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PubMed1989

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells.

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.

Animals↗
PubMed1982

Guinea-pig lymphotoxin-mediated cytotoxicity and activation of endogenous phospholipase A2.

The effect of cations on lysis of target cells (L . P3 cells) by guinea-pig lymphotoxin (GLT) was examined. Depletion of Ca2+ ions from the extracellular environment caused limited cytolysis, whereas addition of Ca2+ led to enhanced lysis. By applying the GLT pulse technique, it was shown that Ca2+ was not required for the initial step of GLT-mediated cytolysis, the step of binding of GLT molecules to target cell membranes. Among other cations tested, La3+ was found to interfere with the cytolytic action. Neither Mg2+ nor Mn2+ was effective. When target cells whose phospholipids were specifically radiolabelled at the C-2 position with [14C]-arachidonic acid were treated with GLT, radioactivity in the phospholipid fraction decreased. On the contrary, that in the free fatty acid, triglyceride and diglyceride fractions increased. This indicates the existence of endogenous phospholipase A2 that can be activated by GLT. Activation of the enzyme was partially inhibited by addition of EGTA with a concomitant decrease of cytolysis. When GLT was added to a crude membrane fraction of the radiolabelled L . P3 cells, liberation of free fatty acids from phospholipids was readily observed, but the increase of triglyceride-associated radioactivity was negligible. In this case, Ca2+ was found to be prerequisite for the GLT-induced activation of the enzyme. The results suggest that GLT-induced cytolysis includes a substantially Ca2+-dependent rate-limiting stage which comes after the binding of GLT to the target cells. Activation of membrane phospholipase A2 may be at least one of the biochemical changes that constitutes the Ca2+-dependent stage, although it remains to be clarified whether the activation of phospholipase A2 is directly responsible for target cell destruction or not.

Animals↗
PubMed2001

Carnitine affects fatty acid metabolism after cardioplegic arrest in neonatal rabbit hearts.

BACKGROUND: Fatty acid (FA) metabolism and the contribution of carnitine to metabolism after cardioplegic arrest still remain unclear, especially in the neonatal heart where beta-oxidation is not a predominant source of adenosine triphosphate. METHODS: FA metabolism and the effects of carnitine administration were evaluated using a newborn (7-day-old) rabbit blood-perfused Langendorff model subjected to cold cardioplegic arrest. The hearts were divided into five groups; (1) perfused with unmodified diluted blood (n = 9), (2) subjected to 180 minutes of cold cardioplegic arrest and reperfused with the blood (n = 9), (3) subjected to the same ischemia and reperfused with the blood containing 40 microM/L (n = 9), (4) 0.5 mM/L (n = 5), and (5) 5 mM/L of carnitine (n = 5). During reperfusion, FA metabolism was assessed by iodine-123-labeled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid, a fatty acid. The myocardial time-radioactivity curve was then determined and a mathematical compartment analysis of the external detection was used to elucidate FA metabolism in the cardiac myocyte. RESULTS: Cold cardioplegic arrest resulted in significantly impaired FA metabolism following reperfusion. Compartment analysis suggested that FA activation in the cytosol and beta-oxidation were impaired. Carnitine supplementation in groups 3 and 4 improved FA metabolism during reperfusion. In contrast, supplementation in group 5 had no beneficial effect on FA metabolism. CONCLUSIONS: These results suggest that FA metabolism is impaired after cold cardioplegic arrest and that carnitine supplementation may improve aerobic metabolism in neonates after open heart surgery.

Animals↗
PubMed1978

[Metabolism of (1-14C)-palmitic acid in the cat's brain].

Following injection into the cerebral ventricles of conscious cats, (1-14C) palmitic acid was rapidly taken up and incorporated into a variety of brain lipids. The peak of uptake of (1-14C) palmitic acid, about 50% of injected radioactive material, into the brain tissue was obtained within the first 24 h following its administration. Thereafter, the radioactivity slowly decreased reaching the least value by the end of the second week. The most heavily labelled lipids were the phospholipids, while the free fatty acids were appreciably labelled. Small percentage of the radioactive material was found in monoglycerides, diglycerides and triglycerides. The least incorporation was into cholesterol esters.

Animals↗
PubMed2004

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae).

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.

Animals↗
PubMed1992

Fatty acid kinetics in aerobic myocardium: characteristics of tracer carbon entry and washout and influence of metabolic demand.

These studies evaluated the kinetics of tracer uptake and washout after step-function labeling with 14C-palmitate. Washout and uptake function curve analysis for total radioactivity (TR) was derived according to the expressions: TR = Fx integral of 0 infinity C(t) x dt and TR = Fx integral of 0 infinity (Css - C(t)) x dt, respectively, with Vc = TR/Ca, where F = coronary flow; Css = steady-state concentration; C(t) = concentration with respect to time; Ca = arterial concentration; and Vc = distribution volumes within the fatty acid pathway. The only radioactive metabolites in venous effluent were fatty acids and 14CO2. The estimated Vc of fatty acids was small (1.2-1.7 ml/g dry wt or 0.4-0.5 mumol/g dry wt) and compatible with labeled substrate trapped in the blood volume. The Vc of 14CO2 was much larger (11.4-15.8 ml/g dry wt or 3.6-4.2 mumol/g dry wt) and correlated with counts contained in the aqueous soluble and fatty acid fractions in tissue. The counts in tissue were distributed between the aqueous soluble fraction (40%), which was rapidly depleted during washout, and a lipid fraction (60%) (triacylglycerols and phospholipids), which was resistant to washout. Distributions in tissue radioactivity between the aqueous soluble and lipid fractions support the notion of a dual pathway in fatty acid oxidation, one arm of which passes through the resident pool of triacylglycerols, which has a long time constant. The presence of this pool may impart an error in estimating fatty acid oxidation by external labeling techniques.

Aerobiosis↗
PubMed1981

Lipid composition and molecular species of sialic acids of a mouse cell line (JLS-V9) and its ouabain-resistant mutant.

Some properties of a mouse cell line (JLS-V9) and its ouabain-resistant mutant clone (JLS-V9OR) were compared. Specific activities of (Na+ + K+)-ATPase (EC 3.6.1.3) of the cell homogenate, particulate fraction and the plasma membrane fraction were 1.12, 1.72 and 8.75 mu mol/h per mg protein, respectively, in the parent cell and 0.97, 1.68 and 9.36 mu mol/h per mg protein in the mutant cell. The half-maximal concentration of ouabain for the inhibition of the (Na+ + K+)-ATPase was 3.4 . 10(-5) M in the parent cell and 4.0 . 10(-4) M in the resistant clone. The contents of phospholipid and cholesterol on a basis of protein in the mutant were 135 and 105% of those in the parent cell. The mutant's monohexosylceramide (HexCer), lactosylceramide (LacCer), trihexosylceramide (GbOse3Cer) and sialyllactosylceramide (GM3) were 111, 145, 274 and 114% of those of the parent cell. Sialic acids of GM3, analyzed by GC-MS, were 98% N-acetylneuraminic and 2% N-glycolylneuraminic acids in the parent cell, whilst 69% N-acetylneuraminic and 31% N-glycolylneuraminic acids in the mutant clone. The in vivo syntheses of these glycolipids were confirmed by the incorporation of radioactive galactose. No significant difference in fatty acid composition was observed between the two cell types. Neutral glycolipids contained mainly 24:0, 24:1, 22:0 and 16:0, whilst in GM3 18:0 and 18:1 predominated. These results show that the lipid content per mg protein is elevated in the ouabain-resistant cell compared to the parent cell.

Animals↗
PubMed1999

Endogenous surfactant metabolism in critically ill infants measured with stable isotope labeled fatty acids.

Little is known about endogenous surfactant metabolism in infants, because radioactive isotopes used for this purpose in animals cannot be used in humans. We developed a novel and safe method to measure the endogenous surfactant kinetics in vivo in humans by using stable isotope labeled fatty acids. We infused albumin-bound [U-13C]palmitic acid (PA) and [U-13C]linoleic acid (LLA) for 24 h in eight critically ill infants (mean+/-SD: weight: 3.7+/-1.3 kg: age: 51.3+/-61.6 d) who required mechanical ventilation. The 13C enrichment of PA and LLA in surfactant phosphatidylcholine (PC), obtained from tracheal aspirates, was measured by gas chromatography combustion interface-isotope ratio mass spectrometry. We measured a significant incorporation of both 13C-PA and 13C-LLA into surfactant PC. PC-PA and PC-LLA became enriched after 8.7+/-4.9 h (range: 3.4-17.3) and 10.0+/-7.2 h (range: 3.0-22.4), respectively; the times at maximum enrichment were 49.2+/-8.9 and 45.6+/-19.3 h, respectively. The fractional synthesis rate of surfactant PC-PA ranged from 0.4 to 3.4% per h, whereas the fractional synthesis rate of PC-LLA ranged from 0.5 to 3.8% per h. The surfactant PC-PA and PC-LLA half-lives ranged from 16.8 to 177.7 and 23.8 to 144.4 h, respectively. This method provides new data on surfactant metabolism in infants requiring mechanical ventilation. We found that synthesis of surfactant from plasma PA and LLA is a slow process and that there were marked differences in PC kinetics among infants. This variability could be related to differences in lung disease and could affect the clinical course of the respiratory failure.

Carbon Isotopes↗
PubMed1981

Effect of essential polyunsaturated fatty acid modifications on prostaglandin production by MDCK canine kidney cells.

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE2 release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9- times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglyceride fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE2 release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE2 accounting for 55 to 68% of the total output from [1-14C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE2 formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. It is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids become highly enriched in arachidonate.

Animals↗
PubMed1975

Studies on the fatty acid binding proteins in cytosol of rat liver.

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.

Animals↗
PubMed2002

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway.

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.

Biological Transport↗
PubMed1988

Utilization of mevalonate by aorta for the synthesis of medium-chain n-fatty acids (C8, C10) and acylglycerols.

[2-14C]Mevalonate was incorporated into acylglycerols by bovine aorta. The majority of the radioactivity was found in diacylglycerols. Hydrolysis, followed by analysis by thin-layer chromatography, paper chromatography, gas chromatography, and gas chromatography-mass spectrometry showed that the radioactivity was greatest in the medium-chain n-fatty acids, caprylic (C8) and capric (C10) acids. Triacylglycerols and diacylglycerols have important functions in the maintenance of the metabolic integrity of organs and their constituent cells. Therefore, medium-chain fatty acids may play significant roles, such as solubilization and transport of cholesterol and regulation of metabolism, as constituents of diacylglycerols. The results suggest that the trans-methylglutaconate shunt of mevalonate metabolism functions in the aorta.

Animals↗
PubMed1993

Transport of 22:6n-3 in the plasma and uptake into retinal pigment epithelium and retina.

Vertebrate retina is highly enriched in 22-carbon polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA, 22:6n-3), which is supplied by the plasma. To study the transport and uptake of 22:6n-3 into the retina, young Long Evans rats were fasted overnight and given an oral dose of palm oil containing [4,5-3H] 22:6n-3 and [9,10-3H] 18:1n-9 (oleic acid). At 1, 6, 12, 24, and 48 hr postgavage, blood, liver, retinal pigment epithelium (RPE) and retina were collected, and plasma lipoprotein fractions were obtained by density-gradient ultracentrifugation. The distribution of radioactivity among the different lipoproteins (dpm%) showed that both fatty acids were mainly associated with VLDL 1 hr after gavage. For 22:6n-3, the majority of radioactivity was associated with LDL at 6 hr postgavage. By 12 hr post administration, the majority of radioactivity was in HDL2 which retained around 40% until the 48-hr time point. For 18:1n-9, a different profile was observed. By 6 hr postgavage, approximately 30% of the radioactivity was found in HDL2, but the percentage of radioactivity decreased to approximately 20% thereafter. The half-life of 22:6n-3 was significantly longer than for 18:1n-9 (P < 0.0005) in plasma, liver, and the lipoprotein fractions. The specific activities of 22:6n-3 and 18:1n-9 in the plasma continuously declined from 1 hr to 48 hr postgavage, while liver showed a peak of specific activity at 6 hr, then gradually declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Activation of lipid metabolism of nuclei and chromatin in thymocytes of rats subjected to chronic low dose intensity gamma-irradiation.

Contents and radioactivity (as incorporation of [2-14C]acetate) of thymocyte nuclear and chromatin lipids were investigated in rats subjected to chronic gamma-irradiation in a dose 10 Gy at the intensity of 12.9 cGy/day. The incorporation of label into total fraction of lipids of nuclei and chromatin and into total phospholipid fraction of thymocyte nuclei increased. However, in the whole cells differences in radioactivity of these fractions were not found. The amount of fatty acids in thymocytes from irradiated rats was decreased but synthesis of fatty acids was activated. The amount of fatty acids in nuclei and chromatin remained unchanged compared with control, whereas radioactivity was significantly higher than in the whole cells. In irradiated thymocytes the amounts of phosphatidylserine in nuclei and phosphatidylserine and phosphatidylinositol in chromatin were increased. The incorporation of [2-14C]acetate into proteins increased in thymocytes of irradiated rats. The role of lipid metabolism in genome functioning is discussed.

Acetates↗
PubMed1996

The biosynthesis of omega 3 fatty acids from 18:2 omega 6 in Artemia spp.

To examine the biosynthesis of omega 3 fatty acids from 18:2 omega 6, reference Artemia (RAC III, Artemia Reference Center, Gent, Belgium) were fed rice bran supplemented with [1(-14)C]18:2 omega 6 for 2 days following 48 hours starvation since cyst hydration and 1, 2, or 3 days following 72 hours of starvation, under axenic conditions. Artemia fatty acids were analyzed by gas chromatography, AgNO3 thin layer chromatography and high performance liquid chromatography and fatty acid fractions were collected for radioisotope counts. No significant differences were observed in the omega 3 fatty acid content of Artemia cultured under axenic and xenic conditions. Radioisotope studies showed that radioactivity from [1(-14)C]18:2 omega 6 was incorporated into other fatty acids, including 18:3 omega 3, 18:4 omega 3 and 20:5 omega 3. The conversion rate was less than 5% for the two day feeding period. In the three day feeding experiment, the amount of radioactivity recovered in both 18:3 omega 3 and 20:5 omega 3 increased by 2.2- and 1.8-fold, respectively, over the three day feeding period. These results demonstrated that Artemia synthesized these omega 3 polyunsaturated fatty acids from 18:2 omega 6.

Animals↗
PubMed1989

Studies on the incorporation and transacylation of various fatty acids in choline and ethanolamine-containing phosphoacylglycerol subclasses in human neutrophils.

The incorporation of eight 14C-labeled fatty acids into human neutrophil phospholipids was investigated and the results were expressed as percent of the total phospholipid associated 14C-labeled substrate incorporated after an initial 15 min labeling and a subsequent 2 h reincubation in fatty acid-free buffer. In all cases, the PC fraction accounted for more than 40% of the total phospholipid radioactivity. The inositol-containing phosphoacylglycerols were also labeled well by all the fatty acids except 22:6(n - 3) and 16:0; however, a greater percentage of [14C]22:6(n - 3) was found in PE than that of any other labeled fatty acid substrate. In all cases, most of the radioactivity in PC after 15 min was in the diacyl subclass. After 2 h, there was a shift of [14C]20:4(n - 6), [14C]20:5(n - 3), [14C]22:6(n - 3) and [14C]18:4(n - 4) into the ether-linked subclass. No such shift was observed for [14C]16:0 or [14C]18:2(n - 6) and, although there was an increase in the percent radioactive 20:3(n - 6) and 20:3(n - 9) in ether-linked PC after 2 h, the total radioactivity in this fraction remained low by comparison. A similar shift in label occurred in the plasmalogenic-linked PE subspecies in cells labeled with [14C]20:4(n - 6), [14C]20:5(n - 3) and [14C]22:6(n - 3).

Acylation↗
PubMed1993

Effect of salt-loading and spontaneous hypertension on in vitro metabolism of [1-14C]linoleic and [2-14C]dihomo-gamma-linolenic acids.

The present study compared the effect of spontaneous hypertension and salt-loading on in vitro metabolism of 18:2n-6 (linoleic acid) and 20:3n-6 (dihomo-gamma-linolenic acid). Ten weanling spontaneously hypertensive rats (SHR) and 10 normotensive Wistar-Kyoto rats (WKY) maintained on a rodent lab chow were given tap water with (n = 5) or without (n = 5) addition of 1% NaCl for 4 weeks. Thereafter, animals were killed and liver microsomes were prepared. Aliquots of microsomes suspended in the phosphate-sucrose buffer containing MgCl2, ATP, CoA, and NADPH were incubated with 0.3 microCi of [1-14C]-18:2n-6 or [2-14C]-20:3n-6 at 37 degrees C for 15 min. The activity of delta 6- and delta 5-desaturases, and the distribution of radioactivity in different lipid fractions and in phospholipid fatty acids were determined. Results showed that both spontaneous hypertension and salt-loading suppressed the desaturation of radiolabeled 18:2n-6 and of 20:3n-6. Incubation of microsomes with [1-14C]-18:2n-6 resulted in 29% of radioactivity being associated with phospholipid fraction, of which 3% was associated with 18:3n-6. Incubation with radiolabeled 20:3n-6 resulted in 30% of the radioactivity being incorporated into phospholipids, of which 50% was associated with 20:4n-6 (arachidonic acid). Salt-loading suppressed the incorporation of radiolabeled fatty acids into phospholipids, more so in SHR than in WKY. Thus, salt-loading not only suppressed the desaturation of 18:2n-6 and 20:3n-6, but also interfered with the acylation of n-6 fatty acids into the phospholipid fraction.

Animals↗