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PubMed1996

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits.

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.

Acylation↗

PubMed1986

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain.

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.

Animals↗

PubMed1977

Improved techniques for studies of adipocyte cellularity and metabolism.

Two methods are described for the study of adipose tissue cellularity and metabolism. In the first, 8 M urea was used to liberate osmium tetroxide-fixed adipocytes from the connective tissue matrix. In the smaller-sized cell ranges there was a significant reduction in apparent adipocyte number of rat, pig, and beef adipose tissue with 8 M urea treatment. This was attributed to solubilization of connective tissue debris that was counted as adipocytes in samples isolated without urea. There was no effect on the larger cell-size fractions with 8 M urea treatment. Eight molar urea had no effect on fixed adipocyte retention of radioactivity. The second method entailed the use of hydrogen peroxide to volatilize the black, osmium tetroxide-fatty acid complex of osmium tetroxide-fixed adipocytes, containing radioactivity, resulting in colorless lipid suitable for liquid scintillation counting. This latter technique permits incubation of unfixed adipose tissue slices with a radioactive substrate, followed by fixation with osmium tetroxide and subsequent separation of the adipocytes, by screening, into the desired size ranges. Adipocytes in various size fractions can then be counted, sized, and then decolorized with hydrogen peroxide in order to quantitate the amount of radioactivity within the adipocytes. There was no loss of radioactivity from the fixed cells with hydrogen peroxide treatment.

Adipose Tissue↗

PubMed2006

Validation of 18F-fluoro-4-thia-palmitate as a PET probe for myocardial fatty acid oxidation: effects of hypoxia and composition of exogenous fatty acids.

UNLABELLED: Fatty acid oxidation (FAO) is the predominant energy-producing pathway in the healthy heart. Abnormalities in FAO are associated with many ischemic and nonischemic disease states. The aim of the present study was to further validate 16-[(18)F]-fluoro-4-thia-palmitate ((18)F-FTP) as a metabolically trapped FAO probe in the isolated perfused rat heart model by examining both the effects of hypoxia and the effects of changes in exogenous fatty acid availability. METHODS: Hearts were excised from Sprague-Dawley rats and perfused in the Langendorff mode with Krebs-Henseleit solution under the following conditions: palmitate at 0.4 mmol/L with 95% oxygen, palmitate at 0.4 mmol/L with 35% oxygen, palmitate at 0.2 mmol/L plus oleate at 0.2 mmol/L with 95% oxygen, and palmitate at 0.2 mmol/L plus oleate at 0.2 mmol/L with 35% oxygen. Hearts were paced at 270 beats per minute, and the rate of left ventricular pressure change (LV dP/dt) was monitored. (18)F-FTP in the perfusion medium was administered for 20 min, and this step was followed by a 20-min washout period without tracer in the perfusion medium. (18)F kinetics in the whole heart were monitored externally, and the time-activity curves were analyzed to determine the fractional trapping rate for (18)F-FTP (FTR(FTP)). A "lumped constant" (LC) was defined as the ratio of FTR(FTP) to the fractional rate of oxidation of fatty acid in the perfusion medium. RESULTS: The kinetic data for (18)F-FTP demonstrated metabolic trapping of (18)F radioactivity that was insensitive to changes in the mixture of fatty acids in the perfusion medium but that was sensitive to the inhibition of mitochondrial FAO by hypoxia. LV dP/dt was reduced 47%-67% in hypoxic hearts relative to hearts with normal oxygenation (controls). FAO rates for palmitate and oleate were similar in group 3 (palmitate alone) and group 4 (palmitate and oleate). FAO was decreased 70%-76% with hypoxia, whereas FTR(FTP) was reduced 86%-88%, demonstrating hypersensitivity of a change in (18)F-FTP retention to FAO inhibition by oxygen deprivation. The (18)F-FTP LC was approximately 2 in myocardium with normal oxygenation and fell to 1.0-1.2 in hypoxic myocardium. CONCLUSION: The results confirm (18)F-FTP to be a metabolically trapped palmitate analog that is capable of indicating rates of myocardial oxidation of exogenous long-chain fatty acids. The heterogeneous nature of fatty acids in plasma does not alter the quantitative analysis of (18)F-FTP kinetics. However, the decreased LC value in hypoxic myocardium suggests the need to develop an understanding of the relationship of (18)F-FTP processing to natural fatty acids at key limiting transport and metabolism processes, analogous to previous studies examining the LC values for radiolabeled deoxyglucose tracers used to estimate the glucose metabolic rate.

Animals↗

PubMed1985

GM2-ganglioside metabolism in cultured human skin fibroblasts: unambiguous diagnosis of GM2-gangliosidosis.

The metabolism of GM2-ganglioside was studied in situ using cultured skin fibroblasts from normal individuals and patients with different forms of GM2-gangliosidosis. [3H]Sphingosine-labeled GM2 was provided in the culture medium to confluent cells in 6-cm petri dishes. After 10 days, the cells were washed free of radioactivity and harvested by trypsinization. The cellular lipids were extracted and analyzed for radioactivity in GM2 and its metabolic products. In fibroblasts from healthy subjects, 50-60% of the total cellular radioactivity was found in the neutral glycosphingolipids, ceramide, sphingomyelin and fatty acids. Degradation of the labeled GM2 progressed rapidly via GM3, ceramide dihexoside and ceramide monohexoside with a build-up of radioactivity mainly in the ceramide pool of the cell. The labeled ceramide is also reutilized for the synthesis of ceramide trihexoside, globoside and sphingomyelin or is converted to fatty acid and incorporated in ester linkages. In contrast, cells from patients with GM2-gangliosidosis representing Tay-Sachs, Sandhoff and AB variant forms of the disease did not metabolize the ingested labeled GM2-like controls. Nearly all of the radioactivity was present in the ganglioside fraction in the lipid extracts from these cells and consisted of unhydrolyzed GM2. High-performance liquid chromatographic analysis of monosialogangliosides from cells grown without added labeled GM2 in the medium indicated accumulation of endogenously synthesized GM2 in cell lines from all patients with GM2 gangliosidosis compared to healthy controls. This approach provides a reliable tool for pre- and post-natal diagnosis of all forms of GM2-gangliosidosis without ambiguity.

Cells, Cultured↗

PubMed1992

Permeability of arachidonic acid metabolites through the round window membrane in chinchillas.

Our previous studies showed that samples of middle ear effusion contain high concentrations of arachidonic acid metabolites (AAMs); both prostaglandins (PGs) and leukotrienes (LTs). Since abnormal levels of AAMs in the perilymph seem to be associated with salicylate ototoxicity and the round window membrane (RWM) is the most likely route of entry from the middle ear to the inner ear, it would be important to know the permeability of AAMs through the RWM. Permeability of AAMs was determined by applying labeled and standard representatives AAMs on the RWM and counting radioactivity or measuring concentrations of AAMs in perilymph at different time intervals. Using chinchillas as experimental animals, samples of perilymph were obtained through a fenestration made at the helicotrema of the cochlea after 0.5, 1, 2, and 4, hours from placing tritiated or standard PGE2, 6-keto-PGF1 alpha, LTB4, LTC4 and 15-hydroxyeicosatetraenoic acid (HETE) over RWMs. Radioactivity in the perilymph was determined by a scintillation counter and concentrations of AAMs were measured by reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay. AAMs, which are twenty carbon fatty acids, were found to cross the RWM readily. Radioactivity and concentrations of AAMs in perilymph were detected in 30 minutes and peaked by one hour from the application of AAMs on the RWM. The results of this study suggest that AAMs such as PGs and LTs present in the middle ear fluid can cross the RWM readily and enter into the perilymphatic space of the cochlea. Sensorineural hearing loss observed in chronic otitis media could be mediated by PGs and LTs in the middle ear effusion crossing over to the inner ear through the RWM.

Adrenal Cortex Hormones↗

PubMed2005

In vitro conjugation of ethanolamine with fatty acids by rat liver subcellular fractions.

Previous studies from our laboratory have shown the enzymic formation of fatty acid (FA) conjugates of xenobiotic alcohols and amines. In the present study, the formation of FA conjugates of a bifunctional compound, ethanolamine was investigated by incubating [1-14C]oleic acid (1 mM) with ethanolamine (25 mM) at 37 degrees C in the presence of various rat liver subcellular fractions. The resultant product (or products) was separated by thin-layer chromatography (TLC) and the radioactivity corresponding to the relative flow of fatty acid amide was determined. Under similar conditions, formation of ethanolamides of palmitic, stearic, linoleic, linolenic, and arachidonic acids were also examined. The formation of ethanolamine conjugate with oleic acid was found to be 16.3 nmol/h/mg protein as compared to 6.7, 6.2, 8.1, 8.3, and 7.6 nmol/h/mg protein for palmitic, stearic, linoleic, linolenic, and arachidonic acids, respectively. The formation of oleoyl ethanolamide was found to be 18.9, 40.1, 65.9, and 0.3 nmol/h/mg protein in postnuclear, mitochondrial, microsomal, and cytosolic fractions, respectively. Mass spectrometric and nuclear magnetic resonance spectroscopic data of the TLC-purified product confirm the formation of oleoyl ethanolamide, and amidation appeared to be a preferred reaction over esterification. The results of this study suggest that the enzyme responsible for the amidation of fatty acids resides mainly in the microsomal fraction of the liver, and that oleic acid is a better substrate than other fatty acids used in the present study.

Analysis of Variance↗

PubMed1971

De novo fatty acid synthesis and elongation of fatty acids by subcellular fractions of lung.

Fatty acid synthesis by subcellular fractions of rabbit lung was studied by measuring the incorporation of either radioactive acetyl coenzyme A or malonyl coenzyme A into long-chain fatty acids. Evidence is presented to support the conclusions that the 95,000 g-supernatant fraction contains the enzymes, i.e., fatty acid synthetase and acetyl coenzyme A carboxylase, necessary for de novo fatty acid synthesis and is capable of synthesizing long-chain fatty acids, probably palmitic acid, under the appropriate conditions. The mitochondrial fraction incorporates the short-chain coenzyme A derivatives into fatty acids predominantly by the elongation pathway. It is suggested that the palmitic acid synthesized in vivo by the de novo fatty acid synthetic pathway, demonstrated in vitro in rabbit lung, may be a source of the lecithin palmitic acid utilized in the synthesis of pulmonary surfactant.

Adenosine Triphosphate↗

PubMed1977

Prostaglandin A2 and 35S uptake in connective tissue.

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartilage, long bone and skull bone than did normal control animals. Administration of prostaglandin A2 stimulated 35S uptake by lung, kidney and aorta while 35S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.

Animals↗

PubMed1988

Long-chain fatty acyl-CoA synthetase of rat adrenal microsomes. Effect of ACTH and epinephrine.

Acyl-CoA synthetase activity with various long-chain fatty acid substrates and its kinetic properties were measured in rat adrenal microsomes. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order eicosa-8,11,14-trienoic acid less than alpha-linolenic acid less than linoleic acid less than palmitic acid. The maximum velocities with these fatty acids decreased in the order linolenic greater than eicosa-8,11,14-trienoic acid greater than palmitic acid. The synthesis of radioactivity palmitoyl-CoA, linoleyl-CoA, alpha-linolenyl-CoA and eicosa-8,11,14-trienoyl-CoA from the respective radioactive substrates decreased in the presence of all the other fatty acids mentioned above. These effects were inversely correlated with their apparent Km values. These results support the idea of a single long-chain fatty acyl-CoA synthetase in the adrenal microsomal fraction for the acid tested. After testing the influence of different hormones, it was shown that the administration of epinephrine, ACTH and dexamethasone caused a significant decrease in the activity of the long-chain fatty acid-CoA synthetase. This inhibition is independent of the one produced by the same hormones on the desaturation of linoleic to gamma-linolenic acid.

Adrenal Glands↗

PubMed1998

Characterization of fatty acid elongase enzymes from germinating pea seeds.

In vitro biosynthesis of radioactive arachidate and behenate was observed when microsomal fractions of germinating pea seeds were incubated with exogenous stearoyl-CoA (18:0-CoA) or arachidoyl-CA (20:0-CoA) in the presence of NADPH, [2-14C]malonyl-CoA and ATP. Characterization of parameters required for optimal stearoyl- and arachidoyl-CoA elongation revealed that, at least, two chain-length-specific elongases are necessary for very-long-chain fatty acid synthesis. Both enzymes were found to be sensitive to the group-selective reagents, p-CMB, NEM, iodoacetate, arsenite and phenylglyoxal. Subcellular fractionation studies indicated that both of these elongases were localized mainly in the endoplasmic reticulum.

Acyl Coenzyme A↗

PubMed2002

14(R,S)-[18F]Fluoro-6-thia-heptadecanoic acid as a tracer of free fatty acid uptake and oxidation in myocardium and skeletal muscle.

14( R, S)-[(18)F]Fluoro-6-thia-heptadecanoic acid ([(18)F]FTHA) is a long-chain fatty acid substrate for fatty acid metabolism. [(18)F]FTHA has been used to study fatty acid metabolism in human heart and skeletal muscle. It has been suggested that the rate of radioactivity accumulation in the myocardium reflects the beta-oxidation rate of free fatty acids (FFAs). However, the net accumulation of FFAs in tissue always represents the sum of FFA oxidation and incorporation into triglycerides. The fraction of [(18)F]FTHA entering directly into mitochondria for oxidation has not been previously measured. Eight anaesthetized pigs were studied with [(18)F]FTHA and positron emission tomography (PET). Immediately after each PET experiment, tissue samples from myocardium and skeletal muscle were taken for the isolation of mitochondria and measurements of radioactivity accumulation, and for intracellular [(18)F]FTHA metabolite analysis. Fractional [(18)F]FTHA uptake rates were calculated both by graphical analysis of PET data and by measuring (18)F in the tissue samples. Fractional [(18)F]FTHA uptake rates based on the analysis of tissue samples were 0.56+/-0.17 ml g(-1) min(-1) and 0.037+/-0.007 ml g(-1) min(-1) for myocardium and skeletal muscle (mean +/- SD), respectively. The myocardial results obtained from the PET data (0.50+/-0.11 ml g(-1) min(-1)) were similar to the values obtained from the tissue samples ( r=0.94, P=0.002). We also found that 89%+/-23% (mean+/-SD, n=7) of the (18)F entered mitochondria in myocardium, as compared with only 36%+/-15% (mean+/-SD, n=7) in skeletal muscle. Intracellular [(18)F]FTHA metabolite analysis showed that a major part of [(18)F]FTHA is metabolized in the mitochondria in the heart. Our data suggest that ~89% of [(18)F]FTHA taken up by the heart enters mitochondria. This supports the hypothesis that [(18)F]FTHA traces FFA beta-oxidation in the heart. In contrast to this, only ~36% of [(18)F]FTHA accumulated in skeletal muscle appears to directly enter mitochondria; the majority is taken up by the other cell fractions, suggesting that in skeletal muscle [(18)F]FTHA traces FFA uptake but not specifically FFA beta-oxidation.

Animals↗

PubMed1983

Fatty acid synthesis in the arrested rabbit heart during ischemia.

In rabbit hearts arrested by a carnosine-buffered cardioplegic solution the incorporation of [1-14C]acetate into lipids was investigated. After 10-20 and 60 min of ischemia the radioactivities of phospholipids, mono-, di-, and triacylglycerols, acyl-CoA, acylcarnitine, and free fatty acids were determined. In the first period of ischemia mainly acylcarnitine was labelled (ca. 50%) and only 25% of [14C]-activity was found in phospholipids which showed the lowest specific activity of all lipid classes. After 60 min of ischemia the percentage of total radioactivity of acylcarnitine and phospholipids was decreased, whereas that of neutral lipids was increased to more than 50%. During the increase of total radioactivity the relative specific activity of all lipids decreased except that of triacylglycerols. Only fatty acids up to chain lengths of 16 carbon atoms were labelled. Lauric and myristic acid had high specific activities. These results indicated de novo synthesis of fatty acids accumulating in triacylglycerols during ischemia of the arrested heart.

Acetates↗

PubMedDate not supplied

Glutamate excretion triggering mechanism: a reinvestigation of the surfactant-induced modification of cell lipids.

Lipid alterations induced by surfactants to trigger glutamate excretion were investigated with an industrial strain of Corynebacterium glutamicum. The lipid composition of this strain was determined for cultures in a synthetic medium and in a complex medium. A distribution of complex lipids between the cell wall and the cell membrane is proposed. Depending on growth conditions, 70-85% of the cell fatty acids had saturated chains in cells grown with surfactants. In the synthetic medium up to 80% of the straight-chain fatty acids may have come from the acylated surfactant added to the induce excretion. In industrial fermentation, the maximal excretion rate corresponded to the highest saturated fatty acid content of cells. It was shown by radioactive labelling in the synthetic medium that the addition of the acylated surfactant induced the degradation of more than 50% of the phospholipids. Some phospholipid synthesis occurred at that time using the surfactant (saturated) fatty acids, but the membrane did nor recover its previous phospholipid content. A model is proposed to explain the mechanism of glutamate excretion triggering by surfactants.

Cell Membrane↗

PubMed1984

Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds.

Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.

Cysteine↗

PubMed1965

THE INCORPORATION OF CARBON DIOXIDE INTO MILK CITRATE IN THE ISOLATED PERFUSED GOAT UDDER.

1. Isolated perfused goat udders supplied with glucose, acetate and amino acids were infused for several hours with NaH(14)CO(3). 2. Lactose, milk-fat fatty acids and glycerol had very little radioactivity. The specific radioactivity (counts./min./mg. of C) of milk citrate was 9-16% that of the carbon dioxide in the perfusion fluid and 19% that estimated for tissue carbon dioxide. The specific radioactivity of tissue citrate resembled that of milk citrate. 3. The radioactivity in citrate was predominantly in C-6, suggesting some carboxylation of alpha-oxoglutarate in addition to carboxylation of C(3) compounds. 4. [1-(14)C]Glutamate was infused in a similar experiment, and milk citrate radioactivity was predominantly in C-1+C-5. 5. The results are discussed in relation to the contribution of glucose and acetate carbon to citrate. The implications of the carboxylation of alpha-oxoglutarate are considered.

Acetates↗

PubMed1992

Incorporation and turnover of eicosapentaenoic and docosahexaenoic acids in human blood platelets in vitro.

Mass changes in the incorporation of linoleic (C18:2), eicosapentaenoic (C20:5) and docosahexaenoic (C22:6) acids in human blood platelet phospholipids were induced by incubating the cells and these fatty acids complexed to albumin. The remodelling of [14C]C18:2, [14C]C20:5 and [14C]C22:6 in classes, subclasses and molecular species of platelet phospholipids was studied in resting and thrombin-stimulated cells. More than 85% of the incorporation was located in phospholipids, representing 5-fold and 2.5-fold increases in the phospholipid C20:5 and C22:6 endogenous content respectively. Thrombin stimulation induced a 30% degradation of 1-acyl-2-C20:5-glycerophosphocholine (GPC) and 1-acyl-2-C22:6-GPC, but did not induce significant release of C18:2 from 1-acyl-2-C18:2-GPC. There was no change in the [14C]fatty acid composition of 1-alkyl-2-acyl-GPC. Thrombin-dependent increases in 1-alkenyl-2-C20:5-glycerophosphoethanolamine (GPE) and 1-alkenyl-2-C22:6-GPE of 2.1-fold and 2.5-fold respectively accounted for the rise in GPE radioactivity and partly compensated for the loss of these fatty acids from 1,2-diacyl-GPC: transfer to 1-alkenyl-2-acyl-GPE was 0.4 and 1.5 nmol/10(9) platelets for C20:5 and C22:6 respectively. [14C]C20:5 and [14C]C22:6 were incorporated into six different species of 1,2-diacyl-GPC, with acylation in the major endogenous forms (C18:1 +C16:0 and C18:0 species) representing 76% and 66% respectively of the total radioactivity present in 1,2-diacyl-GPC. Stimulation by thrombin induced significant release of these fatty acids from the main molecular species of 1,2-diacyl-GPC, but significantly stimulated the synthesis of alkenyl forms of GPE containing C18:1/C22:6 +C16:0/C22:6, C18:0/C22:6 and C18:0/C20:5. C18:0/C18:2, the major endogenous C18:2 molecular species, represented only 10.5% of the incorporation; none of the [14C]C18:2 molecular species was a substrate for transfer towards 1-alkenyl-2-acyl-GPE. It is concluded that when C20:5 and C22:6, but not C18:2, are acylated in 1,2-diacyl-GPC, they participate in thrombin-dependent phospholipid remodelling, and might compete with the turnover and release of arachidonic acid from platelet phospholipids and the subsequent activation of the cells.

Blood Platelets↗

PubMed2002

Fatty acid oxidation in neonatal hepatocytes: effects of sepsis and glutamine.

OBJECTIVE: Little is known about fat use during sepsis during the neonatal period. Intramitochondrial O(2) consumption is inhibited in isolated hepatocytes from suckling septic rats and this impairment is reversed by glutamine. We investigated the effect of neonatal sepsis on fat oxidation and whether glutamine can directly affect fatty acid oxidation. METHODS: Suckling Wistar rats (11 d) received an intraperitoneal injection of 300 microg/kg of lipopolysaccharide (Escherichia coli 055:B5); controls received normal saline. At 2 h, hepatocytes were isolated. Hepatocytes were incubated at 37 degrees C with 0.5 mM [1-(14)C]palmitate or 0.5 mM [1-(14)C]palmitate plus 10 mM glutamine. After 1 h, the perchloric acid-soluble (14)C-radioactivity (representing mainly ketone bodies) and (14)CO(2) were measured. Hepatocyte O(2) consumption from 0.5 mM palmitate was measured with and without 2.5 ng/mL myxothiazol to estimate intramitochondrial O(2) consumption. RESULTS: There were no significant differences in fatty acid oxidation between control and endotoxemic hepatocytes measured as acid-soluble radioactivity (which represents mainly ketogenesis, plus Krebs cycle intermediates), as (14)CO(2) production, or as the sum of acid-soluble radioactivity plus (14)CO(2) generation. Glutamine significantly increased fatty acid oxidation (acid-soluble radioactivity plus (14)CO(2)) in hepatocytes from control and endotoxic animals. CONCLUSIONS: The finding of no significant difference in fatty acid oxidation between hepatocytes from control and endotoxemic rats is surprising given that intramitochondrial O(2) consumption from palmitate is decreased. This may reflect altered use of acetyl-coenzyme A to ketone bodies and Krebs cycle intermediates. Glutamine enhanced fatty acid oxidation from control and endotoxemic hepatocytes, suggesting that it may promote substrate oxidation during endotoxemia.

Animals↗