in relation to its influence on phospholipid metabolism in the liver of rats fed diets containing different amounts of choline chloride (0, 2 and 8 g/kg diet). The time-dependent effect of eritadenine supplementation was also investigated. Eritadenine supplementation (50 mg/kg diet) significantly decreased the phosphatidylcholine (PC):phosphatidylethanolamine (PE) ratio in liver microsomes and the S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio in the liver, in addition to the plasma cholesterol concentration, irrespective of dietary choline levels. There was a significant correlation between the plasma cholesterol concentration and the liver microsomal PC:PE ratio. Although eritadenine caused fatty liver when added to the diets containing 0 or 2 g/kg choline chloride, a high level (8 g/kg) of choline chloride fully prevented the eritadenine-induced fatty liver without diminution of hypocholesterolemic action. Both the PC:PE ratio and the SAM:SAH ratio decreased significantly prior to the decrease in the plasma cholesterol concentration (1 d vs. 2 d after) in response to eritadenine supplementation, supporting the hypothesis that the alteration of hepatic phospholipid metabolism may be a cause of the hypocholesterolemic action of eritadenine. These observations suggest that the essential hypocholesterolemic action of eritadenine might be associated with a modification of hepatic phospholipid metabolism rather than with the PC deficiency, due to the inhibition of PE N-methylation.

Eritadenine-induced alteration of hepatic phospholipid metabolism in relation to its hypocholesterolemic action in rats

The hypocholesterolemic effect of dietary supplementation with eritadenine, a hypocholesterolemic factor present in the Lentinus edodes mushroom, was investigated in relation to its effect on hepatic phospholipid metabolism in rats. The plasma total cholesterol level was significantly decreased by eritadenine supplementation at levels above 8 μmol/kg of diet in a dose-dependent manner, accompanying decreases in both VLDL + LDL and HDL cholesterol levels. Eritadenine supplementation significantly increased the phosphatidylethanolamine (PE) content and inversely decreased the phosphatidylcholine (PC) content of liver microsomes in a dose-dependent manner. There was a highly significant correlation between plasma cholesterol levels and the content or proportion of PC and PE of liver microsomes. Eritadenine supplementation did not decrease the activity of PE N-methyltransferase in liver microsomes but rather increased the activity, possibly because of the increased PE content of liver microsomes. On the one hand, eritadenine had no direct inhibitory effect on the enzyme activity when added to the assay mixture. On the other hand, eritadenine supplementation increased the hepatic S-adenosylhomocysteine (SAH) level and decreased the ratio of S-adenosylmethionine (SAM) to SAH in a dose-dependent manner. The in vivo incorporation of radioactivity of [methyl- 3H]methionine into the PC of liver microsomes and blood plasma was also markedly depressed by dietary eritadenine supplementation at a level of 200 μmol/kg of diet. These results suggest that the hypocholesterolemic action of eritadenine might be elicited through an alteration of the hepatic phospholipid metabolism that resulted from an inhibition of PE N-methylation due to a decreased SAM SAH ratio in the liver.