[Lipidomies is energing: 1st and 2nd lipidomics meeting].
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Disease↗
Biomedical subjects
M Record
Publications and source records attributed to M Record.
Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes: a paracrine signal for preadipocyte growth.
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3T3 Cells↗
Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth.
In the search for the existence of adrenergic regulation of the autocrine/paracrine function of the white adipose tissue, it was observed that conditioned media from isolated adipocytes or dialysates obtained by in situ microdialysis of human subcutaneous adipose tissue increased spreading and proliferation of 3T3F442A preadipocytes. These effects were amplified when an alpha2-adrenergic agonist was present during the obtention of conditioned media and microdialysates. This alpha2-adrenergic-dependent trophic activity was completely abolished by pretreatment of the conditioned media or microdialysates with the lysophospholipase, phospholipase B. Among the different lysophospholipids tested only lysophosphatidic acid (LPA) was able to induce spreading and proliferation of 3T3F442A preadipocytes. Moreover, previous chronic treatment of 3T3F442A preadipocytes with LPA which led to a specific desensitization of LPA responsiveness, abolished the alpha2-adrenergic-dependent trophic activities of the conditioned media and microdialysates. Finally, alpha2-adrenergic stimulation led to a rapid, sustained, and pertussis toxin-dependent release of [32P]LPA from [32P]-labeled adipocytes. Based upon these results it was proposed that in vitro and in situ stimulation of adipocyte alpha2-adrenergic receptors provokes the extracellular release of LPA leading, in turn, to regulation of preadipocyte growth.
3T3 Cells↗
Distinct pathways for tumor necrosis factor alpha and ceramides in human cytomegalovirus infection.
Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-alpha) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-alpha. To investigate the mechanisms involved in the inhibition of HCMV by TNF-alpha, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-alpha versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-alpha. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-alpha. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-alpha is independent of ceramides. In addition, our results suggest that TNF-alpha and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.
Antiviral Agents↗
Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.
Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.
Animals↗
Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D.
By searching into Expressed Sequence Tags databases (dbEST) using Blast X algorithm software and a plant phospholipase D as template, we have identified a cDNA from human brain (Z45777) which encodes for a protein similar to the amino acid region 743-929 of the human phospholipase D1 (PLD1), and a cDNA from human liver (R93485) which encodes for a protein similar to region 815-932 of PLD1. Sequence comparison between cloned phospholipases showed the presence of 3 conserved amino acid sequences: AFVGGIDLAYGRWD (box A), IIGSANINDRS (box B), and YIYIENQFFI (box C). Phylogenic analysis indicated that the cDNA from brain and liver encoded for human isologs of PLD1.
ADP-Ribosylation Factors↗
A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.
We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.
Animals↗
Involvement of vicinal dithiols in differential regulation of fMLP and phorbol ester-activated phospholipase D in stimulated human neutrophils.
In the human neutrophil, the fMLP-activated phospholipase D (PLD) was entirely calcium and tyrosine kinase dependent, but protein kinase C independent. An opposite regulation was observed with phorbol ester PMA, since the phospholipase D activity was mostly calcium insensitive, tyrosine kinase independent, but protein kinase C dependent. The arsenical compound, phenylarsine oxide (PAO), which reacts with vicinal sulhydryl groups, activated twofold at one minute the PMA stimulated-PLD activity, whereas it inhibited half of the fMLP-activated PLD after a time lag of 30 sec. Our data indicate that PAO acted on a mechanism regulating the balance between fMLP-activated and PMA-activated phospholipase D activity. Noteworthy, PAO similarly inhibited fMLP and PMA-induced O2(-)-production.
Arsenicals↗
Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.
Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.
Amidohydrolases↗
Phosphatidylcholine turnover in activated human neutrophils. Agonist-induced cytidylyltransferase translocation is subsequent to phospholipase D activation.
Phosphatidylcholine synthesis and degradation are tightly regulated to assure a constant amount of the phospholipid in cellular membranes. The chemotactic peptide fMLP and the phorbol ester, phorbol 12-myristate 13-acetate, are known to stimulate phosphatidylcholine degradation by phospholipase D in human neutrophils. fMLP alone triggered phosphatidylcholine breakdown into phosphatidic acid, but did not stimulate phosphatidylcholine synthesis or activation of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase. Adding cytochalasin B to fMLP led to some conversion of phosphatidic acid into diglyceride, and fMLP was then able to trigger choline incorporation into phosphatidylcholine, and cytidylyltransferase translocation from cytosol to membranes. Inhibition of phosphatidyl-choline-phospholipase D activation with tyrphostin led to inhibition of choline incorporation. Therefore, phosphatidic acid-derived diglyceride but not phosphatidic acid alone was effective to promote cytidylyltransferase translocation. With phorbol 12-myristate 13-acetate as agonist, and by selective labeling of phosphatidylinositol and phosphatidylcholine, we demonstrated that only phosphatidylcholine-derived diglyceride participated in cytidylyltransferase translocation. Oleic acid stimulated phosphatidylcholine synthesis, but induced a weak increase in diglyceride and a slight cytidylyltransferase translocation, and did not stimulate phospholipase D activity. Our data established that only diglyceride derived from phosphatidylcholine degradation by the phospholipase D/phosphatidate phosphatase pathway are required for agonist-induced cytidylyltransferase translocation and subsequent choline incorporation into phosphatidylcholine.
Biological Transport↗
Alpha 2-adrenergic stimulation promotes preadipocyte proliferation. Involvement of mitogen-activated protein kinases.
Primary cultures of rat preadipocytes were shown to express alpha 2- and beta-adrenoreceptors when maintained in serum-deprived medium. alpha 2-Adrenoreceptor stimulation led to an increase in cell number, whereas beta-adrenoreceptor stimulation was without effect. On 3T3-F442A clones stably transfected with the human alpha 2-C10 gene and expressing a physiologically related number of alpha 2-adrenoreceptors to overexpression, the proliferative effect of alpha 2-adrenoreceptor agonist UK14304 was proportional to the level of alpha 2-adrenoreceptor expressed in individual clones and required alpha-2 adrenoreceptor stimulation. Analysis of the signaling pathway linked to alpha 2-adrenoreceptor activation showed that the mitogenic effect was adenylyl cyclase- and protein kinase C-independent. It was pertussis toxin-sensitive, implying the involvement of pertussis toxin-sensitive G proteins. The activation of the mitogen-activated protein kinase pathway was increased after alpha 2-adrenoreceptor stimulation in both 3T3-F442 clones and rat preadipocytes and inhibited by pertussis toxin treatment. So, catecholamines are involved in the control of white preadipocyte proliferation through the alpha 2-adrenoreceptor activation, linked to the mitogen-activated protein kinase pathway.
3T3 Cells↗
Phosphatidylcholine cycle and regulation of phosphatidylcholine biosynthesis by enzyme translocation.
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Animals↗
Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the 'de novo' pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake.
The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the 'de novo' pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the 'de novo' pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).
Animals↗
Human neutrophil phospholipase D activation by N-formylmethionyl-leucylphenylalanine reveals a two-step process for the control of phosphatidylcholine breakdown and oxidative burst.
A comparative study of real-time kinetics of respiratory burst, monitored by H2O2-dependent chemiluminescence, and phospholipase D (PLD)-mediated phosphatidylcholine breakdown has been undertaken on human neutrophils stimulated by N-formylmethionyl-leucylphenylalanine in the absence of cytochalasin B. The fungal metabolite 17-hydroxywortmannin (HWT), an inhibitor of NADPH oxidase activation, decreases phosphatidic acid (PA) production by 30% at a concentration of 1 nM. Higher concentrations (10 nM-1 microM) inhibit PA formation maximally by 50% as compared with control. In all cases, the inhibition is delayed by 20-30 s after addition of the agonist. Thus the full PA generation is actually the result of an early (HWT-insensitive) and a late (HWT-sensitive) phosphatidylcholine breakdown. However, under all conditions, alkylacylglycerol remains at the basal level. PLD activity is dependent on Ca2+ influx, but is fully inhibited in cells depleted of Ca2+ with EGTA and Quin 2. The effect of HWT on the respiratory burst was investigated by measuring the kinetics of H2O2-induced chemiluminescence. This method allows to distinguish various phases of superoxide ion production: a lag, an increase in H2O2 formation (early phase), the duration of H2O2 production (late phase) and the termination of the oxidative burst. The lag remains constant for all HWT concentrations. A concentration of 10 nM-HWT, which fully inhibits the HWT-sensitive part of PA production, decreases superoxide ion production with a delay of about 20 s after addition of the agonist. Higher HWT concentrations, which have no additional effect on PLD inhibition, equally affect an early and a late phase of the burst. Thus high doses of HWT have a site of action which decreases the whole burst but does not affect the PLD any more. Therefore HWT and Ca2+ provide evidence for a two-step process for PLD activation. Only the delayed PA generation is functionally linked to a late phase of the oxidative burst.
Androstadienes↗
Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells.
Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.7.15), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.
Animals↗
Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells.
Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10(6) cells. Based on [3H]choline incorporation a net synthesis of 22 nmol/h per 10(6) cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10(6) cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.
Animals↗
Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.
Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.
Antineoplastic Agents↗
Subcellular localization of enzymes related to PAF metabolism in Krebs-II ascites cells: evidence for the lack of intracellular PAF transfer activity.
We have investigated various steps in the metabolism of Platelet-Activating Factor (PAF-acether or PAF) at the subcellular level in Krebs-II ascites cells. Microsomes contained an active acetyltransferase located on a heavy-rough domain of the endoplasmic reticulum quite rich in ribosomes, as monitored by [3H]uridine labelling, and which displayed a very high density across the Percoll gradient. This membrane domain, which was separated from all other cellular organelles including peroxisomes, also contained a membrane-bound acetylhydrolase with similar activity as the acetyltransferase. However most part of the cellular acetylhydrolase was located in the cytosol, which was actually devoid of PAF transfer activity, normally involved in transport of the mediator within the cell.
Acetyltransferases↗