BBA - Molecular and Cell Biology of Lipids (v.1736, #2)

Certain organophosphates react with the active site serine residue of neuropathy target esterase (NTE) and cause axonal degeneration and paralysis. Cloning of NTE revealed the presence of homologues in eukaryotes from yeast to man and that the protein has both a catalytic and a regulatory domain. The latter contains sequences similar to the regulatory subunit of protein kinase A, suggesting that NTE may bind cyclic AMP. NTE is tethered via an amino-terminal transmembrane segment to the cytoplasmic face of the endoplasmic reticulum. Unlike wild-type yeast, mutants lacking NTE activity cannot deacylate CDP–choline pathway-synthesized phosphatidylcholine (PtdCho) to glycerophosphocholine (GroPCho) and fatty acids. In cultured mammalian cells, GroPCho levels rise and fall, respectively, in response to experimental over-expression, and inhibition, of NTE. A complex of PtdCho and Sec14p, a yeast phospholipid-binding protein, both inhibits the rate-limiting step in PtdCho synthesis and enhances deacylation of PtdCho by NTE. While yeast can maintain PtdCho homeostasis in the absence of NTE, certain post-mitotic metazoan cells may not be able to, and some NTE-null animals have deleterious phenotypes. NTE is not required for cell division in the early mammalian embryo or in larval and pupal forms of Drosophila, but is essential for placenta formation and survival of neurons in the adult. In vertebrates, the relative importance of NTE and calcium-independent phospholipase A2 for homeostatic PtdCho deacylation in particular cell types, possible interactions of NTE with Sec14p homologues and cyclic AMP, and whether deranged phospholipid metabolism underlies organophosphate-induced neuropathy are areas which require further investigation.
Keywords: Membrane; Homeostasis; Endoplasmic reticulum; Axon; Neurodegeneration; Organophosphate;

The lipid composition of very-low-density lipoprotein (VLDL) in plasma is crucial for human health. A pre-requisite for the alteration of VLDL composition is a co-ordinated understanding of the complex interactions in VLDL assembly. In order to determine the potential effects of changes in substrate availability on VLDL lipid composition, we constructed, parameterized and evaluated a mechanistic mathematical model of the biosynthesis of triglycerides, phospholipids, and cholesterol esters and the assembly of VLDL in human hepatocytes. Using published data on human liver metabolism, the model was also used to provide insight into the complex process of lipid metabolism and to estimate the affinities of different liver enzymes for different fatty acids (FA). For example, we found that Δ6-desaturase is 19 times more selective for C18:3n-3 than C18:2n-6, stearoyl-CoA-desaturase is 2.7 times more selective for C18:0 than C16:0, Δ5-desaturase desaturates C20:4n-3 preferentially over C20:3n-6 and FA elongase preferentially elongates C18:3n-6. The model was also used to predict the plasma free fatty acid (FFA) composition required to generate a prescribed change in plasma lipoprotein FA composition. Furthermore, the model was tested against a published human feeding trial that investigated the effect of changes in dietary FA composition on human plasma lipid FA composition. The model is a useful tool for predicting the effect of changes in plasma FFA composition on plasma lipoprotein lipid FA composition.
Keywords: Lipid; Human; Triglyceride; Enzyme; Phospholipid; Lipoprotein;

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 ≈200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.
Keywords: Actin; 5-lipoxygenase; Cytosolic phospholipase A2; Leukotriene; Polymorphonuclear leukocyte; Latrunculin;

Atorvastatin prevents peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) downregulation in lipopolysaccharide-stimulated H9c2 cells by Anna Planavila; Rosa M. Sánchez; Manuel Merlos; Juan C. Laguna; Manuel Vázquez-Carrera (120-127).
Although abnormalities in cardiac fatty acid metabolism are involved in the development of several cardiac pathologies, the mechanisms underlying these changes are not well understood. Given the prominent role played by peroxisome proliferator-activated receptor β/δ (PPARβ/δ in cardiac fatty acid metabolism, the aim of this study was to examine the effects of nuclear factor (NF)-κB activation on the activity of this nuclear receptor. Embryonic rat heart-derived H9c2 cells stimulated with lipopolysaccharide (LPS) showed a reduction (38%, P  < 0.05) in the mRNA levels of the PPARβ/δ-target gene pyruvatedehydrogenase kinase 4 (PDK4) that was prevented in the presence of the NF-κB inhibitors parthenolide (10 μM) and atorvastatin (10 μM). Electrophoretic mobility shift assay revealed that both parthenolide and atorvastatin significantly decreased LPS-stimulated NF-κB binding activity in H9c2 cardiac cells. LPS-stimulation of H9c2 cardiac cells also led to a 30% reduction (P  < 0.05) in the mRNA levels of PPARγ Coactivator 1 (PGC-1) that was consistent with the reduction in the protein levels of this coactivator. In the presence of either atorvastatin or parthenolide, the reduction in PGC-1 expression was prevented. Co-immunoprecipitation studies showed that LPS-stimulation led to a reduction in the physical interaction between PGC-1 and PPARβ/δ and that this reduction was prevented in the presence of atorvastatin. Finally, electrophoretic mobility shift assay revealed that parthenolide and atorvastatin prevented LPS-mediated reduction in PPARβ/δ binding activity in H9c2 cardiac cells. These results suggest that LPS-mediated NF-κB activation inhibits the expression of genes involved in fatty acid metabolism by a mechanism involving reduced expression of PGC-1, which in turn affects the PPARβ/δ transactivation of target genes involved in cardiac fatty acid oxidation.
Keywords: PGC-1; PPARβ/δ; NF-κB; Atorvastatin;

Linoleic acid (LA, 18:2n-6) has variously been found to increase or inhibit synthesis of 2-series prostaglandins (PGs), derived from arachidonic acid (AA, 20:4n-6). γ-linolenic acid (GLA, 18:3n-6) containing oils are promoted to women for a variety of reproductive problems. Little is known concerning their actual effects on reproduction. We investigated the effects of LA, GLA and AA supplementation (25–100 μM) on basal and oxytocin (OT) stimulated production of 1-, 2- and-3 series PGs by uterine epithelial cells isolated from non-pregnant ewes, used as a model system to study endometrial PG production. PGF isomers were measured using radioimmunoassays following separation by high performance chromatography (HPLC). OT challenge increased the proportion of PGF in relation to PGF and PGF in control medium. LA supplementation decreased all PGF isomer production and reduced responsiveness to OT. GLA increased both absolute and proportional PGF production and slightly enhanced PGF generation. AA increased PGF generation and raised its isometric proportion. Both GLA and AA increased overall PGF output significantly but prevented the cells from responding to OT. These results suggest that consumption of LA and GLA are likely to differentially alter both uterine PG metabolism and responsiveness to OT. This may have implications for the control of a variety of reproductive processes.
Keywords: Polyunsaturated fatty acid; Prostaglandin isomer; Epithelial cell; Uterus; Ewe;

High-density lipoproteins attenuate interleukin-6 production in endothelial cells exposed to pro-inflammatory stimuli by Monica Gomaraschi; Nicoletta Basilico; Francesca Sisto; Donatella Taramelli; Sonia Eligini; Susanna Colli; Cesare R. Sirtori; Guido Franceschini; Laura Calabresi (136-143).
The purpose of the present study was to investigate the ability of high-density lipoproteins (HDL) to attenuate endothelial dysfunction, by assessing down-regulation of cytokine-induced interleukin-6 (IL-6) production in cultured endothelial cells, and measuring plasma IL-6 levels in three groups of healthy individuals with low, average, or high plasma HDL-cholesterol. Human plasma HDL caused a concentration-dependent inhibition of TNFα-induced IL-6 production in human endothelial cells (by 58.5 ± 1.5% at 2 mg of HDL-protein/ml). Reconstituted HDL made with apolipoprotein A-I (apoA-I) and phosphatidylcholine were as effective as plasma HDL, while lipid-free apoA-I or phosphatidylcholine liposomes had no effect. HDL attenuated IL-6 mRNA levels, an effect which occurs through inhibition of p38 MAP kinase. The median plasma IL-6 concentration was significantly higher in subjects with low HDL-cholesterol (2.54 pg/ml) compared with those with average or high HDL-cholesterol (1.31 pg/ml and 1.47 pg/ml, respectively). When all subjects were considered together, a lower HDL-cholesterol was the strongest independent predictor of higher IL-6 (F  = 25.38, P  < 0.001). By inhibiting IL-6 production and lowering plasma IL-6 concentration, HDL may limit the pro-atherogenic effects of both acute and chronic inflammatory states, of which IL-6 is a key orchestrator.
Keywords: High-density lipoprotein; Endothelial function; Interleukin; Atherosclerosis;

Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-α, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (M r  = 14,169) of AtsPLA2-α exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-α showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-α continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-α transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.
Keywords: Phospholipase A2; Phospholipid; Bioactive lipid;

Regulation of intermediary metabolism in rat cardiac myocyte by extracellular glycerol by Ségolène Gambert; Cécile Héliès-Toussaint; Alain Grynberg (152-162).
In the human heart, although all substrates compete for energy production, fatty acids (FA) represent the main substrate for ATP production. In the healthy heart, a balance between FA and carbohydrate utilization ensures that energy supply matches demand. This study was carried out to evaluate, in a model of spontaneously beating neonatal rat cardiomyocytes in culture, the hypothesis that glycerol could play a central role in the metabolic control of the routes involving long chain FAs and may then affect the balance between β-oxidation and glucose oxidation. The intracellular-free glycerol significantly increased with extracellular glycerol concentration (0 to 660 μM). The synthesis of phospholipids was significantly increased in parallel with both extracellular glycerol (1.5 and 14.8 nmol glycerol/mg protein, at 82 and 660 μM of extracellular glycerol, respectively). The oxidation of glycerol increased proportionally to extracellular glycerol concentration (from 1 to 3 nmol glycerol/mg protein, at 82 μM and 660 μM extracellular glycerol, respectively, P  < 0.001). At its maximum, this oxidation represented 15% of the glucose oxidation, which was not affected by glycerol extracellular supply or intracellular availability. Conversely, extracellular glycerol significantly reduced the palmitate oxidation above (−47% at 660 μM glycerol), but not octanoate oxidation. Investigations on the mechanism of the decreased palmitate oxidation reveals a glycerol-dependent increase in malonyl-CoA associated with a significant decrease in CPT-1 activity which accounts for the difference between palmitate and octanoate. These results clearly demonstrate the importance of glycerol in regulating the cardiac metabolic pathways and energy balance.
Keywords: Cardiomyocyte; Glycerol; Palmitate; Oxidation; Energy metabolism;