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.     

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.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

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.     

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.     

Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system

In previous studies it was shown that hepatocellular uptake of fatty acids is mediated by a specific fatty acid binding membrane protein. To determine now directly the driving forces for their entry into hepatocytes, the uptake of a representative long chain fatty acid, [3H]oleate, by basolateral rat liver plasma membrane vesicles was examined. Influx of oleate was stimulated by increasing the Na+ concentration of the medium. In the presence of an inwardly directed Na+ gradient (NaSCN, NaNO3, NaCl) oleate was accumulated during the initial uptake phase (20 s) at a concentration of 1.4-1.9-fold that at equilibrium (overshoot). This activation of influx was not observed after replacement of Na+ by Li+, K+, or choline+. Na+-dependent oleate uptake was significantly stimulated by creation of a negative intravesicular potential, either by altering the accompanying anions or by valinomycin-induced K+ diffusion potentials, suggesting an electrogenic transport mechanism. Na+-dependent fatty acid uptake was temperature dependent, with maximal overshoots occurring at 37 degrees C, and revealed saturation kinetics with a Km of 83.1 nM and Vmax of 2.9 nmol X min-1 X mg protein-1. These studies demonstrate that the carrier-mediated hepatocellular uptake of fatty acids represents an active potential-sensitive Na+-fatty acid cotransport system.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

Palmitate movement across red and white muscle membranes of rainbow trout

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.     

The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis

1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-¹⁴C]stearate, [U-¹⁴C]oleate, [U-¹⁴C]palmitate and [1-¹⁴C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4·2mg./min./kg. body wt. for acetate, and 0·18, 0·28 and 0·42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18–21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [¹⁴C]acetate infusion, milk triglyceride fatty acids of chain length C₄–C₁₄ showed specific radioactivities that were 75–90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C₄–C₁₄ arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.     

Differences in partitioning of meal fatty acids into blood lipid fractions: a comparison of linoleate, oleate, and palmitate

There has been much interest in the health effects of dietary fat, but few studies have comprehensively compared the acute metabolic fate of specific fatty acids in vivo. We hypothesized that different classes of fatty acids would be variably partitioned in metabolic pathways and that this would become evident over 24 h. We traced the fate of fatty acids using equal amounts of [U-(13)C]linoleate, [U-(13)C]oleate, and [U-(13)C]palmitate given in a test breakfast meal in 12 healthy subjects. There was a tendency for differences in the concentrations of the tracers in plasma chylomicron-triacylglycerol (TG) (oleate > palmitate > linoleate). This pattern remained in plasma nonesterified fatty acid (NEFA) and very low-density lipoprotein (VLDL)-TG (P 相似文献   

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

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.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

The physical-chemical basis for sex-related differences in uptake of fatty acids by the liver

The uptake of fatty acids by the liver was shown previously to be a non-catalyzed process, and rates of uptake were correlated to the affinity of the plasma membranes of liver cells for fatty acids. The experiments in this paper were designed to test whether the known differences in uptake and metabolism of free fatty acids by the livers of male and female rats could be understood based on differences in the affinities of the corresponding plasma membranes for these substrates. The relative affinities for palmitate and oleate of 'male' plasma membranes were found to be lower versus 'female' membranes. Measurements of uptake of palmitate from albumin-palmitate complexes by 'male' and 'female' perfused livers showed higher uptake rates by the latter when correlated with the concentration of the complex. However, the rates of uptake were identical when the concentrations of the fatty acid in the plasma membranes of male and female liver cells were the same.