Distribution of newly formed palmitate and stearate among molecular species of choline and ethanolamine phosphatides.

The choline and ethanolamine phosphatides derived from isolated rat livers during perfusion with 75 percent deuterated water (Kuksis, A., Myher, J.J., Marai L., Yeung, S.K.F., Steiman, I. & Mookerjea, S. (1975) Can. J. Biochem. 53, 509-518) were resolved into molecular species by argentation thin-layer chromatography. The time course of percentage replacement of the newly synthesized fatty acids in each molecular species was determined by gas chromatography with mass spectrometry. The results confirmed the earlier postulated differential utilization of palmitic and stearic acids in glycerolipid biosynthesis as well as supported the hypothesis of a precursor-product relationship between the oligoenoic and tetranoic species of both phosphatides. Calculations of half-lives gave values of 14-19 h for palmitoyl oligoenes, 40-50 h for palmitoyl tetraenes, and 22-28 h for palmitoyl hexaenes of both choline and ethanolamine phosphatides. The corresponding stearoyl species had half-lives which ranged from 89 to 200 h. Evidence was obtained for a metabolic heterogeneity among subsets of molecular species recognized on the basis of combinations of new and old glycerol and fatty acids in the same glycerolipid molecule.     

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1–2 μg/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in Cytidylyltransferase activity was found in the 100 000 × g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.     

The metabolism of lipids in mouse pancreatic islets. The oxidation of fatty acids and ketone bodies.

The rate of oxidation of 14C-labelled fatty acids and of ketone bodies was measured in isolated pancreatic islets of obese-hyperglycaemic mice (ob/ob). The following main observations were made. 1. Octanoate, palmitate and oleate were all converted into CO2 by the pancreatic islets. Octanoate was oxidized with the highest rate followed by palmitate and oleate. 2. The rate of oxidation of 0.7 mM-palmitate was 3.1 pmol/h per mug drug weight. This was decreased by 50% in the presence of 16.7 mM-glucose. The rate of palmitate oxidation was also inhibited by 2-bromostearate. The palmitate oxidation showed a concentration-dependent increase, which was most marked between 0.25 and 1.0 mM. 3. Octanoate (5 mM) had no effect on the rate of oxidation of 3.3 mM- glucose. 4. Acetoacetate (5 mM) and D-3-hydroxybutyrate (5 mM) were oxidized at rates of 5.9 and 5.4 pmol/h per mug dry weight respectively. These rates were less than 10% of those found in kidney-cortex slices. The magnitude of the oxidation rates found for fatty acids and for ketone bodies suggest that these substrates represent important metabolic fuels for the pancreatic B-cells.     

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells

Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-kappaB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATP(total)/ADP(total) ratio by 26+/-5% (mean+/-SEM) and 15+/-2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34+/-5%, while oleate stimulated it by 12+/-2%. alpha-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by alpha-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B(1). Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-kappaB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Stereospecific analysis of major glycerolipids of Phycomyces blakesleeanus sporangiophores and mycelium.

The positional distribution of fatty acids was determined in the major groups of glycerolipids from the mycelium and sporangiophores of the fungus Phycomyces blakesleeanus. At the sn-1 positions of the triacylglycerols, in both regions of the fungus, greater than 65% of the fatty acids were 16:0 and 18:1. At the sn-2 positions of the triacylglycerols, 18:1, 18:2 and 18:3 comprised greater than 85% of the sporangial fatty acids and more than 90% of the mycelial fatty acids. Positions sn-3 of the triacylglycerols, from both regions of the fungus, contained approximately 40% of 16:0, approximately 30% of 18:2, and the largest proportions of 18:3 (21%) in the triacyglycerols. The major phosphoglycerides of P. blakesleeanus mycelium and sporangiophores are phosphatidylcholine and phosphatidylethanolamine, and more than 85% of the fatty acids at the sn-1 positions of these phosphatides consisted of 16:0, 18:2, and 18:3. The sn-2 positions of phosphatidylcholine and phosphatidylethanolamine contained approximately 98% unsaturated fatty acids. In the phosphoglycerides of both regions of the fungus, 18:2 and 18:3 constituted greater than 85% of the total fatty acids. Although the mycelium and sporangiophores of P. blakesleeanus had different morphological and physiological characteristics, the major glycerolipids of the two regions had similar stereospecific distributions of fatty acids.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

Role of very low density lipoproteins in the energy metabolism of the rat

The role of very low density lipoproteins (VLDL) in the energy metabolism of conscious, 24-hr fasted rats was studied. VLDL labeled with [2-3H]glycerol and [1-14C]palmitate were infused into the rats, along with [1-13C]palmitate bound to albumin and d-8-glycerol, and various metabolic factors were assessed. The rates of appearance in plasma of fatty acids in VLDL and albumin-bound free fatty acids (FFA) were about equal, on a molar basis, and only a small fraction of the FFA flux was derived from VLDL. The rate of direct oxidation of the fatty acids from VLDL was 4.4 +/- 0.9 mumol of FA/kg X min, as compared with the value of 4.0 +/- 0.42 mumol of FA/kg X min for plasma FFA. Four percent of the plasma glycerol flux was derived from VLDL. Thus, the direct oxidation of fatty acids in VLDL played an important role in the energy metabolism of the rats, accounting for a percentage of the total CO2 production that was equal to the amount that arose from the oxidation of plasma FFA. The oxidation of VLDL-fatty acids did not involve prior entry of the fatty acids into the plasma FFA pool to any significant extent.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Functional properties of EL-4 tumor cells with lipid-altered membranes.

The effect of fatty acid substitution of membrane phosphatides on a number of surface-mediated phenomena in EL-4 cells was examined. Tumor cells were grown in the presence of fatty acids that could be expected, on the basis of their physical properties, either to stiffen or fluidize the plasma membrane. Substitution of EL-4 cell membrane phosphatides with as much as 74% nonadecanoic acid (19:0) had no effect on either conjugation with effector cells or subsequent cytolysis by the effector cells. Substitution with linolenic acid (18:3) or elaidic acid (18:1trans) likewise affected neither conjugation nor cytolysis. Substitution with these fatty acids also had no effect on the susceptibility of EL-4 cells to cytolysis by antibody plus complement. On the other hand, the rate of patching of H-2 surface antigens was very sensitive to substitution by both 19:0 and 18:3. Although not conclusive, these results suggest that alterations of the fluid state of the membrane that affect lateral movements of surface proteins may not affect cytolytic processes.     

Phosphatidyglycerol in rat lung. II. Comparison of occurrence, composition, and metabolism in surfactant and residual lung fractions.

A comparison of the occurrence, fatty acid composition, and metabolism of phosphatidyglycerol and phosphatidylcholine in the surfactant and residual fraction of rat lung has been carried out. The surfactant and residual fractions were separated by discontinuous sucrose density gradient centrifugation. The surfactant fraction was found to contain 69 percent phosphatidylcholine and 7 percent phosphatidylglycerol. The residual fraction contained 46 percent phosphatidylcholine and 3 percent phosphatidylglycerol. Phosphatidylcholine and phosphatidylglycerol were found to contain 85 and 79 percent palmitate in the surfactant fraction and 67 and 68 percent in the residual fraction, respectively. Isolated rat lungs were perfused with medium containing [U-14C]glucose, [9,10-3H]palmitate, and [1-14C]acetate and the incorporation into palmitate isolated from the alpha and beta position of phosphatidylcholine and phosphatidylglycerol was determined. Each radioactive substrate was found to be incorporated into palmitate of phosphatidylcholine equally at the alpha and beta position of the surfactant fraction. In the residual fraction the specific activity of the beta position palmitate was found to be twice that of the alpha position. The incorporation of [9,10-3H]palmitate and [1-14C]acetate into palmitate at the alpha and beta positions of phosphatidylglycerol was similar in both the surfactant and residual fractions. In each case palmitate at the alpha position had approximately twice the specific activity of that at the beta position. The incorporation of [U-14C]glucose into phosphatidylglycerol of the surfactant fraction was, however, greater in palmitate at the beta position than at the alpha. The results show that phosphatidylglycerol is associated with the lung surfactant fraction and suggest that palmitate esterified to the alpha and beta positions of phosphatidylglycerol and phosphatidylcholine occurs at different rates and is dependent upon the precursor source of palmitate.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Effects of culture age and hexoses on fatty acid oxidation by Leishmania major

The effect of culture age on the rate of oxidation of short-, medium, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10-14C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-14C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-14C]palmitate and of [1-14C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in beta-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

Palmitate and oleate metabolism of rainbow trout in vivo

The turnover rates of palmitate and oleate were measured in vivo by continuous infusion of 1-[14C]palmitate and 9,10-[3H]oleate in rainbow trout. Our goals were: (1) to quantify the incorporation of a saturated and of a monounsaturated fatty acid into other classes of plasma lipids (neutral lipids, NL, and phospholipids, PL); and (2) to determine whether they could both be used as tracers to quantify fluxes of total non-esterified fatty acids (NEFA). We found that both acids play very different physiological roles because palmitate is preferentially channeled towards plasma PL, whereas oleate is mainly incorporated in circulating NL. Consequently, palmitate is predominantly involved in membrane PL turnover and oleate in the metabolism of circulating NL that may be used to shuttle oxidative fuel in teleosts. Despite this striking difference in their metabolism, palmitate and oleate have flux rates that are proportional to their relative abundance in plasma NEFA (i.e. they have the same fractional turnover rate). They can therefore both be used as reliable tracers to quantify the kinetics of total NEFA.     

Effects of individual fatty acids on glucose uptake and glycogen synthesis in soleus muscle in vitro

Soleus muscle strips from Wistar rats were preincubated with palmitate in vitro before the determination of insulin-mediated glucose metabolism in fatty acid-free medium. Palmitate decreased insulin-stimulated glycogen synthesis to 51% of control in a time- (0-6 h) and concentration-dependent (0-2 mM) manner. Basal and insulin-stimulated glucose transport/phosphorylation also decreased with time, but the decrease occurred after the effect on glycogen synthesis. Preincubation with 1 mM palmitate, oleate, linoleate, or linolenate for 4 h impaired glycogen synthesis stimulated with a submaximal physiological insulin concentration (300 microU/ml) to 50-60% of the control response, and this reduction was associated with impaired insulin-stimulated phosphorylation of protein kinase B (PKB). Preincubation with different fatty acids (all 1 mM for 4 h) had varying effects on insulin-stimulated glucose transport/phosphorylation, which was decreased by oleate and linoleate, whereas palmitate and linolenate had little effect. Across groups, the rates of glucose transport/phosphorylation correlated with the intramuscular long-chain acyl-CoA content. The similar effects of individual fatty acids on glycogen synthesis but different effects on insulin-stimulated glucose transport/phosphorylation provide evidence that lipids may interact with these two pathways via different mechanisms.