Search

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

Palmitic acid increased the conductivity of BLM from mitochondrial phospholipids when they were dissolved in a mixture of decane and chlorodecane, and was ineffective when phospholipids were dissolved in decane. Lauric acid produced an increase in the membrane conductivity independently of the phospholipid type in the membrane-forming solutions (mitochondrial phospholipids, asolectin, lecithin with cholesterol) and their solvents (decane or decane with chlorodecane). The results show that discrepancies between published data concerning fatty acid effects on the BLM conductivity may be explained by differences in phospholipids, their solvents and fatty acid used.

Unlike other saturated fatty acids, dietary stearic acid does not appear to raise plasma cholesterol. The reason for this remains to be established, although it appears that it must be related to inherent differences in the metabolism of the fatty acid. In the present study, we have looked at the metabolism of palmitic acid and stearic acid, in comparison with oleic acid, by cultured hamster hepatocytes. Stearic acid was taken up more slowly and was poorly incorporated into both cellular and secreted triacylglycerol. Despite this, stearic acid stimulated the synthesis and secretion of triacylglycerol to the same extent as the other fatty acids. Incorporation into cellular phospholipid was lower for oleic acid than for palmitic acid and stearic acid. Desaturation of stearic acid, to monounsaturated fatty acid, was found to be greater than that of palmitic acid. Oleic acid produced from stearic acid was incorporated into both triacylglycerol and phospholipid, representing 13% and 6% respectively of the total after a 4 h incubation. Significant proportions of all of the fatty acids were oxidized, primarily to form ketone bodies, but by 8 h more oleic acid had been oxidized compared with palmitic acid and stearic acid.

Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor. A multifunctional expression plasmid was constructed by cloning the V2 receptor cDNA into the vector pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis experiments without subcloning, and stable and transient expression of the V2 receptor in Ltk- and COS.M6 cells respectively. The conserved cysteine residues Cys-341 and Cys-342 were replaced by serine residues, yielding the single mutants C-341S and C-342S and the double mutant C-341S/C-342S. Functional expression in stably transfected Ltk- cells showed that the affinity of the three mutant receptors for arginine vasopressin was not altered. In contrast with the activation of adenylate cyclase through β2 adrenergic receptors, arginine vasopressin stimulated adenylate cyclase to the same extent and with similar EC50 values in both wild-type and mutant receptors. Transient expression of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an unaltered affinity of the mutant receptor for arginine vasopressin. However, the number of arginine vasopressin-binding sites on the cell surface was reduced by 30%, suggesting that the transport of the mutant receptor to the cell surface was impaired. In addition, the decrease in detectable arginine vasopressin-binding sites on the cell surface following pre-exposure to hormone was reduced, indicating that the sequestration/internalization of the mutant receptor on the cell surface was affected. The present data indicate that palmitoylation of the V2 receptor is important for intracellular trafficking and/or sequestration/internalization but not for agonist binding or activation of the Gs/adenylate cyclase system.

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

Short term treatment of rats with beta,beta′-tetramethylhexadecanedioic acid (MEDICA 16) results in a pronounced decrease in plasma very-low-density-lipoprotein (VLDL) cholesterol and VLDL triacylglycerol, previously ascribed to a decrease in liver VLDL production [Bar-Tana, Rose-Kahn, Frenkel, Shafer and Fainaru (1988) J. Lipid Res. 29, 431-441]. The hypolipidaemic effect of MEDICA 16 was further analysed here by monitoring plasma VLDL clearance and its hepatic uptake. VLDL triacylglycerol and VLDL apolipoprotein (apo) B fractional clearance rates were increased 7-8-fold in MEDICA 16-treated rats. The increase in the fractional clearance rate of plasma VLDL was essentially eliminated by functional hepatectomy. It was accounted for by activation of plasma VLDL uptake by the liver being completed during the first 4 min after the injection of the VLDL label and before commencement of uptake in non-treated animals. The hypolipidaemic effect of MEDICA 16 was accompanied by a 3.5-fold decrease in plasma apoC-III, but plasma apoC-III clearance remained unaffected by MEDICA 16. MEDICA 16-induced premature hepatic uptake of plasma VLDL due to suppression of apoC-III production may thus account for enhancement of plasma VLDL clearance in treated animals.

In the presence of 150 microM BSA, uptake of [3H]oleate by Xenopus laevis oocytes was a saturable function of the unbound oleate concentration (Vmax. 110 +/- 4 pmol/h per oocyte; Km 193 +/- 11 nM unbound oleate). Oleate uptake was three orders of magnitude faster than that of another test substance, [35S]bromosulphophthalein, and was competitively inhibited by 55 nM unbound palmitate (Vmax. 111 +/- 14 pmol/h per oocyte; Km 424 +/- 63 nM unbound oleate) (P < 0.01). Oleate uptake was also inhibited by antibodies to a 43 kDa rat liver plasma-membrane fatty acid-binding protein, a putative transporter of long-chain fatty acids in mammalian cells; uptake of the medium-chain fatty acid [14C]octanoate was unaffected. Immunofluorescence and immunoblotting demonstrated that the antiserum reacted with a single 43 kDa protein on the oocyte surface. Hence a protein related to the mammalian plasma-membrane fatty acid-binding protein may play a role in saturable uptake of long-chain fatty acids by Xenopus oocytes.

The fatty acids palmitic (C16:0), stearic (C18:0), arachidic (C20:0) and arachidonic (C20:4) acids inhibit Ca2+ uptake and enhance Ca2+ efflux measured in vesicles derived from the sarcoplasmic reticulum of skeletal muscle. These effects of the fatty acids are impaired by the Ca(2+)-ATPase ligands Mg2+, Ca2+ and K+, and by drugs that block the leakage of Ca2+ through the Ca(2+)-ATPase such as Ruthenium Red, spermine [de Meis (1991) J. Biol. Chem. 266, 5736-5742] and thapsigargin [de Meis and Inesi (1992) FEBS Lett. 299, 33-35].

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

Treatment of cholesterol-fed male hamsters kept on a diet of purina chow with beta beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) resulted in a progressive hypocholesterolaemic effect, amounting to a 50% decrease in the cholesterol content of all plasma lipoproteins. The decrease in plasma cholesterol could be accounted for by activation of plasma-cholesterol efflux through the liver into the bile mediated by MEDICA 16-induced (a) increase of the number of liver LDL receptors, (b) activation of liver neutral cholesteryl ester hydrolase with a concomitant inhibition of liver acyl-CoA cholesterol acyltransferase, resulting in shifting of the liver cholesteryl ester/free-cholesterol cycle in the direction of free cholesterol, and (c) activation of cholesterol efflux from the liver into the bile. The increase in bile cholesterol output was accompanied by an increase in bile phospholipids but not in bile acids. In contrast with rats, MEDICA 16-treatment of male hamsters did not result in a hypotriacylglycerolaemic effect, inhibition of lipogenesis, nor in a substantial decrease in plasma apolipoprotein C-III content.

1. We describe the acyl-CoA and acyl-carnitine esters which arise from the incubation of well-coupled State 3 rat skeletal-muscle mitochondrial fractions with [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine. 2. Acyl-CoA ester intermediates of chain length 16, 14, 12, 10 and 8 carbons were detected. 3. Although incubations were in steady state in respect of oxygen consumption, 14CO2 production and generation of acid-soluble radioactivity, quantitative analysis of acyl-CoA esters showed that steady state was not achieved in respect of all intermediates. 4. 3-Hydroxyacyl- and 2-enoyl-CoA and -carnitine esters were found under normoxic conditions. 5. Direct measurement of NAD+ and NADH shows that under identical incubation conditions our observations cannot be explained by gross perturbation of the [NAD+]/[NADH] ratio. 6. We hypothesize that there is a small pool of rapidly recycling NAD+ channelled between complex I of the respiratory chain and the newly described mitochondrial-inner-membrane-associated beta-oxidation trifunctional enzyme [Uchida, Izai, Orii and Hashimoto (1992) J. Biol. Chem. 267, 1034-1041].

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium.

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.

1. Sodium pump function has been assessed by measurement of ouabain-sensitive 86Rb uptake in human erythrocytes after incorporation of palmitic, stearic, oleic and linoleic acids into the erythrocyte membrane. 14C-labelled fatty acids were used to measure membrane uptake of these substances. 2. For palmitic, oleic and linoleic acids, up to 1000 nmol of the fatty acid/ml of packed cells can be incorporated without causing significant haemolysis. For stearic acid, 270 nmol/ml of packed cells was incorporated in similar conditions. More than 88% of the fatty acid incorporated could be extracted with a 50 μmol/l fatty-acid-free albumin solution and was, therefore, in a non-esterified form in the erythrocyte membrane. The concentrations of palmitic, stearic, oleic and linoleic acids incorporated in these experiments represent a five- to ten-fold increase above the normal concentrations of these fatty acids in the membrane. 3. Up to 1000 nmol of palmitic, oleic and linoleic acids/ ml of packed cells and up to 270 nmol of stearic acid/ml of packed cells could be incorporated without a significant change in mean ouabain-sensitive 86Rb uptake with respect to control cells. Mean percentage changes in ouabain-sensitive 86Rb uptake for all these experiments were: palmitic acid, 3.7% (sd 11.4, n = 15); stearic acid, 4.0% (sd 5.7, n = 7); oleic acid, −4.8% (sd 19, n = 17); linoleic acid, 2.2% (sd 15.6, n = 19). 4. The demonstration of near-normal sodium pump activity in the presence of greatly elevated membrane levels of these fatty acids makes it extremely unlikely that they act as modulators of sodium pump function in vivo.

1. This study was designed to determine prospectively whether changing alcohol consumption influenced the proportion of plasma linoleic acid independently of diet or smoking habits, and to evaluate changes in the plasma linoleic acid concentration as a potential marker of changes in alcohol consumption. 2. Fasting plasma fatty acid profiles were investigated in 72 male drinkers who were randomly assigned to drink low-alcohol beer or to maintain their usual drinking habits for a period of 4 weeks. 3. At entry to the study, a higher alcohol intake was associated with lower proportions of plasma linoleic acid, arachidonic acid and dihomolinolenic acid and higher proportions of plasma palmitoleic acid, independently of changes in body mass index. Smoking habits were unchanged and there were no major changes in diet during the period of the intervention. 4. Because the plasma palmitoleic acid concentration has been suggested as a possible marker of ‘at risk’ drinking, we investigated plasma fatty acid concentrations as indicators of alcohol intake. The plasma palmitoleic acid concentration was not a useful discriminator. Indices determined using logistic regression and combining plasma apolipoprotein A-II and linoleic acid concentrations gave better discrimination than either variable alone.

Alterations of glucose metabolism and the oxidation of glutamine and palmitate were studied, by using specifically labelled substrates, in freshly isolated Kupffer cells and hepatic endothelial cells after infusion in vivo of human recombinant tumour necrosis factor-alpha (TNF; 7.5 x 10(5) IU/30 min per kg body wt., intravenously). Cells were incubated in a medium containing 5 mM-glucose, 0.4 mM-palmitate, 1 mM-lactate and 0.5 mM-glutamine. Administration of TNF in vivo increased glucose use in Kupffer cells by 70%. Glucose oxidation in the tricarboxylic acid cycle and flux in the Embden-Meyerhof (EM) pathway were elevated by 40 and 80% respectively. Treatment in vitro with 1 microM-phorbol 12-myristate 13-acetate (PMA) resulted in a similar percentage increase in glucose use by Kupffer cells prepared from either saline- or TNF-treated rats. However, PMA increased the activity of the hexose monophosphate shunt (HMS) by 3- and 10-fold in cells isolated from saline- or TNF-infused animals respectively. A phagocyte stimulus in vitro, opsonized zymosan, increased glucose use by 30% and doubled the flux through the HMS in Kupffer cells from saline-infused animals. The activity of the HMS in response to zymosan was increased by 400% after TNF treatment. In endothelial cells, basal glucose utilization was not altered by TNF treatment. PMA increased HMS activity in endothelial cells to a similar degree after saline or TNF infusion. Zymosan, however, increased HMS activity only in endothelial cells from TNF-treated rats. Oxidation of palmitate or glutamine was not affected by TNF treatment either under basal conditions or after challenge in vitro. Our data indicate that, after phagocytosis in vitro or protein kinase C activation, glucose use and flux through the HMS increase in Kupffer cells. This is accompanied by increased glycolytic flux, with no changes in glucose oxidation in the tricarboxylic acid cycle. After TNF exposure, followed by a secondary stimulus, the enhanced glucose use by Kupffer cells is primarily channelled through the HMS pathway. These data suggest that the increased glucose use in vivo by Kupffer cells found after immune-stimulated conditions may subserve primarily the increased need for NADPH and HMS intermediates.

The mechanisms by which noradrenaline, lipolytic agents and long-chain fatty acids stimulate glucose transport were investigated in rat brown adipocytes. Glucose transport was evaluated with tracer D-[U-14C]glucose and cell respiration was measured polarographically. Noradrenaline increased basal oxygen consumption (8-10-fold) and glucose transport (4-5-fold) in a dose-dependent manner, with a maximal stimulation at 100 nM. The stimulatory effects of noradrenaline on respiration and glucose transport were selectively mimicked by dibutyryl cyclic AMP (DBcAMP), 3-isobutyl-1-methylxanthine, cholera toxin and physiological concentrations of palmitic acid. Cytochalasin B completely blocked the effects of these agents on glucose transport. The beta-adrenergic antagonist propranolol inhibited noradrenaline-induced glucose transport, but did not affect the action of DBcAMP, palmitic acid or cholera toxin on this process. The specific inhibitor of mitochondrial carnitine palmitoyltransferase, 2-tetradecylglycidic acid (McN 3802) (50 microM), inhibited the stimulatory effects of noradrenaline (100 nM) and palmitic acid (0.5 mM) on both glucose transport and mitochondrial respiration. Significantly, McN 3802 failed to affect insulin (1 nM) action under identical experimental conditions. These results demonstrate that (a) the stimulatory effects of noradrenaline on brown-adipocyte respiration and glucose transport can be dissociated from those induced by insulin, and (b) noradrenaline increases glucose transport indirectly, by activating adenylate cyclase via beta-adrenergic pathways and by stimulating mitochondrial oxidation of fatty acids.

This paper discusses the regulation of the hydrolysis of phosphatidylcholine in Swiss 3T3 cells.

A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a ‘hysteretic’ pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied.

Rat ventral prostate incorporated (1-14C)acetate, (1-14C)palmitate and (1-14C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO2 whereas no differences were observed in the radioactivity incorporated into CO2 from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO2 oxidation.

31P n.m.r. analysis of control and diabetic hearts perfused for 1 h with a glucose buffer showed constant and normal levels of phosphocreatine and ATP. Supplementing the buffer with 0.5, 1.2 or 2.0 mM-palmitic acid had little or no effect on high-energy-phosphate levels in control hearts. In contrast, increases in palmitate concentration produced significant decreases in ATP in diabetic hearts, despite normal and constant levels of phosphocreatine. This 31P n.m.r. study suggests a defect in phosphocreatine metabolism in the perfused diabetic heart that might be related to creatine kinase kinetics.

1. The specific binding of thyroid-stimulating hormone to isolated human thyroid membranes was examined under a variety of conditions. 2. In phosphate-saline buffer (in the presence of 0.14 M-NaCl) on increasing the temperature the binding of the hormone is increased, the plots of bound/free hormone against temperature showing a distinct break around 30 degrees C. 3. Detailed analysis showed that the increased binding is associated with an increase in the number of binding sites. 4. The motional characteristics of three membrane-bound fluorescent probes, 2-(9-anthroyl)palmitic acid, 12-(9-anthryl)stearic acid and N-1-naphthyl-N-phenylamine, were also examined as a function of temperature by measuring both fluorescence polarizations and lifetimes. 5. The results indicated that the ‘fluidity’ of membrane lipids also increased with temperature. The temperature-dependence of this property also shows a change at about 30 degrees C. 6. Bivalent cations decreased both membrane fluidity and hormone binding. 7. Similar correlations were found between the binding of adrenocorticotrophic hormone and the fluidity of the plasma membranes obtained from adrenal-cortical cells, with the discontinuity occurring in this case at 23 degrees C. 8. The possibility of lipid mobility being important in controlling hormone-receptor function is discussed.

Perifused fat-cells showed similar values for acylglycerol glycerol synthesis from glucose with insulin and for the effects of added palmitate to those in normal incubations and those reported in the literature. Fatty acid synthesis was lower in perifused cells compared with normal incubations, and there was a net release of fatty acids only with the perifused fat-cells. Hence fluxes of metabolites were different in the two incubation systems, and the perifusion system enables the investigation of the flux of metabolites under conditions which may more closely resemble those in vivo.

1. Rats were treated for 4 weeks with liquid diets that contained, on the basis of energy content, 35% fat, 18% protein and 47% carbohydrate (high-fat diet) or 35% fat, 18% protein, 11% carbohydrate and 36% ethanol (high-fat/ethanol diet). 2. The livers were perfused with 1mm-[1-14C]palmitate and with 0, 10mm- or 80mm-ethanol. The oxidation and esterification of palmitate was measured. Two subcellular pools of triacylglycerol were separated; one contained triacylglycerol from cytoplasmic lipid droplets and the other contained triacylglycerol from the endoplasmic reticulum and Golgi apparatus. 3. In the presence of ethanol, liver from rats fed on the high-fat diet esterified about 70% of the [1-14C]palmitate taken up compared with 90% in liver from rats fed chow (containing 11% fat on the basis of energy content). Compared with chow diet the high-fat diet did not potentiate the effect of ethanol on storage of [1-14C]palmitate in hepatic triacylglycerol. The relation between the fat content of the diet and the degree of fatty liver induced by by ethanol [Lieber & DeCarli (1970) Am. J. Clin. Nutr.23, 474–478] is discussed. 4. The ethanol-containing diet increased the hepatic content of triacylglycerol 4-fold and the increase was exclusively found in the fraction suggested to contain lipid from cytoplasmic lipid droplets. The ethanol-induced fatty liver, perfused with ethanol, esterified and oxidized palmitate at rates that were quite similar to the rates found in high-fat control livers perfused without ethanol. This suggests that the fatty liver had adapted to the presence of ethanol with respect to palmitate metabolism. 5. O2 and ethanol uptake by the livers were not affected by the ethanol-containing diet.

Bovine heart cytochrome c oxidase was reconstituted in phospholipid vesicles, and the effect of different non-esterified fatty acids (NEFA) was studied on its proton pump and on the proton permeability of the vesicles. Neither parameter appeared to be affected by concentrations of NEFA known to uncouple oxidative phosphorylation (10 microM). Also the permeability for K+ was not affected by them. The fatty acids caused an increase in the rate of electron transfer in the absence, but not in the presence, of uncoupler and/or valinomycin [diminution of the respiratory-control index (RCI)]. The RCI of 8.7-7.5 was decreased to about 4.5 in the presence of 0.27-10 microM-NEFA. Oleic acid was not effective at the above concentrations. Subunit III-depleted enzyme preparations gave vesicles with an RCI of about 5.5, which was decreased to 4.5 in the presence of NEFA. With both native and subunit III-depleted oxidase the RCI was never decreased to the value of 1 by NEFA, as happens with classical protonophores.

1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed.

The influence of 48 h starvation on glucose-induced changes of palmitate metabolism and insulin release in isolated rat islets was investigated. (1) Islet insulin response to 20 mM-glucose was abolished after 48 h starvation, and it was restored by 0.25 mM-2-bromostearate, an inhibitor of fatty acid oxidation. (2) The increase in glucose concentration from 3 to 20 mM was accompanied by a 50% decrease in the oxidation rate of 0.5 mM-[U-14C]palmitate in control (fed) islets, and a concomitant increase (100%) in its incorporation into triacylglycerol and phospholipid fractions. (3) Starvation induced a higher basal (3 mM-glucose) rate of palmitate oxidation, which was resistant to inhibition by 20 mM-glucose. The latter also failed to increase palmitate incorporation into islet triacylglycerols and phospholipids. (4) 2-Bromostearate (0.25 mM) strongly inhibited the high oxidation rate of palmitate in islets of starved rats, and allowed a normal stimulation of its incorporation rate into islet lipids by 20mM-glucose. (5) The results suggest that starvation restricts islet esterification of fatty acids by inducing a higher rate of their oxidative degradation that is insensitive to regulation by glucose.

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.

Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats.

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.

1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids.

The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart. Infusion of the long-chain fatty acid palmitate resulted in both an inactivation of the branched-chain 2-oxo acid dehydrogenase and an inhibition of the measured metabolic flux through this enzyme complex. Pyruvate addition also caused both an inactivation and an inhibition of the flux through the complex. On the other hand, the medium-chain fatty acid octanoate caused an activation of and a stimulation of flux through the branched-chain 2-oxo acid dehydrogenase when the perfusion conditions before octanoate addition maintained the enzyme complex in its inactive state. When the enzyme complex was activated before octanoate infusion, this fatty acid caused a significant inhibition of the flux through the branched-chain 2-oxo acid dehydrogenase reaction. Inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.

1. Rat liver mitochondria oxidizing [16-14C]palmitoylcarnitine accumulate saturated long-chain thiester intermediates which may be detected by radio-g.1.c.2. Time-courses of intermediate accumulation display no product-precursor relationships and the end product, measured as [14C]citrate, is produced without a detectable initial lag. 3. A short pulse of [16-14C]palmitoylcarnitine followed by unlabelled palmitoylcarnitine showed that the observed intermediates(at least in the greater part)were not the direct precursors of [14C]citrate. 4. The quantity of saturated intermediates depended on the total accumulated flux of acyl units through the pathway provided that some mitochondrial CoA and unused substrate remained. 5. In the presence of rotenone and carnitine, 2-unsaturated, 3-unsaturated and 3-hydroxy intermediates were formed as well as saturated intermediates…

Tumour necrosis factor (TNF) did not stimulate lipolysis in isolated rat adipocytes, though preincubation with TNF increased adrenaline-stimulated fatty acid release. Glycogenolysis, gluconeogenesis and ketogenesis in isolated rat hepatocytes were not influenced by TNF in short-term (30-60 min) incubations. TNF stimulated 14CO2 production from [U-14C]glucose in rat hemidiaphragm preparations, but lactate production and alanine release were not significantly altered. It is concluded that TNF does not regulate short-term metabolism in adipocytes, hepatocytes and muscle preparations in the manner of a catabolic hormone.

The structures of the diabolic acid-containing phospholipids of Buryrivibrio S2 grown in the presence of palmitic acid have been investigated. Generally they consist of two conventional bacterial phospholipid or galactolipid structures linked by esterification through a single diabolic acid residue. The main lipid consists of the butyroyl ester of sn-1-alkenylglycero-3-phospho-1′-sn-glycerol joined in this way to the butyroyl ester of sn-1-alkenyl-3-galactosylglycerol by esterification of the vacant 2-hydroxy groups of the alkenyl-substituted glycerol molecules. A lipid that possess a palmitoyl group rather than one of the butyroyl groups of the latter structure has also been detected. The lipid occurring in the second highest concentration consists of two molecules of sn-1-alkenylglycero-3-phospho-sn-1′-glycerol butyroyl ester linked through diabolic acid in a similar manner to the main lipid. Other lipids with the latter structure either minus a butyroyl group or with palmitoyl group, instead of one of the buryroyl groups, exist as minor components.

1. Studies were carried out to test the assumptions involved in the calculation of free fatty acid (FFA) and triglyceride transport rates from the plasma radioactivity curves after the intravenous administration of labelled fatty acid or of pre-labelled lipoprotein triglyceride. 2. Plasma FFA equilibrates reversibly with the interstitial lymph. This makes it necessary to use either a pulse injection, with analysis of the results in terms of a two-compartment model, or continuous infusion of the label in determining the FFA flux. 3. The recycling of labelled fatty acid due to direct hydrolysis of plasma triglyceride accounted for less than 5% of the total plasma FFA flux and did not appreciably affect the plasma triglyceride disappearance curve after endogenous labelling with 14C-labelled fatty acid. Turnover values calculated from triglyceride in unfractionated plasma represent mainly the very-low-density lipoprotein triglyceride transport. The distribution space of plasma triglyceride was found to be equal to the plasma volume. 4. Our findings suggest a complex relation between plasma triglyceride concentration and its transport kinetics. The plasma triglyceride concentration is determined by the rates of influx and efflux, and this concentration also regulates its own production and removal. Evidence obtained from the endogenous labelling technique and after experimental hypertriglyceridaemia suggests the application of enzyme kinetic models to characterize plasma triglyceride transport.

The metabolic fate of 14C derived from radioactively labelled dietary precursors was determined in immature (18- and 25-day-old) lean and obese Zucker rats. This included measurement of 14C incorporated into body lipid, non-essential amino acids and expired CO2. Before weaning (18 days) there was no phenotypic difference between the fates of [14C]palmitate and [14C]-glucose. However, after weaning (25 days) all the precursors studied exhibited an increase in the fraction incorporated into lipid in the obese rat as compared with the lean animal. This was reflected in the fate of acetyl-CoA in the tricarboxylic acid cycle. There was little phenotypic difference in the fraction of leucine or valine catabolized. The results presented here suggest that the high rate of lipogenesis found in the obese rat is supported by carbon from all the dietary precursors studied. It is also argued that the decreased protein deposition found in the obese rat is not caused by the high rate of lipogenesis removing precursors for protein synthesis, as has been suggested elsewhere [Cleary, Vasselli & Greenwood (1980) Am. J. Physiol. 238, E284-E292].