Cellular Glycerolipids Research Articles

Remodeling of Leaf Cellular Glycerolipid Composition under Drought and Re-hydration Conditions in Grasses from the Lolium-Festuca Complex.

Drought tolerant plant genotypes are able to maintain stability and integrity of cellular membranes in unfavorable conditions, and to regenerate damaged membranes after stress cessation. The profiling of cellular glycerolipids during drought stress performed on model species such as Arabidopsis thaliana does not fully cover the picture of lipidome in monocots, including grasses. Herein, two closely related introgression genotypes of Lolium multiflorum (Italian ryegrass) × Festuca arundinacea (tall fescue) were used as a model for other grass species to describe lipid rearrangements during drought and re-hydration. The genotypes differed in their level of photosynthetic capacity during drought, and in their capacity for membrane regeneration after stress cessation. A total of 120 lipids, comprising the classes of monogalactosyldiacyloglycerol, digalactosyldiacyloglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, diacylglicerol, and triacylglicerol, were analyzed. The results clearly showed that water deficit had a significant impact on lipid metabolism in studied forage grasses. It was revealed that structural and metabolic lipid species changed their abundance during drought and re-watering periods and some crucial genotype-dependent differences were also observed. The introgression genotype characterized by an ability to regenerate membranes after re-hydration demonstrated a higher accumulation level of most chloroplast and numerous extra-chloroplast membrane lipid species at the beginning of drought. Furthermore, this genotype also revealed a significant reduction in the accumulation of most chloroplast lipids after re-hydration, compared with the other introgression genotype without the capacity for membrane regeneration. The potential influence of observed lipidomic alterations on a cellular membrane stability and photosynthetic capacity, are discussed.HIGHLIGHTS A higher drought tolerance of grasses could be associated with an earlier lipidome response to a stress signal and with a membrane regeneration after stress cessation accompanied by a turnover of chloroplast lipids

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.)

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC–MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.

The stearoyl-acyl-carrier-protein desaturase promoter (Des) from oil palm confers fruit-specific GUS expression in transgenic tomato

The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5′ deletions of the Des promoter (Des1–4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between −590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

The stearoyl–acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven single-nucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.

Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.

Progress in the study on mammalian diacylgycerol acyltransgerase (DGAT) gene and its biological function

Diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) is a microsomal enzyme that plays a central role in the metabolism of cellular glycerolipids. DGAT catalyzes the final step in triacylglycerol (TAG) biosynthesis by converting diacylgycerol (DAG) and fatty acyl-coenzyme A (CoA) into triacylglycero1. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. Therefore, DGAT is not only an key factor for control triglycerides and fatty acids, but also may play a key modulatory role in animal fat deposition.

The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves.

The role of phosphatidylcholine (PC) in fatty acid exchange and desaturation was examined and compared with that of monogalactosyldiacylglycerol (MGDG) in Brassica napus leaves using (14)C-labelling in vivo. Data are presented which indicate that in the chloroplast newly formed saturated (palmitic acid, 16:0) and monounsaturated (oleic acid, 18:1) fatty acid is incorporated into MGDG and desaturated in situ. In the non-plastidic compartments, however, newly formed fatty acid is exchanged with polyunsaturated fatty acid in PC, the probable major site of subsequent desaturation. The unsaturated fatty acid is released to the acyl-CoA pool, which is then used to synthesize diacylglycerol (DAG) containing a high level of unsaturated fatty acid. This highly unsaturated DAG may be the source for the biosynthesis of other cellular glycerolipids. The generally accepted pathway in which PC is synthesized from molecular species of DAG containing 16:0 and 18:1 followed by desaturation of the 18:1 to linoleic (18:2) and linolenic (18:3) acids is questioned.

The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves

The role of phosphatidylcholine (PC) in fatty acid exchange and desaturation was examined and compared with that of monogalactosyldiacylglycerol (MGDG) in Brassica napus leaves using 14C-labelling in vivo. Data are presented which indicate that in the chloroplast newly formed saturated (palmitic acid, 16:0) and monounsaturated (oleic acid, 18:1) fatty acid is incorporated into MGDG and desaturated in situ. In the non-plastidic compartments, however, newly formed fatty acid is exchanged with polyunsaturated fatty acid in PC, the probable major site of subsequent desaturation. The unsaturated fatty acid is released to the acyl-CoA pool, which is then used to synthesize diacylglycerol (DAG) containing a high level of unsaturated fatty acid. This highly unsaturated DAG may be the source for the biosynthesis of other cellular glycerolipids. The generally accepted pathway in which PC is synthesized from molecular species of DAG containing 16:0 and 18:1 followed by desaturation of the 18:1 to linoleic (18:2) and linolenic (18:3) acids is questioned.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid.

When rat hepatocytes were cultured for 24 h in the absence of exogenous fatty acid, the amount of very-low-density lipoprotein (VLDL) triacylglycerol (TAG) secreted (114 +/- 14 micrograms/mg of cell protein) could not be accounted for by the mass of TAG lost from the cells (29 +/- 6.1 micrograms/mg of cell protein) during this period (n = 12). Of the balance (85 +/- 14 micrograms/mg; 94 +/- 15 nmol/mg), a maximum of only 37 nmol/mg of cell protein of TAG could be accounted for by fatty acids synthesized de novo. When labelled exogenous oleate (initial concentration, 0.75 nM) was present in the culture medium, the net gain in cellular plus VLDL TAG (253 +/- 38 micrograms/mg of cell protein per 24 h) was greater than that contributed by the exogenous fatty acid (155 +/- 18.2 micrograms/mg of cell protein, n = 5). Again, the balance (98.8 +/- 18.2 micrograms/mg of cell protein per 24 h) was too great to be accounted for by fatty acid synthesis de novo. In experiments in which cellular glycerolipids were prelabelled with [9, 10(n)-3H]oleic acid, following removal of the labelled fatty acid, there was a net increase in labelled cellular plus VLDL TAG over the next 24 h. That cellular phospholipids are the source of a substantial part of the excess TAG synthesized is supported by the following evidence. (1) The loss of prelabelled cellular phospholipid during culture was greater than could be accounted for by secretion into the medium. (2) During culture of cells prelabelled with 1,2-di-[l-14C]palmitoyl phosphatidylcholine, a substantial amount of label was secreted as VLDL TAG. (3) In pulse-chase experiments, the kinetics of labelled phospholipid turnover were consistent with conversion into a non-phospholipid pool. The enzymology involved in the transfer of phospholipid fatty acids into TAG is probably complex, but the present results suggest that this pathway may represent an important route by which extracellular fatty acids are channelled into VLDL TAG.

Metabolism of gammalinolenic acid in human neutrophils.

Gammalinolenic acid (GLA), when provided as a dietary supplement, has been reported to improve clinical symptoms of several inflammatory disorders. The goal of the current study was to examine the metabolism of GLA and its relationship to arachidonic acid (AA) in the human neutrophil. Initial studies indicated that neutrophils provided GLA in vitro rapidly elongate it (by two carbons) to dihomogammalinolenic acid (DGLA). The bulk of this newly formed DGLA is incorporated into neutral lipids and specifically triacylglycerides. Neutrophils from volunteers supplemented with GLA as borage oil also had elevated quantities of DGLA but not GLA, when compared with neutrophils from volunteers not consuming the GLA supplement. To determine whether DGLA could be mobilized from cellular glycerolipids, neutrophils were stimulated with ionophore A23187 and fatty acid levels were determined. DGLA and AA were both released during stimulation, and the quantities of DGLA mobilized increased threefold after in vitro GLA supplementation. Exogenously provided DGLA was converted to one major metabolite during cell stimulation; this product migrated on reverse-phase HPLC with the 15-lipoxygenase product, 15-hydroxy-eicosa-trienoic acid (15-HETre). Both 15-HETre and DGLA (provided exogenously) inhibited the formation of leukotriene B4, (LTB4) and 20-hydroxy-leukotriene B4 (20-OH-LTB4). The IC50 for 15-HETre inhibition of both LTR, and 20-OH-LTB4 in A23187-stimulated neutrophils was 5 microM. This inhibition could be reversed by removing the compounds from the cells. Taken together, these data reveal that there are enzymes within the human neutrophil that metabolize GLA or its elongation product DGLA, and that the metabolism of GLA and AA may interact at a number of critical junctures.

Differential roles for triglyceride and phospholipid pools of arachidonic acid in human lung macrophages.

Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.

Endogenous epoxyeicosatrienoyl-phospholipids. A novel class of cellular glycerolipids containing epoxidized arachidonate moieties.

By chromatographic and mass spectral techniques we document in rat liver the presence of new classes of glycerophospholipids which contain an epoxyeicosatrienoate moiety, esterified to the glycerol-sn-2 position. These novel lipids are formed in vivo from endogenous precursors and under physiological conditions. Chromatographic resolution followed by hydrolysis and regioisomeric analysis showed that they consist of the 8,9-, 11,12-, and 14,15-epoxyeicosatrienoyl derivatives of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Their relative concentrations (micromoles of oxidized lipid/mol of phospholipid) were 70, 85, and 106 for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, respectively. Chiral analysis of the fatty acids at sn-2 revealed an enantioselective preference for 8(S),9(R)-, 11(S),12(R)-, and 14(R),15(S)-epoxyeicosatrienoates in all three lipid classes. The data suggest a multienzyme process initiated by cytochrome P-450 epoxidation of arachidonic acid followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and stereoselective lysolipid acylation. These results provide a molecular basis for a potential physiological role of cytochrome P-450 in the biosynthesis of unique cellular glycerolipids and, consequently, in the control of cell membrane structure and function.

Retroconversion and δ4 desaturation of docosatetraenoate (22:4( n−6)) and docosapentaenoate (22:5( n−3)) by human cells in culture

This study has investigated the metabolic modification of [3- 14C]docosatetraenoate (22:4( n−6)) and [3- 14C]docosapentaenoate (22:5( n−3)) by human cells in culture. Fetal skin fibroblasts converted as much as 20% of the incoporated [ 14 C]22:4(n−6) to [ 14 C]20:4(n−6) within 6 h and 41% within 48 h. Retroconversion of incorporated [ 14 C]22:5(n−3) was less than 13% at all time points. Chain shortening of [ 14 C]22:4(n−6) was also 2–6-fold greater than that of [ 14 C]22:5(n−3) in retinoblastoma and vascular endothelial cells. Fibroblasts, vascular endothelial cells and retinoblastoma cells all elongated substantially more [ 14C]eicosapentaenoate than [ 14C]arachidonate to the respective C 22 fatty acids. Within 3–4 days, fibroblasts incubated with either [ 14 C]20:5(n−3) or [ 14 C]22:5(n−3) had the same ratio of radiolabeled C 22:C 20 fatty acids in cellular glycerolipids. By contrast, the cells incubated with [ 14 C]22:4(n−6) or [ 14 C]22:4(n−6) did not reach a common C 22/C 20 equilibrium by 5 days. Although fibroblasts were found to desaturate [ 14 C]22:5(n−3) , a substantial lag time was observed; [ 14 C]22:5(n−3) was 2% at 48h and 20% at 96h. By contrast, synthesis of [ 14 C]22:6(n−3) by retinoblastoma cells was 51% within 6 h and > 90% at 96 h. Desaturation of [ 14 C]22:4(n−6) was observed in retinoblastoma cells, but not in fibroblasts. These results thus suggest that the ratio of C 22C 20 polyunsaturated fatty acids in cells is regulated by the relative rates of retroconversion and chain elongation, with the net effect of the two processes favoring C 20 for n−6 and C 22 for the n−3 fatty acids. Furthermore, although fibroblasts desaturate [ 14 C]22:5(n−3) , the process appears to be qualitatively different from that of retinoblastoma cells.

Differential effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells.

The effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells were examined to identify fatty acid specific changes in lipid metabolism that might indicate a basis for the hypolipidemic effect attributed to eicosapentaenoic acid and related n-3 fatty acids. Cellular glycerolipid synthesis, as determined by [3H]glycerol incorporation, increased in a concentration-dependent manner in cells incubated 4 h with either eicosapentaenoic acid or oleic acid at concentrations between 10 and 300 microM. [3H]Glycerol-labeled triglyceride was the principal lipid formed and increased approximately fourfold with the addition of 300 microM oleic acid or eicosapentaenoic acid. Both fatty acids also produced a 20-40% increase in the total cellular triglyceride mass. Although both fatty acids increased triglyceride synthesis to similar extents, eicosapentaenoic acid-treated cells secreted 40% less [3H]glycerol-labeled triglyceride than cells fed oleic acid. Cellular synthesis of [3H]glycerol-labeled phosphatidylethanolamine and phosphatidylcholine was also reduced by 40% and 30%, respectively, in cells given eicosapentaenoic acid versus cells given oleic acid. Similar results were obtained in determinations of radiolabeled oleic acid and eicosapentaenoic acid incorporation. At a fatty acid concentration of 300 microM, incorporation of radiolabeled eicosapentaenoic acid into cellular triglycerides was greater than the incorporation obtained with radiolabeled oleic acid, while the reverse relationship was observed for the formation of phosphatidylcholine from the same fatty acids. Eicosapentaenoic acid is as potent as oleic acid in inducing triglyceride synthesis but eicosapentaenoic acid is a poorer substrate than oleic acid for phospholipid synthesis. The intracellular rise in de novo-synthesized triglyceride in eicosapentaenoic acid-treated cells without corresponding increases in triglyceride secretion suggests that eicosapentaenoic acid is less effective than oleic acid in promoting the transfer of de novo-synthesized triglyceride to nascent very low density lipoproteins.

Chain elongation of polyunsaturated fatty acids by vascular endothelial cells: Studies with arachidonate analogues

This study has utilized radiolabeled analogues of arachidonic acid to study the substrate specificity of elongation of long-chain polyunsaturated fatty acids. Human umbilical vein endothelial cells were incubated for 2-72 hr in medium supplemented with 0.9-2.6 microM [14C]fatty acid, and cellular glycerolipids were analyzed by gas-liquid chromatography with radioactivity detection. Elongation of naturally occurring C20 polyunsaturated fatty acids occurred with eicosapentaenoate (20:5(n-3] greater than Mead acid (20:3(n-9] greater than arachidonate (20:4(n-6]. Chain length markedly influenced the extent of elongation of 5,8,11,14-tetraenoates (18:4 greater than 19:4 greater than 20:4 greater than 21:4); effects of initial double bond position were also observed (6,9,12,15-20:4 greater than 4,7,10,13-20:4. Neither 5,8,14- nor 5,11,14-20:3 was elongated to the extent of 5,8,11-20:3. Differences between polyunsaturated fatty acids were observed both in the initial rates and in the maximal percentages of elongation, suggesting that the content of cellular C20 and C22 fatty acids may represent a balance between chain elongation and retroconversion. Umbilical vein endothelial cells do not exhibit significant desaturation of either 22:4(n-6) or 22:5(n-3). By contrast, incubation with 5,8,11,14-[14C]18:4(n-4) resulted in formation of both [14C]20:5(n-4) and [14C]22:5(n-4). The respective time courses for the appearances of [14C]22:5(n-4) and [14C]20:5(n-5) suggests delta 6 desaturation of [14C]22:4(n-4) rather than delta 4 desaturation of [14C]20:4(n-4).

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1- 14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[ 14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1- 14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1- 14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [ 14C]linolenate. Exogenous [1- 14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[ 14C]linolenoyl molecular species of phosphatidylcholine as those species with two [ 14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1- 14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the twopathway scheme of glycerolipid synthesis in plants.

Synthesis and release of fatty acids by human trophoblast cells in culture

In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from [14C]acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from [14C]acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. [14C]acetate was also incorporated into cellular triacylglycerol, phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with [1-14C]oleate; trophoblast cells then released 14C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the 14C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release.

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells.

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.

Elongation of arachidonic and eicosapentaenoic acids limits their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells

This study has examined the thrombin-stimulated release of polyunsaturated fatty acids from endothelial glycerolipids. Human umbilical vein endothelial cells were incubated with 1.25 μM [ 14C]arachidonate or [ 14C]eicosapentaenoate and then exposed to thrombin in buffered saline plus albumin. After an incorporation period of 0.5 h, the thrombin-stimulated release of the two radiolabeled fatty acids was quite similar. By contrast, after 24 h of fatty acid incorporation, the thrombin-stimulated release of radiolabeled fatty acid from cells incubated with [ 14C]eicosapentaenoate was only 25–30% of that from cells with [ 14C]arachidonate. Analysis of cellular glycerolipids indicated that 23 and 72%, respectively, of the incorporated [ 14C]arachidonate and [ 14C]eicosapentaenoate had been elongated to 22-carbon fatty acids in 24 h. Both 20- and 22-carbon 14C-labeled fatty acids were released to albumin in the medium in control incubations. Addition of thrombin stimulated the release of [ 14C]arachidonate and [ 14C]eicosapentaenoate, but not of their respective elongation products. Furthermore, endothelial cells incorporated exogenous [ 14C]docosatetraenoate into cellular glycerolipids but did not release it in response to thrombin. Thus, the thrombin-stimulated release of polyunsaturated fatty acids from vascular endothelial cells is highly selective for arachidonate and eicosapentaenoate. These results suggest that the extensive elongation of eicosapentaenoate by these cells serves to remove n-3 polyunsaturated fatty acids from the pool of cellular acyl groups which are released in response to thrombin and are thus made available for metabolism by cyclooxygenase and lipoxygenase enzymes.