Fatty acid incorporation is decreased in astrocytes cultured from alpha-synuclein gene-ablated mice

Because alpha-synuclein may function as a fatty acid binding protein, we measured fatty acid incorporation into astrocytes isolated from wild-type and alpha-synuclein gene-ablated mice. alpha-Synuclein deficiency decreased palmitic acid (16:0) incorporation 31% and arachidonic acid [20:4 (n-6)] incorporation 39%, whereas 22:6 (n-3) incorporation was unaffected. In neutral lipids, fatty acid targeting of 20:4 (n-6) and 22:6 (n-3) (docosahexaenoic acid) to the neutral lipid fraction was increased 1.7-fold and 1.6-fold, respectively, with an increase in each of the major neutral lipids. This was consistent with a 3.4- to 3.8-fold increase in cholesteryl ester and triacylglycerol mass. In the phospholipid fraction, alpha-synuclein deficiency decreased 16:0 esterification 39% and 20:4 (n-6) esterification 43% and decreased the distribution of these fatty acids, including 22:6 (n-3), into this lipid pool. alpha-Synuclein gene-ablation significantly decreased the trafficking of these fatty acids to phosphatidylinositol. This observation is consistent with changes in phospholipid fatty acid composition in the alpha-synuclein-deficient astrocytes, including decreased 22:6 (n-3) content in the four major phospholipid classes. In summary, these studies demonstrate that alpha-synuclein deficiency significantly disrupted astrocyte fatty acid uptake and trafficking, with a marked increase in fatty acid trafficking to cholesteryl esters and triacylglycerols and decreased trafficking to phospholipids, including phosphatidylinositol.     

Brain arachidonic acid incorporation is decreased in heart fatty acid binding protein gene-ablated mice

Heart fatty acid binding protein (H-FABP) is expressed in neurons, but its role in brain fatty acid incorporation and metabolism is poorly defined. We examined the effect of H-FABP gene ablation on brain incorporation of arachidonic ([1-(14)C]20:4n-6) or palmitic ([1-(14)C]16:0) acid in vivo. Analysis of brain mRNA confirmed gene ablation and demonstrated no compensatory changes in the levels of other FABP mRNA in the gene-ablated mice. In brains from H-FABP gene-ablated mice, the incorporation coefficient for [1-(14)C]20:4n-6 was reduced 24%, while that for [1-(14)C]16:0 was unaffected. Within the organic and aqueous fractions, significantly more [1-(14)C]20:4n-6 was distributed into the aqueous fraction, suggesting a disruption in the metabolic targeting of 20:4n-6 in these mice. There was less incorporation of [1-(14)C]20:4n-6 into total phospholipids and a marked reduction (51%) in the level of incorporation into the choline glycerophospholipids (ChoGpl). Because FABP can influence steady-state lipid mass, brain individual lipid masses were measured. The brain total phospholipid mass was reduced 17% by gene ablation, ascribed to a 27% and 32% reduction in the masses of ChoGpl and sphingomyelin, respectively. Plasmalogen subclass masses were also reduced, suggesting that H-FABP may augment brain plasmalogen synthesis. In gene-ablated mice, the phosphatidylinositol 20:4n-6 level was reduced 25%, while the proportion of total n-6 fatty acids was reduced in the major phospholipid classes. Thus, these results demonstrate for the first time that H-FABP expression influences brain 20:4n-6 uptake and trafficking as well as steady-state brain lipid levels.     

Heart fatty acid uptake is decreased in heart fatty acid-binding protein gene-ablated mice

Cell culture systems have demonstrated a role for cytoplasmic fatty acid-binding proteins (FABP) in lipid metabolism, although a similar function in intact animals is unknown. We addressed this issue using heart fatty acid-binding protein (H-FABP) gene-ablated mice. H-FABP gene ablation reduced total heart fatty acid uptake 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6 compared with controls, respectively. Similarly, the amount of fatty acid found in the aqueous fraction was reduced 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6, respectively. Less [1-(14)C]16:0 entered the triacylglycerol pool, with significant redistribution of fatty acid between the triacylglycerol pool and the total phospholipid pool. Less [1-(14)C]20:4n-6 entered each lipid pool measured, but these changes did not alter the distribution of tracer among these pools. In gene-ablated mice, significantly more [1-(14)C]16:0 was targeted to choline and ethanolamine glycerophospholipids, whereas more [1-(14)C]20:4n-6 was targeted to the phosphatidylinositol (PtdIns) pool. H-FABP gene ablation significantly increased PtdIns mass 1.4-fold but reduced PtdIns 20:4n-6 mass 30%. Consistent with a reported effect of FABP on plasmalogen mass, ethanolamine plasmalogen mass was reduced 30% in gene-ablated mice. Further, 20:4n-6 mass was reduced in each of the three other major phospholipid classes, suggesting H-FABP has a role in maintaining steady-state 20:4n-6 mass in heart. In summary, H-FABP was important for heart fatty acid uptake and targeting of fatty acids to specific heart lipid pools as well as for maintenance of phospholipid pool mass and acyl chain composition.     

The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response

α-Synuclein (Snca) is an abundant small cytosolic protein (140 amino acids) that is expressed in the brain, although its physiological role is poorly defined. Consistent with its ubiquitous distribution in the brain, we and others have established a role for Snca in brain lipid metabolism and downstream events such as neuroinflammation. In astrocytes, Snca is important for fatty acid uptake and trafficking, where its deletion decreases 16:0 and 20:4n-6 uptake and alters targeting to specific lipid pools. Although Snca has no impact on 22:6n-3 uptake into astrocytes, it is important for its targeting to lipid pools. Similar results for fatty acid uptake from the plasma are seen in studies using whole mice coupled with steady-state kinetic modeling. We demonstrate in gene-ablated mice a significant reduction in the incorporation rate of 20:4n-6 into brain phospholipid pools due to reduced recycling of 20:4n-6 through the ER-localized long-chain acyl-CoA synthetases (Acsl). This reduction results in a compensatory increase in the incorporation rate of 22:6n-3 into brain phospholipids. Snca is also important for brain and astrocyte cholesterol metabolism, where its deletion results in an elevation of cholesterol and cholesteryl esters. This increase may be due to the interaction of Snca with membrane-bound enzymes involved in lipid metabolism such as Acsl. Snca is critical in modulating brain prostanoid formation and microglial activities. In the absence of Snca, microglia are basally activated and demonstrate increased proinflammatory cytokine secretion. Thus, Snca, through its modulation of brain lipid metabolism, has a critical role in brain inflammatory responses.     

Acyl-CoA synthetase activity links wild-type but not mutant alpha-synuclein to brain arachidonate metabolism

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wild-type mouse or human alpha-synuclein, but not by A30P, E46K, and A53T forms of alpha-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools were markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that alpha-synuclein bound free 20:4n-6 (Kd = 3.7 microM) but did not bind 20:4n-6-CoA. These data suggest alpha-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that alpha-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity, although mutant forms of alpha-synuclein fail to restore this activity.     

Dynamics of Docosahexaenoic Acid Metabolism in the Central Nervous System: Lack of Effect of Chronic Lithium Treatment

Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h^–1, respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 mol.g^–1), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A₂ activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithium's action is an arachidonic acid-selective phospholipase A₂.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

Alpha-synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unknown. In the present study, we examined the effect of Snca gene ablation on brain 22:6n-3 metabolism. We determined 22:6n-3 uptake and incorporation into brain phospholipids by infusing awake, wild-type and Snca-/- mice with [1-14C]22:6n-3 using steady-state kinetic modeling. In addition, because Snca modulates 20:4n-6-CoA formation, we assessed microsomal Acsl activity using 22:6n-3 as a substrate. Although Snca gene ablation does not affect brain 22:6n-3 uptake, brain 22:6n-3-CoA mass was elevated 1.5-fold in the absence of Snca. This is consistent with the 1.6- to 2.2-fold increase in the incorporation rate and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n-3-CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n-3, Kd = 1.0 +/- 0.5 micromol/L, it did not bind 22:6n-3-CoA. These effects of Snca gene deletion on 22:6n-3 brain metabolism are opposite to what we reported previously for brain 20:4n-6 metabolism and are likely compensatory for the decreased 20:4n-6 metabolism in brains of Snca-/- mice.     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

Essential fatty acid uptake and esterification in primary culture of rat hepatocytes

Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.     

Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vivo.

The in vivo effects of ethanol on lipid synthesis in Escherichia coli have been examined. Under conditions which uncoupled fatty acid synthesis from phospholipid synthesis, ethanol decreased the amount of saturated fatty acids synthesized but had little effect on the selectivity of their incorporation into phospholipids. In the absence of fatty acid degradation and unsaturated fatty acid synthesis, E. coli was still able to adapt its membrane lipids to ethanol, while the inhibition of total fatty acid synthesis eliminated this response. During growth in the presence of ethanol, strain K1060 (an unsaturated fatty acid auxotroph) incorporated an increased amount of exogenous heptadecanoic acid (17:0) to compensate for the reduction in palmitic acid (16:0) available from biosynthesis. Thus, our results indicate that the reduced levels of saturated fatty acids observed in the phospholipids of E. coli following growth in the presence of ethanol result primarily from a decrease in the amounts of saturated fatty acids available for phospholipid synthesis.     

Somatostatin effects on the cyclic AMP system and lipid metabolism in mouse brain

The effect of somatostatin on cyclic AMP-protein kinase system and lipid metabolism was studied in mouse brain. Subcutaneous injection of the peptide decreased the cyclic AMP and cyclic GMP levels (70% and 60% respectively) as well as protein kinase and triglyceride lipase activities (30%). Cyclic AMP binding protein activity was not affected. Experiments carried out with [¹⁴C]acetate as precursor of lipids seem to indicate that somatostatin blocks the fatty acid turnover. On the other hand, the general decrease of³²P incorporation into all phospholipids by somatostatin suggests that the peptide interferes with the precursor uptake into phospholipids. The findings reported here indicate that somatostatin has a role on brain metabolism and further add more data in support for its neuromodulating action.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.     

In Vivo Fatty Acid Incorporation into Brain Phospholipids in Relation to Signal Transduction and Membrane Remodeling

A method and model are described to quantify in vivo turnover rates and half-lives of fatty acids within brain phospholipids. These kinetic parameters can be calculated by operational equations from measured rates of incorporation of intravenously injected fatty acid radiotracers into brain phospholipids. To do this, it is necessary to determine a dilution factor , which estimates the contribution to the brain precursor acyl-CoA pool of fatty acids released from phospholipids through the action of PLA₁, or PLA₂. Some calculated fatty acid half-lives are minutes to hours, consistent with active participation of phospholipids in brain function and structure. The fatty acid method can be used to identify enzyme targets of drugs acting on phospholipid metabolism. For example, a reduced brain turnover of arachidonate by chronic lithium, demonstrated in rats by the fatty acid method, suggests that this agent, which is used to treat bipolar disorder, has for its target an arachidonate-specific PLA₂. In another context, when combined with in vivo imaging by quantitative autoradiography in rodents or positron emission tomography in macaques or humans, the fatty acid method can localize and quantify normal and modified PLA₂-mediated signal transduction in brain.     

The relationship between valinomycin-induced alterations in membrane phospholipid fatty acid turnover, membrane potential, and cell volume in the human erythrocyte

The relationship between alterations in transmembrane potential, cell volume, and phospholipid fatty acid turnover has been examined in human erythrocytes by treating the cells with the monovalent cation ionophore valinomycin. Valinomycin increases the cellular uptake of tetra[3H]phenylphosphonium ion by erythrocytes, indicating membrane hyperpolarization, and causes net loss of potassium chloride and water from the cells leading to a decrease in cell volume. Treatment of erythrocytes with valinomycin also enhances incorporation of [9, 10-(3)H]oleic acid into phospholipids, primarily diacylphosphatidylethanolamine. After replacing intracellular chloride with sulfate and treating cells with the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate, exposure to valinomycin results in uptake of tetra[3H]phenylphosphonium ion and stimulation of [9, 10-(3)H]oleic acid incorporation, but, because anion efflux is prevented, no decrease in cell volume occurs. When tetra[3H]phenylphosphonium ion uptake is also prevented by suspending these cells in 125 mM KCl to dissipate the transmembrane potassium gradient, valinomycin still enhances [9, 10-(3)H] oleic acid incorporation into phospholipid. These results suggest that the presence of valinomycin in the membrane directly alters phospholipid fatty acid turnover and that some of the effects of this ionophore on cellular function previously attributed to alterations in transmembrane potential or cellular potassium content may instead be due to altered phospholipid turnover. Since it is possible that valinomycin may directly perturb phospholipid fatty acid turnover in other cells, the possibility that valinomycin-induced alterations in cellular function are due to altered phospholipid turnover rather than membrane hyperpolarization or altered potassium content should be considered in the interpretation of studies employing this ionophore.     

Metabolism of palmitate in perfused rat liver. Effect of low and high ethanol concentrations at various concentrations of palmitate in the perfusion medium

1. The effect of ethanol on the metabolism of [1-(14)C]palmitate in rat liver was investigated in a single-pass perfusion system at concentrations of 10mm- or 80mm-ethanol and 0.2mm- or 1mm-palmitate. 2. After the perfusion the hepatic lipid was isolated in subcellular fractions. The two major fractions contained triacylglycerol from cytoplasmic lipid droplets and from endoplasmic reticulum plus Golgi apparatus respectively. 3. In experiments with 0.2mm-palmitate perfusion with 10mm- or 80mm-ethanol did not measurably increase the esterification, and the oxidation was markedly decreased and the fatty acid uptake was not affected. 4. Perfusion with ethanol, at 1mm-palmitate, increased the fatty acid uptake, increased esterification and decreased oxidation. The effects of 10mm- and 80mm-ethanol were similar. The incorporation of [1-(14)C]palmitate into triacylglycerol in cytoplasmic lipid droplets was not affected statistically significantly by ethanol. Ethanol increased the incorporation of [1-(14)C]palmitate into di- and tri-acylglycerol in the membranous fraction. Estimated chemically, the contents of di- and tri-acylglycerol were only slightly affected by ethanol. These results suggest that the effect of ethanol was to increase the turnover of fatty acids in triacylglycerol rather than to increase its accumulation. 5. The results indicate that an increased concentration of fatty acids is more important for the formation of acute fatty liver in fed rats than are the direct effects of ethanol on hepatic fatty acid metabolism.     

Identification of Two Molecular Species of Rat Brain Phosphatidylcholine that Rapidly Incorporate and Turn Over Arachidonic Acid In Vivo

Abstract: In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [³H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2–10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [³H]arachidonate (>87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn -2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn -1 position. However, 10–15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn -2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn -1 position. Analysis demonstrated that tracer was present in both the 16:1–20:4 and 18:2–20:4 PtdCho species at specific activities 10–40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of <10 min in 16:1–20:4 and 18:2–20:4 PtdCho and 1–3 h in 16:0–20:4, 18:0–20:4, and 18:1–20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1–20:4 and 18:2–20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.     

Developmental changes in fatty acid biosynthesis and composition in the house cricket,Acheta domesticus

Incorporation of [1-¹⁴C] acetate into various phospholipid and triacylglycerol fatty acids showed cyclic fluctuations in fatty acid biosynthesis that were similar for all of the major fatty acids in both male and female house crickets, Acheta domesticus, during development. All three stadia showed low levels of biosynthesis near ecdysis followed by increased synthesis to a peak at midstadium. In the phospholipid fraction, the incorporation of newly synthesized saturated fatty acids, 16:0 and 18:0, predominated near ecdysis, while at midstadium linoleic acid was the most actively synthesized fatty acid. In the triacylglycerol fraction, 18:0 and 18:1 predominated throughout the entire stadium. In contrast to the large fluctuations in fatty acid biosynthesis, the fatty acid compositions of the phospholipid and triacylglycerol fractions did not change within a stadium. However, significant differences were demonstrated between the stages and were associated primarily with differences between nymphal and adult stadia. Males and females differed in the proportions of 16:0 and 18:2 incorporated into phospholipids with females showing a greater proportion of 18:2 and a corresponding smaller proportion of 16:0 than males. The greater proportion of linoleic acid in females and in adults in general compared to nymphs and the predominance of the incorporation of newly synthesized linoleic acid into the phospholipid fraction of all stadia are consistent with the importance of this fatty acid in a number of biological roles.