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Activation of peroxisome proliferator-activated receptor alpha in human endothelial cells increases plasminogen activator inhibitor type-1 expression.

OBJECTIVE: To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor 1 (PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVEC were exposed to fatty acids and prostaglandin J(2) in varying concentrations with fresh media. RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs. Transient co-transfection of PAI-1 promoter and PPARalpha gene or PPARgamma gene to ECV304 was performed. RESULTS: PPARalpha, PPARdelta and PPARgamma mRNA in HUVECs were detected by RT-PCR. Treatment of HUVECs with PPARalpha and PPARgamma activators-linolenic acid, linoleic acid, oleic acid and prostaglandin J(2), but not with stearic acid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner. Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARalpha DNA in HUVECs through a transient gene transfection assay, although the mRNA expression of the 3 subtypes of PPAR with their activators were not changed compared with controls. CONCLUSIONS: HUVECs express PPARs. PPARs activators may increase PAI-1 expression in endothelial cells (EC). Although PPARs expression was not enhanced after being stimulated by their activators in EC, the functionally active PPARalpha is probably involved in regulating PAI-1 expression in EC.

Cells, Cultured↗

Evidence for the possible involvement of protein kinase C in the activation of non-specific phospholipase A2 in human neutrophils.

In this article, we provide mass data on released fatty acids in neutrophils stimulated with A23187 in the presence and absence of phorbol 12-myristate 13-acetate (PMA). A23187 alone caused a highly selective accumulation of non-esterified arachidonic acid (AA) relative to other saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids). However, the absolute levels of non-esterified arachidonic acid, oleic acid and linoleic acid were significantly increased (by twofold) in response to the action of A23187 in the presence of PMA. We also noted a further twofold increase in the non-esterified arachidonic acid in the presence of BW755C, a dual inhibitor of cyclooxygenase and lipoxygenases, with no apparent change in the levels of oleic and linoleic acids and other saturated fatty acids. These results suggest that PMA potentiates not only the release of AA but also the release of oleic and linoleic acids from neutrophil phosphoglycerides. This is possibly due to the activation of non-specific phospholipase A2 (PLA2). Our data also suggest a possible role for the 5-lipoxygenase metabolites in PMA-induced synergism with regard to the release of AA through AA-specific and PMA-sensitive PLA2. Protein kinase C appears, therefore, to play a role in the regulation of both AA-specific and non-specific PLA2 in human neutrophils.

Arachidonic Acid↗

[Isolation and identification of two new pyrone compounds from the culture of Herictum erinaceus].

Four crystalline and one liquid compounds were isolated from the solid cultured extract of Hericium erinaceus, Two of them are new, named herein III and IV. Their chemical structures were determined as 6-methyl-2,5-dihydroxymethyl-gamma-pyranone (III) and 2-hydroxymethyl-5-alpha-hydroxy-ethyl-gamma-pyranone (IV). The others are 4-chloro-3,5-dimethoxybenzoic-O-arabitol ester, 4-chloro-3,5-dimethoxybenzoic methyl ester and 4-chloro-3,5-dimethoxybenzoic acid. In addition, a mixture of palmic and stearic acid, a mixture of behenic acid and tetracosanic acid and a mixture of 5 alpha-ergostan-3-one, 5 alpha-stigmasten-22-en-3-one and 5 alpha-stigmastan-3-one were also isolated.

Basidiomycota↗

Biosynthesis of 18(RD)-hydroxyeicosatetraenoic acid from arachidonic acid by microsomes of monkey seminal vesicles. Some properties of a novel fatty acid omega 3-hydroxylase and omega 3-epoxygenase.

Microsomes of seminal vesicles of the cynomolgus monkey were incubated with [14C]5,8,11,14-eicosatetraenoic (arachidonic) acid and NADPH for 40 min at 37 degrees C and the products were characterized. Prostaglandins F2 alpha and E2 were the two main metabolites (approximately 52% of radioactivity), while 18(R)-hydroxy-cis-5,8,11,14-eicosatetraenoic acid (18(R)-HETE) was identified as the main, less polar product (approximately 13%). Significant biosynthesis of the 19-hydroxy or 20-hydroxy metabolites of arachidonic acid could not be detected. The formation of 18(R)-HETE was further investigated in the presence of a prostaglandin synthesis inhibitor, diclofenac sodium. The omega 3-hydroxylation was only partly supported by substituting NADH for NADPH. The hydroxyl oxygen of 18(R)-HETE was derived from the atmosphere and the omega 3-hydroxylation was inhibited by proadifen and partly inhibited by carbon monoxide. These findings suggest that 18(R)-HETE is formed by a cytochrome P-450 (P-450 omega 3). Linoleic acid and 8,11,14-eicosatetraenoic acid were also substrates of the enzyme, but stearic acid was not metabolized. 5,8,11,14,17-Eicosatetraenoic acid was oxygenated under these conditions mainly to 17,18-dihydroxy-5,8,11,14-eicosatetraenoic acid, presumably formed from 17(18)-epoxy-5,8,11,14-eicosatetraenoic acid by hydrolysis. The seminal microsomes thus seem to possess both omega 3-hydroxylase and omega 3-epoxygenase activity. These seminal vesicles also contain prostaglandin E 19-hydroxylase (Oliw, E.H., Kinn, A.-C., and Kvist, U. (1988) J. Biol. Chem. 263, 7222-7227). The presence of arachidonate omega 3-hydroxylase and prostaglandin E 19-hydroxylase was assessed in microsomes of adult and juvenile monkey livers. Arachidonic acid was metabolized extensively to diols (via epoxides), but 18-HETE could not be detected. In contrast, prostaglandin E1 was slowly hydroxylated mainly to 19-hydroxyprostaglandin E1 by both adult male and female juvenile hepatic microsomes. The results indicate that P-450 omega 3 of seminal vesicles might be a tissue-specific enzyme.

Animals↗

Biologically active ether lipids: incorporation of long-chain precursors into 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N-trimethyl)homoserines and other lipids of Chlorella fusca.

The lipids of Chlorella fusca are composed of the ester lipids typical of photosynthetically active cells. In addition, there occurs a class of less common ether lipids, the biologically active 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N- trimethyl)homoserines, at a level of about 1.3% of total lipids. The acyl moieties of the total lipids include saturated as well as mono-, di- and tri-unsaturated species with chain lengths of 16 and 18 carbon atoms, the major constituents being palmitic and oleic acids. In both the diacylglycerophosphocholines, i.e., the major class of ester phospholipids, and the diacylglycero-4'-O-(N,N,N-trimethyl)homoserines palmitic acid is located predominantly at position 1 of the glycerol backbone, whereas oleic acid is almost equally distributed between positions 1 and 2; palmitoleic and polyunsaturated fatty acids are esterified preferentially at position 2. Incubation of C. fusca cultures with 14C-labeled fatty acids leads to their rapid incorporation into various lipid classes. Oleic and palmitic acids are incorporated at a faster rate than stearic acid (18:1 greater than 16:0 much greater than 18:0). 1,2- and 1,3-Diacylglycerols are the most prominent intermediates of early metabolism of the exogenous fatty acids. In the course of time, a steady decrease of radioactive 1,2-diacylglycerols is observed that is accompanied by an increase in labeled triacylglycerols, diacylglycerophosphocholines, and diacylglycero-O-(N,N,N-trimethyl)homoserines. The stereospecific distribution of acyl moieties in the diacylglycero-O-(N,N,N-trimethyl)homoserines in C. fusca indicates that these ether lipids are derived from 1,2-diacylglycerol intermediates. This notion is supported by the finding that during incubation with radioactively labeled fatty acids the formation of diacylglycero-O-(N,N,N-trimethyl)homoserines parallels the biosynthesis of both diacylglycerophosphocholines and diacylglycerophosphoethanolamines, two classes of phospholipids which are known to be derived from 1,2-diacylglycerols. The mechanism of the formation of the ether bond, however, is as yet unknown. Incubation of C. fusca cultures with 14C-labeled fatty acids or alcohols leads to the formation of fair proportions of wax esters that are labeled in both the acyl and the alkyl moieties, indicating that in these algae fatty acids and alcohols are interconverted. 14C-Labeled long-chain alcohols are not incorporated into the alkyl moieties of ether lipids, whereas labeled 1-O-alkylglycerols are used, though to a very small extent, as precursors of ether phospholipids.

Chlorella↗

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations.

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.

Antioxidants↗

Eicosapentaenoic acid inhibits cholesterol esterification in cultured parenchymal cells and isolated microsomes from rat liver.

The effects of eicosapentaenoic acid on synthesis and secretion of cholesterol and cholesterol ester by cultured rat hepatocytes were studied. In the presence of eicosapentaenoic acid cellular cholesterol esterification was decreased by 50-75% compared to oleic acid as measured by radioactive precursors and mass. Secretion of cholesterol ester was reduced by 50-60% in the presence of eicosapentaenoic acid as evaluated by radiolabeled fatty acids, mevalonolactone, and mass measurement. Oleic, palmitic, and stearic acid increased, whereas eicosapentaenoic and docosahexaenoic acid decreased synthesis and secretion of cholesterol ester as compared to a fatty acid-free control. Cellular and secreted free cholesterol were unaffected by eicosapentaenoic acid in comparison with oleic acid. The reduced cholesterol esterification was observed within 1 h and lasted for at least 20 h. Eicosapentaenoic acid caused lower cholesterol esterification than oleic acid in the concentration range 0.2-1.0 mM fatty acid and reduced the stimulatory effect of oleic acid on cholesterol ester formation. Cholesterol esterification and release of cholesterol ester were markedly increased by 25-hydroxycholesterol in the presence of eicosapentaenoic acid as well as oleic acid. Experiments with liver microsomes revealed that radioactive eicosapentaenoic acid and eicosapentaenoyl-CoA were poorer substrates (7-30%) for cholesterol esterification than oleic acid and oleoyl-CoA. Reduced formation of cholesterol ester was also observed when eicosapentaenoyl-CoA was given together with labeled oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linolenoyl-CoA, and arachidonoyl-CoA had no inhibitory effect. In conclusion, eicosapentaenoic acid reduced cellular cholesterol esterification by inhibiting the activity of acyl-CoA:cholesterol acyltransferase. The lowered cholesterol esterification caused by eicosapentaenoic acid secondly decreased secretion of very low density lipoprotein cholesterol ester.

Animals↗

[Biohydrogenation of erucic acid (22:1 n-9 cis) in artificial rumen. I). Effect of octadecapolyenoic fatty acids and the incubation period].

Normally dietary octadecapolyenoic fatty acids are anaerobically hydrogenated in ruminants, both "in vivo" in the rumen and "in vitro" with ruminal content. Here it has been investigated in artificial rumen the process concerning the erucic acid (22:1 n-9 cis) compared with and in presence of C 18 polyunsaturated acids at various incubation times. The results have shown that C 18 polyunsaturated acids hydrogenation with conversion into hydrogenation intermediates and stearic acid does always occur in contrast with erucic acid where it is never revealable, unrelated to the incubations conditions applied.

Animals↗

[Group and fatty acid composition of the lipids in yeasts of the genus Candida].

The group and fatty acid composition of lipids from 62 species and strains of Candida yeast was investigated. No strict correlation between the group composition of lipids and the yeast species was found but certain consistencies were seen. For instance, C. humicola yeast showed a higher ability for biomass and lipid accumulation. In their group composition triglycerides were predominant while polar lipids + monoglycerides and sterol esters + hydrocarbons were in small quantities as compared to other yeast species. Besides, C. humicola yeast contained no pigments. The C. guilliermondii strains contained low amounts of free fatty acids. The lipid fraction of all C. tropicalis yeast studied contained pigments. The yeast with increased amounts of triglycerides showed a comparatively high content of biomass and lipids. C. guilliermondii yeast had a high content of oleic acid and a low content of linoleic acid. All the yeast contained small quantities of stearic acid.

Candida↗

Normal cations and abnormal membrane lipids in the red blood cells of dogs with familial stomatocytosis-hypertrophic gastritis.

Examination of the red blood cells (RBCs) of eight dogs with familial stomatocytosis-hypertrophic gastritis (FS-HG), a multiorgan disease associated with hemolytic anemia, hereditary stomatocytosis (HSt), and hypertrophic gastritis resembling Ménétrier's disease in man, showed abnormal osmotic fragility, normal mean corpuscular volume, slightly increased cell water, and normal cation content and cation fluxes. Cholesterol was decreased in RBC and increased in plasma. In both RBCs and plasma, total phospholipid (PL) was normal, phosphatidylcholine (PC) decreased, and sphingomyelin increased. The palmitic acid content of PC was increased, and the stearic acid content of PC was decreased. Sodium dodecyl sulfate electrophoresis of RBC membrane proteins was normal. These findings have not been described previously in HSt. They suggest that in FS-HG, abnormal composition of the PL in RBCs secondary to abnormal PL in plasma causes defective membrane function and stomatocytic shape-change. This conclusion was supported by a shortened half-life of 51Cr-labeled RBCs from normal dogs after transfusion in dogs with FS-HG. It was concluded (1) that not all hereditary forms of stomatocytosis are necessarily associated with an intrinsic structural defect of the RBC membrane, but that the change in shape of RBC may also be induced by abnormal composition of the plasma; (2) that stomatocytosis may be caused by loss of membrane surface area rather than by the increased cation uptake such as has been shown in some human kindreds with HSt, (3) that FS-HG is a disorder of lipid metabolism, and by consequence, (4) that abnormal lipid metabolism might be involved in the pathogenesis of Ménétrier's disease.

Anemia, Hemolytic↗

Dibutyrate derivatization of monoacylglycerols for the resolution of regioisomers of oleic, petroselinic, and cis-vaccenic acids.

Dibutyrate derivatives of monoacylglycerols of oleic, petroselinic, and cis-vaccenic acids were prepared by diesterification of monoacylglycerols with n-butyryl chloride. The resulting triacylglycerols were analyzed by gas chromatography (GC) with a 65% phenyl methyl silicone capillary column and separated on the basis of both fatty acid composition and regiospecific position. The petroselinic acid derivatives eluted first, followed sequentially by the oleic and cis-vaccenic acid derivatives, with the sn-2 positional isomer eluting before the sn-1 (3) isomer in each case. Separation of the peaks was almost baseline between petroselinic and oleic acids as well as between oleic and cis-vaccenic acids. To assess the accuracy of the method, mixtures of triolein, tripetroselinin, and tri-cis-vaccenin in various known proportions were partially deacylated with the use of ethyl magnesium bromide and derivatized and analyzed as above. The results showed that this method compares favorably to the existing methods for analysis of oleic, petroselinic, and cis-vaccenic fatty acids by GC with respect to peak separation and accuracy, and it also provides information on the regiospecific distribution of the fatty acids. The method was applied to basil (Ocimum basilicum) and coriander (Coriandrum sativum) seed oils. cis-Vaccenic, oleic, and linoleic acids were mainly distributed at the sn-2 position in basil seed oil, and higher proportions of linolenic, palmitic, and stearic acids were distributed at the sn-1(3) position than at the sn-2 position. In coriander seed oil, petroselinic acid was mainly distributed at the sn-1 (3) position, and both oleic and linoleic acids were mostly located at the sn-2 position, whereas palmitic, stearic, and cis-vaccenic acids were located only at the sn-1 (3) position.

Chromatography, Gas↗

Apoptotic effects of dichloro stearic and dichloro myristic acid in human hepatoma cells (HepG2).

Chlorinated fatty acids represent the major fraction of extractable organically bound chlorine in fish. After dietary intake such fatty acids may accumulate in adipose tissue, and even be transferred from mother to child via breast milk. We have previously reported that 9,10-dichloro stearic acid and 5,6-dichloro myristic acid inhibited cell growth. The aim of the present work was to investigate whether the growth inhibitory effect of these modified fatty acids might involve apoptosis. Human hepatoma cells (HepG2) were cultured for 4 days before addition of chlorinated fatty acids, and then cultured for another day before harvested. Morphological analysis was mainly done by light microscopy. In addition, fluorescence microscopy and electrophoretic analysis of DNA were carried out. The effect of 0.3 and 0.6 mmol/l was studied. Both chlorinated fatty acids seemed to cause a concentration-dependent increase in the relative abundance of pycnotic and broken nuclei, as well as nuclear fragments, with the strongest effect of dichloro stearic acid. Apoptosis by the chlorinated fatty acids was however less than that of docosahexaenoic acid, a known apoptosis effector. In conclusion, chlorinated fatty acids seem to possess the ability to cause apoptosis.

Apoptosis↗

Erythrocyte membrane fatty acid profile and saturation index in gallbladder carcinogenesis: a case-control study.

BACKGROUND: Gallbladder cancer is a common neoplasm of biliary tract, with an unknown etiology. PATIENTS AND METHODS: This study was carried out to evaluate the changes in the membrane fatty acid profile and saturation index in patients with gallbladder cancer. The study sample consisted of 50 newly diagnosed cases of gallbladder cancer and 50 patients undergoing cholecystectomy for cholelithiasis were recruited as controls. The fatty acid estimation was carried out by high performance liquid chromatography (HPLC). Statistical analysis was carried out by student 't' test and one-way ANOVA. Pearson's correlation coefficient was also obtained. RESULTS: A significant lowering of erythrocyte membrane stearic acid (p = 0.000), arachidonic acid (p = 0.001), and saturation index (p = 0.001) was observed in patients with gallbladder cancer compared to controls. A significant inverse relation was observed between stearic and oleic acid (r = -0.269 p = 0.007). CONCLUSION: Results of the present study suggest an increase in the delta 9 desaturation in cancer patients compared to controls however, a possible role of biliary stasis due to altered gallbladder motility or derangements of signal transduction secondary to altered membrane lipid bilayer cannot be ruled out.

Journal Article↗

Role of fatty acids in growth-promoting effect of serum albumin on hamster cells in vitro.

Dialyzed serum albumin had considerable growth-promoting effect on cultivated hamster cells. This effect was virtually lost on removal of the fatty acids, and it was completely restored by recombination of the fatty acid-free albumin with the isolated and purified fatty acids. The role of albumin itself appeared to be largely that of a carrier of fatty acids, protecting the cells against toxic effects of fatty acids in free solution. This conclusion was based on two observations: Fatty acids in the absence of albumin were growth-inhibitory except in extremely dilute solutions, and beta-lactoglobulin, a protein possessing, like albumin, the ability to bind and release fatty acids, could replace albumin in the presence of fatty acids with similar growth-promoting effect. Examination of individual molecular types of fatty acids showed that all unsaturated acids tested were growth-promoting, whereas the saturated acids were growth-inhibiting, with the exception of stearic acid in low concentrations. Although the possibility of a mitotic triggering effect was not excluded, the fatty acids presumably stimulated growth by providing substrate for cellular metabolism, since there was a direct relationship between the degree of growth stimulation and the duration of exposure of cells to the fatty acids.

Cell Division↗

[14C]ceramide synthesis by sphingolipid ceramide N-deacylase: new assay for ceramidase activity detection.

Sphingolipid ceramide N-deacylase (SCDase) is an enzyme which hydrolyzes the N-acyl linkage between fatty acid and sphingosine in ceramide of various glycosphingolipids and sphingomyelin. Recently the enzyme was found to catalyze the hydrolysis and its reverse reaction under different conditions. We report here an innovative method for synthesis of radioisotope-labeled ceramide with high specific activity using the reverse hydrolysis reaction of SCDase. More than 80% of free fatty acid was transferred to sphingosine when 1 nmol [14C]stearic acid and 2 nmol sphingosine were incubated with 5 microU SCDase at 37 degrees C for 20 h in 10 microliters of 25 mM phosphate buffer, pH 7.0, containing 0.3% Triton X-100. Free [14C]fatty acid and sphingosine were easily separated from synthesized [14C]ceramide by using a Sep-Pak Plus Silica and a Sep-Pak CM cartridge, respectively. We also developed a sensitive assay method for ceramidase using the [14C]ceramide prepared. The method consists of separation of the [14C]fatty acid released from [14C]ceramide by the action of enzyme on thin-layer chromatography followed by analysis and quantification with an imaging analyzer (BAS1000). This method was sensitive and qualitative and enabled the detection of ceramidase activity in invertebrates for the first time as well as in several human cancer cell lines.

Amidohydrolases↗

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus.

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A2 activation but not palmitic or stearic acids. Of greater interest L-glutamate, N-methyl-D-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of arachidonic acid in amounts over basal levels of 200-300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.

Animals↗

Effect of early postnatal dietary sterculate on the fatty acid composition of rat liver and brain lipids.

Pregnant rats were fed a high carbohydrate diet containing either 1% trilinolein or 1% trilinolein with 0.2% methyl sterculate from 18 day gestation to 21 day postpartum. The pups were weaned at 21 days and continued on the same diet for an additional 10 days. The microsomal stearyl CoA desaturase activities of the liver were effectively inhibited. Liver triglycerides showed increases in the saturated fatty acids concentrations at the expense of the corresponding monoenes. The concentration of cis 6-7 octadecenoic acid was elevated. In liver phospholipids, the concentration of stearic acid was increased without a corresponding decrease in the oleic acid content. A drastic decrease in the nervonic acid (24:1, n-9) concentration of liver sphingomyelin was observed. The lipids of the brain did not contain sterculic acid, and brain desaturase activity was unaffected. There was no significant change in the concentration of monoenoic acids from 16:1 to 22:1. However, nervonic acid was decreased by 32%. These results suggest that brain nervonic acid may be derived from a precursor other than oleic acid.

Animals↗