Modification by clofibric acid of acyl composition of glycerolipids in rat liver. Possible involvement of fatty acid chain elongation and desaturation

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) to rats induced a marked change in acyl composition of hepatic glycerolipids; a considerable increase in the proportion of octadecenoic acid (18:1) was accompanied by a marked decrease in the proportion of octadecadienoic acid (18:2). Among the glycerolipids, the changes in the proportions of 18:1 and 18:2 were the most marked in phosphatidylcholine. The change in the acyl composition of phosphatidylcholine paralleled the change in free fatty acid composition in microsomes. The treatment of rats with clofibric acid resulted in a 2.3-fold increase in activity of microsomal palmitoyl-CoA chain elongation and a 4.8-fold increase in activity of stearoyl-CoA desaturation. The activities of acyl-CoA synthetase, 1-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase in hepatic microsomes were increased approx. 3-, 1.7- and 3.6-times, respectively, by the treatment of rats with clofibric acid. These findings are discussed with respect to the role of fatty acid modification systems in the regulation of acyl composition of phosphatidylcholine.     

Alterations in fatty acyl composition can selectively affect amino acid transport in Saccharomyces cerevisiae

The fatty acid composition of yeast lipid was manipulated by using auxotrophic strain of S.cerevisiae, KD115, which requires unsaturated fatty acid (UFA) for its growth. It was possible to specifically enrich the yeast with different fatty acyl residues. As compared to wild type strain (S288C), the uptake of amino acids viz., L-alanine, glycine, L-glutamic acid, L-valine in KD115 was drastically reduced, however, the uptake of L-leucine and L-lysine was not affected by the change in lipid unsaturation. Kinetic studies revealed that KT and Jmax values for L-alanine were altered whereas for L-lysine they remained unaffected by UFA modification. Furthermore, unsaturation index for wild type cells was found to be fairly constant while it was variable in KD115 supplemented with different UFAs. It is observed that the variation in amino acid permeases activity which was affected by fluctuations in fatty acyl composition corresponds more to degree of unsaturation rather than growth stage of KD115.     

Immunosuppression induced by attenuated Salmonella. Evidence for mediation by macrophage precursors

An attenuated aro A- strain of Salmonella typhimurium, SL3235, was previously shown to afford excellent protection in C3HeB/FeJ mice against challenge by virulent Salmonella and cross-protection against Listeria monocytogenes. In the present study, the immunologic status of immunized mice was evaluated by studying their ability to mount in vivo and in vitro plaque-forming cell responses to SRBC. SL3235-immunized mice exhibited marked suppression in their ability to generate anti-SRBC plaque-forming cell responses. Suppression was active and mediated by soluble factors as demonstrated by the capacity of immune cells to suppress the responses of normal cells in co-culture and across a membrane in transwell plates. Depletion of T cells from immune spleens did not alleviate the suppressive activity of the remaining cells. Depletion of splenic adherent cells resulted in partial alleviation of suppressive activity, demonstrating that mature macrophages are partly responsible for mediating the observed suppression. A sequential multi-step depletion procedure resulted in marked enrichment of a second suppressor cell population that was Mac1+, Thy-1.2-, surface Ig-, J11d-, non-adherent, non-phagocytic, and esterase negative. When cultured in vitro in the presence of L cell-conditioned medium, but not in the presence of Con A supernatant, these cells matured into typical macrophages within 72 h of culture. The cell population enriched for macrophage precursors (75%) retained the suppressive capacity of unfractionated splenocytes. Our data indicate that, in addition to the well-described involvement of mature macrophages in suppressing immune responses, bacterial infection may induce appearance of macrophage precursors that may also play an important regulatory role in the immune system.     

Evidence against calcium as a mediator of mitochondrial dysfunction during apoptosis induced by arachidonic acid and other free fatty acids

Apoptosis is often accompanied by activation of phospholipase A(2), causing release of free fatty acids (FFAs), which in turn are thought to contribute to the loss of mitochondrial transmembrane potential (Deltapsi(m)). In these experiments, we asked whether calcium plays a role as an intermediate in this process. A total of 14 FFAs were compared for their ability to cause loss of Deltapsi(m) and for their ability to affect levels of intracellular calcium. Among the FFAs, unsaturated FFAs tended to induce apoptosis while saturated FFAs did not. Arachidonic acid (AA) was most damaging, causing loss of Deltapsi(m) and cell death in 8-10 h while linoleic acid, gamma-linolenic acid, and docosapentaenoic also strongly induced apoptosis. Effects of the FFAs on levels of intracellular calcium were very different. Many caused strong calcium responses; however, the ability to induce a strong calcium response was not predictive of ability to induce apoptosis, and overall, we did not find a correlation between apoptosis and calcium induction. Also, verapamil and TMB-8 were able to block the calcium response, but these inhibitors did not prevent loss of Deltapsi(m), indicating that the calcium response is not necessary for FFA-induced loss of Deltapsi(m). In contrast, we found that cyclosporine A could inhibit the AA-induced loss of Deltapsi(m) with both whole cells and isolated mitochondria, confirming that the antimitochondrial effects of FFA can stem from direct effects on the mitochondrial permeability transition pore. Finally, we show that the strong apoptosis-inducing activity of AA may stem from its ability to selectively induce its own release.     

Essential fatty acid for the mosquito Culex pipiens: arachidonic acid.

A nutrient associated with animal-derived phospholipids has previously been found essential for newly-emerged adults of the mosquito Culex pipiens to fly and survive more than a few days. Pure arachidonic acid was completely effective in supporting the emergence of viable flying adults; in combination with synthetic dipalmitoyl lecithin, which slightly improves larval growth rate without inducing adult flight, it wholly adequately replaces animal phospholipids. Linoleic and linolenic acids, which have satisfied the needs of all insects hitherto shown to require an essential fatty acid, were ineffective for C. pipiens, with or without synthetic lecithin. An optimal effect on adult flight was obtained with 0.05 mg of arachidonic acid per 100 ml of dietary medium, a concentration much lower than the linoleic/linolenic concentrations needed by other insects with an essential fatty acid requirement. The relationship of this unique mosquito fatty acid requirement to the essential fatty acid needs of both vertebrates and insects in general is discussed.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with Mycobacterium bovis (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE2. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE2. The major metabolites of resident cells were PGE2 and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

Fatty acyl esters potentiate fatty acid induced activation of protein kinase C

Purified rat pancreas protein kinase C (PKC) is activated by unsaturated free fatty acids (oleic and arachidonic). The ethyl esters of these fatty acids are ineffective as enzyme activators. However, when the ethyl esters are added in combination with a free fatty acid, there is significant enhancement of enzyme activation. Nearly optimal PKC activation was obtained when non-activating ethyl oleate or ethyl arachidonate was added to sub-optimally activating concentrations of oleic or arachidonic acids. In addition to the ethyl esters, 1-monooleylglycerol also had a potentiating effect on PKC activation by oleic acid. However, the degree of activation observed in the presence of a free fatty acid and an acyl ester of the fatty acid quantitatively never surpassed that produced by sn-1,2-dioleylglycerol. Our findings indicate that significant PKC activation can be achieved by presenting the enzyme with an environment which we believe approximates the structural characteristics of the endogenous activator, sn-1,2-diacylglycerol.     

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.     

Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing

In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.     

Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling.

Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a coated-pit pathway by this HA receptor. We conclude that liver endothelial cell HA receptors are recycled during the continuous endocytosis and processing of HA.     

Role of arachidonic acid in platelet aggregation induced by S. typhimurium endotoxin

The platelet aggregation reaction was used to assess the influence of arachidonic acid (AA), endotoxin (E) S. typhimurium and ADP on platelet aggregation properties. All the three substances induced platelet aggregation. A higher degree of aggregation was attained by the application of E combined with AA and ADP as compared with the effects produced by E and ADP alone. Prolonged incubation of platelet-rich plasma (PRP) samples with E led to an essential decrease of the aggregation degree on ADP addition. Incubation of PRP samples with E and ADP did not evoke any analogous decrease in the platelet aggregation degree. The data obtained indicate that AA stimulates platelet aggregation induced by E and ADP.     

The effect of fatty acid exposure on the biosynthesis of glycerolipids by cultured hepatocytes

Incubation of hepatocyte monolayers with oleate or palmitate (1.0 mM) for 2-48 h, increased (20 to 80%) the incorporation of [1,3-14C]glycerol and palmitate into triacyglycerol but not phosphatidylcholine. The effect of fatty acids on liver cell triacylglycerol formation correlated well (r = 0.990) with a simultaneous rise (2-4-fold) in phosphatidate phosphatase (EC 3.1.3.4) activity. Phosphatidate phosphatase activity and triacylglycerol biosynthesis are also increased (2-fold) after hepatocyte monolayers are incubated for 24 h with cyclic GMP in the absence of fatty acids. Fatty acid-dependent increases in liver cell triacylglycerol formation and phosphatidate phosphatase activity are not blocked by cycloheximide. Phosphatidylcholine biosynthesis was also elevated in homogenates of liver cells exposed (24-48 h) to 1.0 mM oleate when exogenous CDPcholine was added to the incubation mixture. Apparently, the phosphatidate phosphatase-dependent rise in diacylglycerols that occurs after fatty acid exposure is primarily shunted into triacylglycerols because liver cell CDPcholine content is not correspondingly increased, and high levels of diacylglycerol acyltransferase (EC 2.3.1.20) and fatty acyl-CoA derivatives are present.     

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis

In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.