BBA - Molecular and Cell Biology of Lipids (v.1771, #5)

Evidence for oxylipin synthesis and induction of a new polyunsaturated fatty acid hydroxylase activity in Chondrus crispus in response to methyljasmonate by Emmanuel Gaquerel; Cécile Hervé; Christophe Labrière; Catherine Boyen; Philippe Potin; Jean-Pierre Salaün (565-575).
Signaling cascades involving oxygenated derivatives (oxylipins) of polyunsaturated fatty acids (PUFAs) are known to operate in response to external stimuli. The marine red alga Chondrus crispus uses both oxygenated derivatives of C18 (octadecanoids) and C20 (eicosanoids) PUFAs as developmental or defense hormones. The present study demonstrates that methyljasmonate (MeJA) triggers a cascade of oxidation of PUFAs leading to the synthesis of prostaglandins and other oxygenated fatty acids. As a result of a lipoxygenase-like activation, MeJA induces a concomitant accumulation of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 13-oxo-9Z,11E-octadecadienoic acid (13-oxo-ODE) in a dose-dependent manner in C. crispus. Furthermore, MeJA increases the level of mRNA encoding a gluthatione S-transferase and induces the activity of a new enzyme catalyzing the regio- and stereoselective bisallylic hydroxylation of polyunsaturated fatty acids from C18 to C22. The enzyme selectively oxidized the omega minus 7 carbon position (ω-7) and generated the stereoselective (R)-hydroxylated metabolites with a large enantiomeric excess. The enzyme specificity for the fatty acid recognition was not dependent of the position of double bonds but at least requires a methylene interrupted double bond 1,4-pentadiene motif involving the ω-7 carbon.
Keywords: Plant; Red alga; Eicosanoid; (ω-7)-hydroxylase; Allylic oxidation; Chirality; Defense;

n-3 and n-6 Polyunsaturated fatty acids induce the expression of COX-2 via PPARγ activation in human keratinocyte HaCaT cells by Gérald Chêne; Marc Dubourdeau; Patricia Balard; Laure Escoubet-Lozach; Claudine Orfila; Antoine Berry; José Bernad; Marie-Françoise Aries; Marie Charveron; Bernard Pipy (576-589).
Polyunsaturated fatty acids (PUFA) n-3 inhibit inflammation, in vivo and in vitro in keratinocytes. We examined in HaCaT keratinocyte cell line whether eicosapentaenoic acid (EPA) a n-3 PUFA, gamma-linoleic acid (GLA) a n-6 PUFA, and arachidic acid a saturated fatty acid, modulate expression of cyclooxygenase-2 (COX-2), an enzyme pivotal to skin inflammation and reparation. We demonstrate that only treatment of HaCaT with GLA and EPA or a PPARγ ligand (roziglitazone), induced COX-2 expression (protein and mRNA). Moreover stimulation of COX-2 promoter activity was increased by those PUFAs or rosiglitazone. The inhibitory effects of GW9662 and T0070907 (PPARγ antagonists), on COX-2 expression and on stimulation of COX-2 promoter activity by EPA and GLA suggest that PPARγ is implicated in COX-2 induction. Finally, PLA2 inhibitor methyl arachidonyl fluorophosphonate blocked the PUFA effects on COX-2 induction, promoter activity and arachidonic acid mobilization suggesting involvement of AA metabolites in PPAR activation. These findings demonstrate that n-3 and n-6 PUFA increased PPARγ activity is necessary for the COX-2 induction in HaCaT human keratinocyte cells. Given the anti-inflammatory properties of EPA, we suggest that induction of COX-2 in keratinocytes may be important in the anti-inflammatory and protective mechanism of action of PUFAs n-3 or n-6.
Keywords: Phospholipase A2; Inflammatory response; Nuclear receptor;

Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: Involvement of lipid peroxidation by Rosa Martínez; Mercedes Lacort; José Ignacio Ruiz-Sanz; M. Begoña Ruiz-Larrea (590-599).
Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [3H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with α-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.
Keywords: Apolipoprotein B; Triacylglycerol synthesis, diacylglycerol: acylCoA acyltransferase; Lipid peroxidation; VLDL;

Comparative lipidomics analysis of cellular development and apoptosis in two Taxus cell lines by Song Yang; Bin Qiao; Shu-Huan Lu; Ying-Jin Yuan (600-612).
A comparative lipidomics approach was employed to investigate the changes in membrane phospholipids during the procession of cellular development and apoptosis of two plant cell lines, Taxus cuspidata and Taxus chinensis var. mairei. Analysis of lipids by LC/ESI/MSn showed more than 90 phospholipid molecular species and indicated significant differences in the abundance throughout a 3-week period. Phosphatidic acid (PA), phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) were three important lipid groups that were responsible for the discrimination between the apoptotic T. chinensis var. mairei and living T. cuspidata cells. Continuous increase of phospholipase D (PLD) activity led to PA production in apoptotic T. chinensis var. mairei cells suggesting that the PLD activation and PA formation mediated the apoptosis. Comparison of the profiles of phosphatidylbutanol (PtdBut) with those of PC or phosphatidylethanolamine (PE) indicated that PC rather than PE was the major substrate of PLD in vivo. These results suggest that the alternation of membrane phospholipids may regulate apoptosis, triggering an increase in taxol production of T. chinensis var. mairei cells.
Keywords: Comparative Lipidomics; Apoptosis; Taxus cell; Phospholipase D; Phosphatidic acid; Metabolomics;

Pro-inflammatory action of LDL(−) on mononuclear cells is counteracted by increased IL10 production by Sònia Benítez; Cristina Bancells; Jordi Ordóñez-Llanos; Jose Luis Sánchez-Quesada (613-622).
Objective: LDL(−) is a minor LDL subfraction that induces inflammatory factor release by endothelial cells. Since LDL(−) is present in plasma, its interaction with leucocytes, a cell type involved in atherosclerosis phenomena, is feasible; therefore, the aim of the current study was to evaluate LDL(−) effect on lymphocytes and monocytes isolated from human plasma. Methods and Results: Mononuclear cells were incubated with LDL(+) and LDL(−) and expression and release of several inflammatory mediators were analyzed by protein membrane assay, ELISA and real-time RT-PCR. LDL(−) induced a significantly increased production versus LDL(+) in MCP1, GROβ, GROγ, IL6, IL8 and IL10 in monocytes as well as in lymphocytes. These induced molecules are inflammatory, except for IL10 which is considered an anti-inflammatory cytokine. Therefore, the role of IL10 was evaluated in experiments where exogenous IL10 or antibodies anti-IL10 or anti-IL10 receptor were added. IL10 addition diminished the release of the other factors induced by LDL(−) near to basal production both at protein and RNA level. In contrast, the antibody anti-IL10 increased inflammatory cytokine release around two-fold, whereas the antibody anti-IL10 receptor produced a lower effect. Conclusions: LDL(−) promoted inflammatory cytokine production in leucocytes; however, it also induced IL10 that minimized this effect. Therefore, IL10 developed a significant role in counteracting the LDL(−) inflammatory action.
Keywords: LDL(−); Cytokine; Monocyte; Lymphocyte; IL10;

Predominant expression of lysosomal N-acylethanolamine-hydrolyzing acid amidase in macrophages revealed by immunochemical studies by Kazuhito Tsuboi; Li-Ying Zhao; Yasuo Okamoto; Nobukazu Araki; Masaki Ueno; Haruhiko Sakamoto; Natsuo Ueda (623-632).
Bioactive N-acylethanolamines, including anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory substance), and N-oleoylethanolamine (an anorexic substance) are enzymatically hydrolyzed to fatty acids and ethanolamine. Fatty acid amide hydrolase plays a major role in this reaction. In addition, we cloned cDNA of an isozyme termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)” [K. Tsuboi, Y.-X. Sun, Y. Okamoto, N. Araki, T. Tonai, N. Ueda, Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase, J. Biol. Chem. 280 (2005) 11082–11092]. Previous biochemical analyses suggested the expression of NAAA in macrophage cells and various rat tissues including lung and brain. To clarify the physiological significance of NAAA, here we immunochemically studied NAAA for the first time. We developed an antibody specific for rat NAAA, and by Western blotting revealed that NAAA is glycosylated and subjected to specific proteolysis. In alveolar macrophages isolated from rat lung, NAAA was immunocytochemically localized in lysosomes. In the whole lung tissue, only alveolar macrophages were immunostained for NAAA. Conformably, the mRNA and protein levels and activity of NAAA in alveolar macrophages were much higher than those in the whole lung tissue. In brain, intraventricular macrophages were positively stained with anti-NAAA antibody, while microglia appeared to be negative. These results strongly suggested the importance of macrophages as an expression site of NAAA in rat tissues.
Keywords: N-acylethanolamine; Anandamide; Ceramidase; Endocannabinoid; Lysosome; Macrophage;

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol®, medium chain glycerides and PEG esters by Sylvie Fernandez; Vincent Jannin; Jean-David Rodier; Nicolas Ritter; Bruno Mahler; Frédéric Carrière (633-640).
Labrasol® is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol® by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol® excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol®. These lipases were found to hydrolyze the main components of Labrasol® (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol® is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol® taken orally.
Keywords: Digestive enzyme; Lipase; Lipid excipient; Lipolysis; Oral drug delivery;