Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Effect of even- and odd-numbered saturated fatty acids on glycerolipid synthesis in rat liver slices

Effect of even- and odd-numbered saturated fatty acids, ranging from lauric to stearic acids, was studied on the de novo synthesis of glycerolipids in rat liver slices. For all fatty acids tested, a marked synthesis of saturated glycerolipids was observed except for phosphatidylethanolamine. When compared at the fixed concentration (2 mM), myristic acid caused a peak synthesis of saturated glycerolipids, and the presence of longer or shorter even- and odd-numbered fatty acids resulted in their lesser formation. The formation of saturated species of triacylglycerol and phosphatidylcholine closely followed the mode of synthesis of saturated diacylglycerols, though dipentadecanoyl-and dipalmitoylglycerols appeared to be less converted to the corresponding saturated triacylglycerols in comparison to the other saturated diacylglycerols. Very little formation of saturated diacylglycerols occurred when lauric, tridecanoic and stearic acids were tested. The majority of lauric and tridecanoic acids incorporated into saturated diacylglycerols was shown to be chain-elongated prior to esterification.     

Metabolism of n-3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells

The metabolism of the linolenic acid family (n-3) of fatty acids, e.g., linolenic, eicosapentaenoic, and docosahexaenoic acids, in cultured smooth muscle cells from rabbit aorta was compared to the metabolism of linoleic and arachidonic acids. There was a time-dependent uptake of these fatty acids into cells for 16 hr (arachidonic greater than docosahexaenoic, linoleic, eicosapentaenoic greater than linolenic), and the acids were incorporated mainly into phospholipids and triglycerides. Eicosapentaenoic and arachidonic acids were incorporated more into phosphatidylethanolamine and phosphatidylinositol plus phosphatidylserine and less into phosphatidylcholine than linolenic and linoleic acids. Docosahexaenoic acid was incorporated into phosphatidylethanolamine more than linolenic and linoleic acids and into phosphatidylinositol plus phosphatidylserine less than eicosapentaenoic and arachidonic acids. Added linolenic acid accumulated mainly in phosphatidylcholine and did not decrease the arachidonic acid content of any phospholipid subfraction. Elongation-desaturation metabolites of linoleic acid did not accumulate. Cells treated with eicosapentaenoic acid accumulated both eicosapentaenoic and docosapentaenoic acids mainly in phosphatidylethanolamine and the arachidonic acid content was decreased. Added docosahexaenoic acid accumulated mainly in phosphatidylethanolamine and decreased the content of both arachidonic and oleic acids. The following conclusions are drawn from these results. The three n-3 fatty acids are utilized differently in phospholipids. The arachidonic acid content of phospholipids is reduced by eicosapentaenoic and docosahexaenoic acids, but not by linolenic acid. Smooth muscle cells have little or no desaturase activity, but have significant elongation activity for polyunsaturated fatty acids.     

Alterations in Cerebral and Microvascular Prostaglandin Synthesis by Manipulation of Dietary Essential Fatty Acids

Sprague-Dawley rats were fed one of three purified diets--10% corn oil, 10% hydrogenated coconut oil, or 10% linseed oil--through two generations. At 60-80 days of age the animals were sacrificed. The fatty acyl composition of phosphatidylcholine, phosphatidylethanolamine, plasmalogen phosphatidylethanolamine, and combined phosphatidylinositol/phosphatidylserine from cerebral cortex and isolated cerebral microvessels was determined. Brain slice prostaglandin F2 alpha or microvascular prostacyclin synthesis was also measured. Major changes were noted in the fatty acid profiles, most dramatically in the phosphatidylethanolamine and ethanolamine plasmalogen fractions, with an active rise in docosahexaenoic acid resulting from linseed oil feeding. A depression in prostaglandin F2 alpha synthesis was seen in brain slices of hydrogenated coconut oil- and linseed oil-fed rats. Such a depression was also observed in microvascular prostaglandin synthesis at basal and stimulated levels but not in control incubations. The potential importance of these findings to cerebral microcirculation and hemostasis is discussed.     

Regulation of ethanolamine and choline phosphatide biosynthesis in isolated rat intestinal villus cells

The effects of ethanolamine, choline, and different fatty acids on phospholipid synthesis via the CDP-ester pathways were studied in isolated rat intestinal villus cells. The incorporation of [14C]glucose into phosphatidylethanolamine was stimulated severalfold by the addition of ethanolamine and long-chained unsaturated fatty acids, while the addition of lauric acid inhibited the incorporation of radioactivity into phosphatidylethanolamine. At concentrations of ethanolamine higher than 0.2 mM, phosphoethanolamine accumulated, but the concentrations of CDP-ethanolamine and the incorporation of radioactivity into phospatidylethanolamine did not increase further. The incorporation of [14C]glucose into phosphatidylcholine responded in a way similar to that of phosphatidylethanolamine, except that a 10-fold higher concentration of choline was required for maximal stimulation. CCC inhibited the incorporation of choline into phosphatidylcholine. In contrast with hepatocytes, villus cells did not form phosphatidylcholine via phospholipid N-methylation. The data indicate that, in intestinal villus cells, the cytidylyltransferase reactions are rate limiting in the synthesis of phosphatidylethanolamine and probably also of phosphatidylcholine. The availability of diacylglycerol and its fatty acid composition may also significantly affect the rate of phospholipid synthesis.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Effects of Fatty Acids,Lipase Activator,Phospholipids and Related Substances on the Lipase Production by Candida paralipolytica

Unsaturated long-chain fatty acids such as oleic, linoleic and ricinoleic acid except for elaidic acid were effective on the lipase production by Candida paralipolytica, but saturated fatty acids were not effective. When elaidic and myristic acids were dissolved in n-hexadecane to be dispersed in a liquid state, they became to be effective on the lipase production. These results suggest that long-chain fatty acids of a liquid state are effective on the lipase production. Lipase activator A and phosphatidylethanolamine stimulated the effect of fatty acids on the lipase production. Effects of sterols and surface-active substances on the lipase production were also reported.

A weak tributyrin-hydrolyzing activity, in the absence of fatty acids, was found in the yeast cell.     

Interaction of rat alpha-fetoprotein and albumin with polyunsaturated and other fatty acids: determination of apparent association constants

The interaction of fatty acids with rat alpha-fetoprotein and albumin was measured using a partition equilibrium method. alpha-Fetoprotein (AFP) displays one high-affinity binding site for fatty acids and albumin near two binding sites. The AFP association constants for most fatty acids were similar to those of albumin (in the 10(7) M-1 range) whereas for docosahexaenoic acid it was 9.7 x 10(8) M-1, about 50-fold higher than that corresponding to albumin. This difference justifies docosahexaenoic acid in fetal or neonatal serum being mainly bound to AFP and can indicate a highly specific role of AFP in the transport of this fatty acid.     

Influence of saturated long-chain N-acylethanolamines on lipid composition and heart contractility of isolated rat heart under ischemia-reperfusion

The heart contractility and changes of lipid composition of isolated rat heart (n = 26) under total ischemia and ischemia-reperfusion was studied. The effect of N-stearoyl-ethanolamine under these conditions was investigated. N-stearoyl-ethanolamine leads to remodelling of fatty acyl chain composition of myocardial phospholipids: to drastic fall of polyunsaturated fatty acyl chains (18:2w6, 20:3w6, 20:4w6, 22:5w3, 22:5w6, 22:6w3 and 22:6w6) and enhancement of 18:0. This can be caused by N-stearoyl-ethanolamine-induced suppression of polyunsaturated fatty acids synthesis. Naturally occurring minor lipids--N-acyl phosphatidylethanolamine and its derivative N-acylethanolamine were detected in isolated rat heart under ischemia-reperfusion. It is notable that approximately 12% of total N-acylethanolamines were composed by anandamide. Treatment of N-acyl phosphatidylethanolamine by phospholipase D with subsequent fatty acyl chain analysis demonstrates that fatty acid composition of both N-acyl chains of N-acyl phosphatidylethanolamine and free N-acylethanolamine are similar and their main fatty acyl chains are 16:0, 18:0 and 20:4w6. It was shown that exogenous N-stearoyl-ethanolamine did not alter the levels of endogenous N-acyl phosphatidylethanolamine and N-acylethanolamine, but caused the decrease of lyso-phosphatidylcholine and phosphatidylglycerol levels. The rate of heart contractility and heart relaxation was found to increase during the early period of reperfusion. N-stearoyl-ethanolamine prevents this alteration and exerts a negative inotropic effect. It is concluded that membrane protective properties of N-stearoyl-ethanolamine at least partly depend on its ability to inhibit decrease amount of arachidonic and docosahexaenoic acids, to modulate the fatty acyl chains of cardiac phospholipids and to decrease the level of lyso-phosphatidylcholine.     

Myristic acid stimulates endothelial nitric-oxide synthase in a CD36- and an AMP kinase-dependent manner

Dietary free fatty acids have been reported to have various effects on the endothelium including the generation of nitric oxide. The goal of the current study was to determine the mechanism whereby free fatty acid causes an increase in nitric oxide synthesis. The specific hypothesis tested was that free fatty acid association with CD36, a class B scavenger receptor, induces the activation of endothelial nitric-oxide synthase (eNOS). A human microvascular endothelial cell line and a transfected Chinese hamster ovary cell system were used to determine which free fatty acids stimulate eNOS. Surprisingly, only myristic acid, and to a lesser extent palmitic acid, stimulated eNOS. The stimulation of eNOS was dose- and time-dependent. Competition experiments with other free fatty acids and with a CD36-blocking antibody demonstrated that the effects of myristic acid on eNOS required association with CD36. Further mechanistic studies demonstrated that the effects of myristic acid on eNOS function were not dependent on PI 3-kinase, Akt kinase, or calcium. Pharmacological studies and dominant negative constructs were used to demonstrate that myristic acid/CD36 stimulation of eNOS activity was dependent on the activation of AMP kinase. These data demonstrate an unexpected link among myristic acid, CD36, AMP kinase, and eNOS activity.     

Myristic acid increases dense lipoprotein secretion by inhibiting apoB degradation and triglyceride recruitment

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B-100 (apoB-100) levels. To identify mechanisms at the level of production, rat hepatoma cells, McA-RH7777, were incubated with [(35)S]methionine and either fatty acid-BSA complexes or BSA alone. There were increases in labeled apoB-100 secretion with saturated fatty acids palmitic and myristic (MA) (153 +/- 20% and 165 +/- 11%, respectively, relative to BSA). Incubation with polyunsaturated docosahexaenoic acid (DHA) decreased secretion to 26 +/- 2.0%, while monounsaturated oleic acid (OA) did not change it. In pulse-chase studies, MA treatment resulted in reduced apoB-100 degradation, in agreement with its promotion of secretion. In triglyceride (TG) studies, synthesis was stimulated equally by OA, MA, and DHA, but TG secretion was relatively decreased with MA and DHA. With OA, the majority of newly secreted apoB100-lipoproteins was d < or = 1.006, but with MA, they were much denser (1.063 < d). Furthermore, the relative recruitment of newly synthesized TG to lipoproteins was impaired with MA. We conclude that mechanisms for effects of specific dietary fatty acids on plasma lipoprotein levels may include changes in hepatic production. In turn, hepatic production may be regulated by specific fatty acids at the steps of apoB-100 degradation and the recruitment of nascent TG to lipoprotein particles.     

Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. II. Effect on the rat brain fatty acid composition

The effect of N-acylethanolamines mixture (NAE) with saturated and unsaturated acyl chains on the fatty acid composition of the rat brain under chronic morphine dependence was investigated. Long-term administration of NAE reduced morphine-induced decrease of mono- and polyunsaturated fatty acids in the rat brain crude lipid extract. Furthermore, NAE restored the acyl chain spectrum, especially the content of docosahexaenoic acid in essential phospholipids--phosphatidylcholine and phosphatidylethanolamine. Pharmacological activity of NAE depended on a dose.     

Influence of dietary selenium and vitamin E on the levels of fatty acids in brain and liver tissues of lambs

In this study, the effects of dietary vitamin E, selenium, and their combination on the levels of fatty acid composition of the brain and liver tissues were examined. In brain tissue, the amounts of most fatty acids increased in vitamin E, combination and selenium groups compared with control group values. While the proportions of myristic, pentadecanoic, palmitic, linoleic, and total saturated fatty acids were decreased in vitamin E, Se and combination groups, eicosapentaenoic, total unsaturated and MUFA were increased in the same groups. In addition, the proportions arachidonic, eicosapentaenoic, total unsaturated, ω6 and MUFA in the combination group were higher than in the control group. In liver tissue, the amounts of myristic, pentadecanoic, palmitic, eicosedienoic, eicosapentaenoic, docosahexaenoic, ω3 and PUFA were higher in the combination group than in the control group. Also the proportions of eicosapentaenoic, docosahexaenoic acids in supplemented groups were higher than those in the control group. We conclude that dietary vitamin E and selenium have an influence on the levels of fatty acids in the brain and liver. Copyright © 1999 John Wiley & Sons, Ltd.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Influence of dietary source of docosahexaenoic and arachidonic acids on their incorporation into membrane phospholipids of red blood cells in term infants

Here we studied whether the chemical structure of dietary arachidonic and docosahexaenoic acids in full-term infant diets affects their incorporation into erythrocyte membrane phospholipids. From birth to 3 months, infants were fed breast milk (n = 9) or formula milk containing arachidonic acid and docosahexaenoic acid provided by egg phospholipids (n = 10) or by low-eicosapentaenoic acid fish oil and fungal triglycerides (n = 10). We compared the fatty acid composition of erythrocyte phosphatidylethanolamine, phosphatidylcholine and sphingomyelin before and after administration of the experimental diet. At 3 months, infants on formula milk showed lower concentrations of docosahexaenoic acid (in phosphatidylcholine and in phosphatidylethanolamine) and arachidonic acid (in phosphatidylcholine) than those receiving breast milk. We conclude the incorporation of the two fatty acids into erythrocyte phospholipids depends mainly on the lipid composition of the diet received rather than the chemical form in which they are delivered.     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

The phospholipids, particularly phosphatidylethanolamine, of brain gray matter are enriched with docosahexaenoic acid (22:6n-3). The importance of uptake of preformed 22:6n-3 from plasma compared with synthesis from the alpha-linolenic acid (18:3n-3) precursor in brain is not known. Deficiency of 18:3n-3 results in a compensatory increase in the n-6 docosapentaenoic acid (22:5n-6) in brain, which could be formed from the precursor linoleic acid (18:2n-6) in liver or brain. We studied n-3 and n-6 fatty acid incorporation in brain astrocytes cultured in chemically defined medium using delipidated serum supplemented with specific fatty acids. High performance liquid chromatography with evaporative light scattering detection and gas liquid chromatography were used to separate and quantify cell and media lipids and fatty acids. Although astrocytes are able to form 22:6n-3, incubation with 18:3n-3 or eicosapentaenoic acid (20:5n-3) resulted in a time and concentration dependent accumulation of 22:5n-3 and decrease in 22:6n-3 g/g cell fatty acids. Astrocytes cultured with 18:2n-6 failed to accumulate 22:5n-6. Astrocytes secreted cholesterol esters (CE) and phosphatidylethanolamine containing saturated and monounsaturated fatty acids, and arachidonic acid (20:4n-6) and 22:6n-3. These studies suggest conversion of 22:5n-3 limits 22:6n-3 synthesis, and show astrocytes release fatty acids in CE.     

Lipid composition of Zymomonas mobilis: effects of ethanol and glucose

Zymomonas mobilis is an alcohol-tolerant microorganism which is potentially useful for the commercial production of ethanol. This organism was found to contain cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine as major phospholipids. Vaccenic acid was the most abundant fatty acid, with lesser amounts of myristic, palmitic, and palmitoleic acids. No branched-chain or cyclopropane fatty acids were found. Previous studies in our laboratory have shown that ethanol induces the synthesis of phospholipids enriched in vaccenic acid in Escherichia coli (L. O. Ingram, J. Bacteriol. 125:670-678, 1976). The fatty acid composition of Z. mobilis, an obligately ethanol-producing microorganism, represents an extreme of the trend observed in E. coli. In Z. mobilis, vaccenic acid represents over 75% of the acyl chains in the polar membrane lipids. Glucose and ethanol had no major effect on the fatty acid composition of Z. mobilis. However, both glucose and ethanol caused a decrease in phosphatidylethanolamine and phosphatidylglycerol and an increase in cardiolipin and phosphatidylcholine. Ethanol also caused a dose-dependent reduction in the lipid-to-protein ratios of crude membranes. The lipid composition of Z. mobilis may represent an evolutionary adaptation for survival in the presence of ethanol.     

Ethynylestradiol alters lipid composition and phosphatidylethanolamine metabolism in red blood cells

Treatment of female rats with ethinylestradiol at a dose of 60 micrograms/rat, daily for 21 days, produced marked changes in red blood cell lipids. Cholesterol was decreased by 22% and total phospholipids were increased by 13%, resulting in a 31% decrease in the cholesterol to phospholipid ratio. The mass distribution of phosphatidylcholine and phosphatidylethanolamine relative to total phospholipids was unchanged. Whereas control red cells incorporated preferentially fatty acids in phosphatidylcholine, ethinylestradiol stimulated their incorporation specifically in phosphatidylethanolamine, where increases occurred with palmitic acid (+75%), oleic acid (+68%) and arachidonic acid (+31%). Incorporation in phosphatidylcholine was unaffected with any of the 3 fatty acids. The stimulation of fatty acid incorporation in phosphatidylethanolamine is likely to reflect an estrogen-dependent increase in turnover rate of fatty acids in this phospholipid. Such alterations in lipid composition and fatty acid incorporation in red cell phospholipids may have significant effects on membrane function.     

Effects of adrenoceptor blockade on cardiac hypertrophy and myocardial phospholipids.

Catecholamines have been proposed as a stimulus for the hypertrophic response to pressure overload of the heart and could also mediate the membrane lipid changes associated with cardiac hypertrophy. To address both of these possibilities, cardiac hypertrophy was induced by aortic constriction in the presence or absence of chronic alpha- or beta-adrenoceptor blockade. Heart weights and heart weight to body weight ratios in aortic-constricted rats of the adrenoceptor-blocked and vehicle-treated groups were elevated to the same extent when compared with values in sham-operated rats of each group. Analysis of the fatty acyl composition of the major phospholipid classes revealed that similar changes occurred in vehicle-treated, alpha-blocked, and beta-blocked aortic-constricted rats when compared with respective groups of sham-operated rats. Specifically, linoleic acid was reduced in the phosphatidylcholine, phosphatidylethanolamine (PE), and cardiolipin (CL) fractions in all groups of aortic-constricted rats. This reduction was accompanied by increased docosahexaenoic acid, arachidonic acid, or palmitic acid in phosphatidylcholine; docosahexaenoic acid in phosphatidylethanolamine; and oleic acid in cardiolipin fractions. Adrenoceptor blockade did not prevent or attenuate the major changes in the fatty acyl composition of phospholipids or the increase in heart weight associated with aortic constriction. This suggests that a change in the level of adrenoceptor stimulation is not the stimulus for cardiac hypertrophy or the observed alterations in phospholipid composition in the pressure-overloaded rat heart.