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BBA - Molecular and Cell Biology of Lipids (v.1821, #9)

Editorial Board (pp. i).

Modulation of excitability, membrane currents and survival of cardiac myocytes by N-acylethanolamines by Oleg I. Voitychuk; Valentyna S. Asmolkova; Nadiya M. Gula; Ganna V. Sotkis; Sehamuddin Galadari; Frank C. Howarth; Murat Oz; Yaroslav M. Shuba (pp. 1167-1176).

N-Acylethanolamines (NAE) are endogenously produced lipids playing important roles in a diverse range of physiological and pathological conditions. In the present study, using whole-cell patch clamp technique, we have for the first time investigated the effects of the most abundantly produced NAEs, N-stearoylethanolamine (SEA) and N‐oleoylethanolamine (OEA), on electric excitability and membrane currents in cardiomyocytes isolated from endocardial, epicardial, and atrial regions of neonatal rat heart. SEA and OEA (1–10μM) attenuated electrical activity of the myocytes from all regions of the cardiac muscle by hyperpolarizing resting potential, reducing amplitude, and shortening the duration of the action potential. However, the magnitudes of these effects varied significantly depending on the type of cardiac myocyte (i.e., endocardial, epicardial, atrial) with OEA being generally more potent. OEA and to a lesser extent SEA suppressed in a concentration-dependent manner currents through voltage-gated Na⁺ (VGSC) and L-type Ca²⁺ (VGCC) channels, but induced variable cardiac myocyte type-dependent effects on background K⁺ and Cl⁻ conductance. The mechanisms of inhibitory action of OEA on cardiac VGSCs and VGCCs involved influence on channels' activation/inactivation gating and partial blockade of ion permeation. OEA also enhanced the viability of cardiac myocytes by reducing necrosis without a significant effect on apoptosis. We conclude that SEA and OEA attenuate the excitability of cardiac myocytes mainly through inhibition of VGSCs and VGCC-mediated Ca²⁺ entry. Since NAEs are known to increase during tissue ischemia and infarction, these effects of NAEs may mediate some of their cardioprotective actions during these pathological conditions.► N-acylethanolamines (NAEs), SEA and OEA, attenuate cardiac excitability. ► NAEs inhibit voltage-gated sodium and calcium channels. ► NAEs enhance the viability of cardiac myocytes mainly via suppression of necrosis. ► OEA is more potent than SEA. ► NAEs may induce cardioprotection during ischemia and infarction.

Keywords: Abbreviations; AP; action potential; APD; action potential duration; NAE; N-acylethanolamines; SEA; N-stearoylethanolamine; OEA; N-oleoylethanolamine; PM; plasma membrane; VGCC; voltage-gated Ca; ²⁺; channel; VGSC; voltage-gated Na; ⁺; channelCardiac myocyte; Endocannabinoid; Excitability; Ion channel

TNF-α inhibits PPARβ/δ activity and SIRT1 expression through NF-κB in human adipocytes by Lucía Serrano-Marco; Chacon Matilde R. Chacón; Maymo-Masip Elsa Maymó-Masip; Emma Barroso; Salvado Laia Salvadó; Martin Wabitsch; Garrido-Sanchez Lourdes Garrido-Sánchez; Francisco J. Tinahones; Xavier Palomer; Joan Vendrell; Vazquez-Carrera Manuel Vázquez-Carrera (pp. 1177-1185).

The mechanisms linking low-grade chronic inflammation with obesity-induced insulin resistance have only been partially elucidated. PPARβ/δ and SIRT1 might play a role in this association. In visceral adipose tissue (VAT) from obese insulin‐resistant patients we observed enhanced p65 nuclear translocation and elevated expression of the pro‐inflammatory cytokines TNF‐α and IL‐6 compared to control subjects. Inflammation was accompanied by a reduction in the levels of SIRT1 protein and an increase in PPARβ/δ mRNA levels. Stimulation of human mature SGBS adipocytes with TNF‐α caused similar changes in PPARβ/δ and SIRT1 to those reported in obese patients. Unexpectedly, PPAR DNA‐binding activity and the expression of PPARβ/δ‐target genes was reduced following TNF‐α stimulation, suggesting that the activity of this transcription factor was inhibited by cytokine treatment. Interestingly, the PPARβ/δ ligand GW501516 prevented the expression of inflammatory markers and the reduction in the expression of PPARβ/δ‐target genes in adipocytes stimulated with TNF‐α. Consistent with a role for NF‐κB in the changes caused by TNF‐α, treatment with the NF‐κB inhibitor parthenolide restored PPAR DNA‐binding activity, the expression of PPARβ/δ‐target genes and the expression of SIRT1 and PPARβ/δ. These findings suggest that the reduction in PPARβ/δ activity and SIRT1 expression caused by TNF‐α stimulation through NF‐κB helps perpetuate the inflammatory process in human adipocytes.► SIRT1 was reduced and PPARβ/δ increased in adipose tissue from obese patients. ► Stimulation of human adipocytes with TNF-α caused similar changes. ► TNF-α effects were inhibited by an NF-κB inhibitor and the PPARβ/δ agonist GW501516. ► These TNF-α effects can help perpetuate the inflammatory process in human adipocytes.

Keywords: Abbreviations; CPT-1b; carnitine palmitoyl transferase 1; HDL; high-density lipoprotein; IL-6; interleukin-6; MCP-1; monocyte chemoattractant protein-1; NF-κB; nuclear factor-κB; PDK4; pyruvate dehydrogenase kinase 4; PGC-1α; PPARγ co-activator 1α; PPAR; peroxisome proliferator-activated receptor; SIRT1; sirtuin 1; SAT; subcutaneous adipose tissue; TNF-α; tumour necrosis factor-α; VAT; visceral adipose tissuePPARβ/δ; TNF-α; NF-κB; SIRT1

Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate by Cecilia I. Lopez; Pelletan Leonardo E. Pelletán; Laila Suhaiman; Gerardo A. De Blas; Nicolas Vitale; Luis S. Mayorga; Silvia A. Belmonte (pp. 1186-1199).

Acrosomal exocytosis involves a massive fusion between the outer acrosomal and the plasma membranes of the spermatozoon triggered by stimuli that open calcium channels at the plasma membrane. Diacylglycerol has been implicated in the activation of these calcium channels. Here we report that this lipid promotes the efflux of intraacrosomal calcium and triggers exocytosis in permeabilized human sperm, implying that diacylglycerol activates events downstream of the opening of plasma membrane channels. Furthermore, we show that calcium and diacylglycerol converge in a signaling pathway leading to the production of phosphatidylinositol 4,5-bisphosphate (PIP₂). Addition of diacylglycerol promotes the PKC-dependent activation of PLD1. Rescue experiments adding phosphatidic acid or PIP₂ and direct measurement of lipid production suggest that both PKC and PLD1 promote PIP₂ synthesis. Inhibition of different steps of the pathway was reverted by adenophostin, an agonist of IP₃-sensitive calcium channels, indicating that PIP₂ is necessary to keep these channels opened. However, phosphatidic acid, PIP₂, or adenophostin could not trigger exocytosis by themselves, indicating that diacylglycerol must also activate another factor. We found that diacylglycerol and phorbol ester stimulate the accumulation of the GTP-bound form of Rab3A. Together our results indicate that diacylglycerol promotes acrosomal exocytosis by i) maintaining high levels of IP₃ – an effect that depends on a positive feedback loop leading to the production of PIP₂ – and ii) stimulating the activation of Rab3A, which in turn initiates a cascade of protein interactions leading to the assembly of SNARE complexes and membrane fusion.► Diacylglycerol (DAG)-induced acrosomal exocytosis requires PKC and PLD1 activation. ► DAG promotes a continuous production of phosphatidylinositol 4,5-bisphosphate (PIP₂). ► PIP₂ is required for IP₃ production and intraacrosomal calcium efflux. ► DAG activates Rab3A leading to the assembly of SNARE complexes and membrane fusion.

Keywords: Diacylglycerol; Regulated exocytosis; Phospholipase D; Phosphatidic acid; Phosphatidylinositol 4,5-bisphosphate; Acrosomal exocytosis

Oxidized phospholipid content destabilizes the structure of reconstituted high density lipoprotein particles and changes their function by Subhabrata Kar; Mitulkumar A. Patel; Rajan K. Tripathy; Priyanka Bajaj; Unnati V. Suvarnakar; Abhay H. Pande (pp. 1200-1210).

High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein component that facilitates various biological activities of HDL particles. Increase in Ox-PL content of HDL particles makes them ‘dysfunctional’ and such modified HDL particles not only lose their athero-protective properties but also acquire pro-atherogenic and pro-inflammatory functions. The details of Ox-PL‐induced alteration in the molecular properties of HDL particles are not clear. Paraoxonase 1 (PON1) is an HDL-associated enzyme that possesses anti-inflammatory and anti-atherogenic properties; and many of the athero-protective functions of HDL are attributed to the associated PON1. In this study we have characterized the physicochemical properties of reconstituted HDL (rHDL) particles containing varying amounts of Ox-PL and have compared their PON1 stimulation capacity. Our results show that increased Ox-PL content (a) modifies the physicochemical properties of the lipid domain of the rHDL particles, (b) decreases the stability and alters the conformation as well as orientation of apoA-I molecules on the rHDL particles, and (c) decreases the PON1 stimulation capacity of the rHDL particles. Our data indicate that the presence of Ox-PLs destabilizes the structure of the HDL particles and modifies their function.► Oxidized phospholipid (Ox-PL) plays an important role in atherosclerosis. ► Ox-PL content alters the physicochemical properties of HDL particles. ► Ox-PL content destabilizes the structure of HDL particles and modifies their function. ► Understanding Ox-PL-induced changes in the properties of HDL is crucial.

Keywords: Abbreviations; apoA-I; apolipoprotein A-I; ATR-FTIR; attenuated total reflectance Fourier transformed infrared; Chol; free cholesterol; CVD; cardio vascular disease; DPH; 1,6-diphenyl-1,3,5-hexatriene; Gnd-HCl; guanidine hydrochloride; GP; generalized polarization; Laurdan; 2-dimethylamino-(6-lauroyl)-naphthalene; MDA; malondialdehyde; MLV; multi lamellar vesicles; Ox-PAPC; oxidized 1-palmitoyl-2-arachidonyl-; sn; -glycero-3-phosphocholine; rPON 1; recombinant paraoxonase 1; PAGE; polyacrylamide gel electrophoresis; PAPC; 1-palmitoyl-2-arachidonyl-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; rHDL; reconstituted HDL; RCT; reverse cholesterol transport; tOx-PLs; truncated oxidized phospholipids; Trp; tryptophan; TBARS; thiobarbituric acid reactive substancesApolipoprotein A-I; ATR-FTIR; Paraoxonase 1; Ox-PL; Fluorescence spectroscopy; rHDL

Inhibitory effect of phospholipids on P-glycoprotein: Cellular studies in Caco-2, MDCKII mdr1 and MDCKII wildtype cells and P-gp ATPase activity measurements by S. Simon; R. Schubert (pp. 1211-1223).

Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character.► Phospholipids are screened with regard to P-glycoprotein(P-gp)-inhibitory effects. ► In vitro P-gp inhibition is examined in Caco-2- and MDCKII mdr1 and wildtype cells. ► 8:0 phosphatidylcholine (PC), 10:0 PC and cis-22:6 PC cause P-gp inhibition. ► 8:0 and 10:0 PC probably inhibit P-gp directly in their monomeric state.

Keywords: P-glycoprotein inhibition by phospholipid; Transport study; Calcein accumulation assay; ATPase activity; Caco-2; MDCKII

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A₂α dependent PGE₂ via both PKA and PKB pathways by Liz Kisslov; Nurit Hadad; Marina Rosengraten; Rachel Levy (pp. 1224-1234).

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂ via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.► The regulation of human colorectal cancer cells - HT-29 proliferation by cPLA2α. ► The proliferation process is regulated by both PKA and PKB pathways. ► PGE₂ via EP induces CREB phosphorylation by the PKA pathway. ► PGE₂ via EP regulates b-catenin, Ecaderin and cyclin D1 localization by PKB pathway.

Keywords: Cytosolic phospolipase A; ₂; α; PGE; ₂; PKA; PKB/Akt; CREB; β-Catenin

Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increase turnover of n-6 docosapentaenoic acid in rat-brain phospholipids by Miki Igarashi; Hyung-Wook Kim; Fei Gao; Lisa Chang; Kaizong Ma; Stanley I. Rapoport (pp. 1235-1243).

Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-¹⁴C]DPAn-6 was infused intravenously for 5min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2–7.7 fold, while turnover rates were increased 2.1–4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. (196 words)► Dietary n-3 PUFA increased DPAn-6 in rat brain. ► The diet increased DPA incorporation and turnover rates in rat brain. ► The change may be associated with increased cytosolic cPLA₂ IVA. ► These could contribute to behavioral and functional abnormalities by the diet.

Keywords: Abbreviations; AA; arachidonic acid; DPA; docosapentaenoic acid (22:5n-6); DHA; docosahexaenoic acid (22:6n-3); EPA; eicosapentaenoic acid (20:5n-3); FAME; fatty acid methyl ester; GC; gas chromatography; LA; linoleic acid (18:2n-6); α-LNA; α-linolenic acid (18:3n-3); PUFA; polyunsaturated fatty acid; PL; phospholipids; TG; triacylglycerol; EtnGpl; ethanolamine glycerophospholipid; ChoGpl; choline glycerophospholipid; PtdSer; phosphatidylserine; PtdIns; phosphatidylinositol; cPLA; ₂; cytosolic phospholipase A; ₂; sPLA; ₂; secretory PLA; ₂; iPLA; ₂; calcium independent PLA; ₂; COX; cyclooxygenase; LOX; lipoxygenaseDocosapentaenoic; Docosahexaenoic; Turnover; Brain; Diet; PUFA

Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana by Thuy T.P. Doan; Frédéric Domergue; Ashley E. Fournier; Sollapura J. Vishwanath; Owen Rowland; Patrick Moreau; Craig C. Wood; Anders S. Carlsson; Mats Hamberg; Per Hofvander (pp. 1244-1255).

Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta. ► In vitro characterization of purified AtFAR6. ► Expression of AtFAR6, AtFAR6-YFP fusion in Nicotiana leaves. ► Besides fatty alcohols as end products, AtFAR6 released fatty aldehydes, fatty acids. ► Both 16:0-CoA and 16:0-ACP serves as substrates at approximate the same efficiency. ► AtFAR6 is the first FAR produces and accumulates C16:0-alcohol within chloroplasts.

Keywords: Fatty alcohol; Fatty aldehyde; Fatty acyl-CoA reductase (FAR); Chloroplast; Chloroplast transit peptide

Glycerophospholipid profile in oncogene-induced senescence by Cristina Cadenas; Sonja Vosbeck; Eva-Maria Hein; Birte Hellwig; Alice Langer; Heiko Hayen; Dennis Franckenstein; Buttner Bettina Büttner; Seddik Hammad; Rosemarie Marchan; Matthias Hermes; Silvia Selinski; Rahnenfuhrer Jörg Rahnenführer; Begüm Peksel; Torok Zsolt Török; Vigh László Vígh; Jan G. Hengstler (pp. 1256-1268).

Alterations in lipid metabolism and in the lipid composition of cellular membranes are linked to the pathology of numerous diseases including cancer. However, the influence of oncogene expression on cellular lipid profile is currently unknown. In this work we analyzed changes in lipid profiles that are induced in the course of ERBB2-expression mediated premature senescence. As a model system we used MCF-7 breast cancer cells with doxycycline-inducible expression of NeuT, an oncogenic ERBB2 variant. Affymetrix gene array data showed NeuT-induced alterations in the transcription of many enzymes involved in lipid metabolism, several of which (ACSL3, CHPT1, PLD1, LIPG, MGLL, LDL and NPC1) could be confirmed by quantitative realtime PCR. A study of the glycerophospholipid and lyso-glycerophospholipid profiles, obtained by high performance liquid chromatography coupled to Fourier-transform ion cyclotron resonance-mass spectrometry revealed senescence-associated changes in numerous lipid species, including mitochondrial lipids. The most prominent changes were found in PG(34:1), PG(36:1) (increased) and LPE(18:1), PG(40:7) and PI(36:1) (decreased). Statistical analysis revealed a general trend towards shortened phospholipid acyl chains in senescence and a significant trend to more saturated acyl chains in the class of phosphatidylglycerol. Additionally, the cellular cholesterol content was elevated and accumulated in vacuoles in senescent cells. These changes were accompanied by increased membrane fluidity. In mitochondria, loss of membrane potential along with altered intracellular distribution was observed. In conclusion, we present a comprehensive overview of altered cholesterol and glycerophospholipid patterns in senescence, showing that predominantly mitochondrial lipids are affected and lipid species less susceptible to peroxidation are increased.► Expression of genes involved in lipid metabolism is altered in senescence. ► Glycerophospholipid profile of senescent cells reveals multiple alterations. ► Shorter and more saturated acyl chains are observed in senescence. ► Cholesterol levels are increased and localization is altered in senescence. ► Increased membrane fluidity and altered mitochondria are observed in senescence.

Keywords: ERBB2; Senescence; Glycerolipids; Cholesterol; Membrane fluidity; Mitochondria

The endogenous regulator 24( S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1) by Eser J. Zerenturk; Ika Kristiana; Saloni Gill; Andrew J. Brown (pp. 1269-1277).

The oxysterol 24( S),25-epoxycholesterol (24,25EC) can affect cholesterol metabolism at multiple points. Previously, we proposed that 24,25EC has an especially significant role in fine-tuning cholesterol synthesis, since it parallels cholesterol production, and without it, acute cholesterol synthesis is exaggerated. 24,25EC is structurally similar to desmosterol, a substrate for the enzyme 3β-hydroxysterol ∆²⁴-reductase (DHCR24, also called Seladin-1) which catalyzes a final step in cholesterol synthesis. In this study, we reveal a novel mode by which 24,25EC can regulate cholesterol synthesis, by interfering with DHCR24, resulting in the rapid accumulation of the substrate desmosterol, at the expense of cholesterol. This effect was independent of DHCR24 protein levels, and was observed in multiple mammalian cell-lines, including those of hepatic and neuronal origin. Conversely, overexpression of DHCR24 blunted the inhibition by 24,25EC. We also determined that the specificity of this effect was restricted to certain side-chain oxysterols, notably those oxygenated at C-25. Importantly, endogenous levels of 24,25EC, manipulated by genetic and pharmacological methods, were sufficient to reduce DHCR24 activity. Together, our work introduces a novel role for 24,25EC in cholesterol homeostasis, through its rapid inhibition of cholesterol synthesis at DHCR24. Also, our work provides new insights into a little studied area, the post-transcriptional regulation of DHCR24, an important enzyme in human health and disease.► 24,25EC regulates DHCR24, with accumulation of desmosterol, decreasing cholesterol. ► DHCR24 inhibition was specific to C-25 oxygenated side-chain oxysterols. ► Manipulating endogenous levels of 24,25EC was sufficient to reduce DHCR24 activity. ► DHCR24, an important enzyme in human health, is post-transcriptionally regulated.

Keywords: Abbreviations; 24,25EC; 24(; S; ),25-epoxycholesterol; Arg-TLC; argentation thin layer chromatography; CD; methyl-β-cyclodextrin; CHO; Chinese hamster ovary; DOS; 2,3(; S; ):22(; S; ),23-dioxidosqualene; DHCR24; 3β-hydroxysterol ∆; ²⁴; -reductase; FAD; flavin adenine dinucleotide; FCS; fetal calf serum; HMGR; 3-hydroxy-3-methylglutaryl-coenzyme A reductase; LDL; low-density lipoprotein; LPDS; lipoprotein-deficient serum; LXR; liver X receptor; MOS; 2,3(; S; )-monooxidosqualene; NCS; newborn calf serum; OSC; 2,3-oxidosqualene cyclase; PBS; phosphate buffered saline; Seladin-1; Selective Alzheimer's Disease Indicator-1; SM; squalene monooxygenase; SREBP; Sterol Regulatory Element Binding ProteinCholesterol; 24(S),25-epoxycholesterol; DHCR24; Seladin-1; Oxysterol; Alzheimer's disease

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A₂-VIA (iPLA₂β)-knockout mice by Yewon Cheon; Hyung-Wook Kim; Miki Igarashi; Hiren R. Modi; Lisa Chang; Kaizong Ma; Deanna Greenstein; Mary Wohltmann; John Turk; Stanley I. Rapoport; Ameer Y. Taha (pp. 1278-1286).

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.► The role of iPLA2β in brain was examined by using [1-¹⁴C]DHA in iPLA₂β knockout mice. ► iPLA₂β knockout mice exhibit disturbances in brain phospholipid metabolism. ► iPLA₂β knockout mice reduce DHA incorporation and DHA content in brain. ► Genetic iPLA₂β deficiency reorganizes lipid-metabolizing enzyme expression in brain. ► These changes may be relevant to neurologic defects in humans with iPLA₂β mutations.

Keywords: Abbreviations; AA; arachidonic acid; ChoGpl; choline glycerophospholipid; COX; cyclooxygenase; cPLA; ₂; cytosolic phospholipase A; ₂; (Group IVA PLA; ₂; ); DHA; docosahexaenoic acid; DHA-CoA; docosahexaenoyl-CoA; EtnGpl; ethanolamine glycerophospholipid; FAME; fatty acid methyl ester; GC; gas chromatography; iPLA; ₂; Ca; 2; ⁺; -independent phospholipase A; ₂; (Group VIA PLA; ₂; ); LOX; lipoxygenase; PUFA; polyunsaturated fatty acid; PtdIns; phosphatidylinositol; PtdSer; phosphatidylserine; sPLA; ₂; secretory phospholipase A; ₂; sn; stereospecifically numbered; TLC; thin layer chromatographyMouse; DHA; Turnover; Incorporation; Lipid; PLA2G6

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14⁺/CD16⁻ circulating monocytes by Thomas Salvary; Ségolène Gambert-Nicot; Marie-Claude Brindisi; Nicolas Meneveau; François Schiele; Seronde Marie-France Séronde; Luc Lorgis; Marianne Zeller; Yves Cottin; Jean-Pierre Kantelip; Philippe Gambert; Siamak Davani (pp. 1287-1294).

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63±3 vs 13±1%, p<0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63±3 vs 41±2%, p<0.001), with less distribution in caveolae (13±1 vs 34±2%, p<0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41±2 vs 66±3%, p<0.001) and caveolae (34±2 vs 18±1%, p<0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins.► Description of a new methodology to separate membrane lipid microdomains. ► Applying this methodology to monocytes from healthy and myocardial infarction patients. ► Analyzing the differences in membrane reorganization between these groups. ► Myocardial infarction redistributes cholesterol to caveolae. ► Pravastatin restored original conditions.

Keywords: Lipid raft; Caveolae; Monocyte; Myocardial infarction; Statin

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium by Takumi Ohdate; Yoshiharu Inoue (pp. 1295-1305).

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H₂O₂ is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes ( GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1∆ cells, and subsequently, the growth of gpx1∆ cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1^C36S was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.► S. cerevisiae has three glutathione peroxidase homologues. ► The expression of Gpx1 was induced when cells were cultured in oleic acid medium. ► Gpx1 was present in the peroxisomal matrix. ► Deficiency in Gpx1 impaired growth and peroxisome formation in oleic acid medium. ► Redox regulation of Gpx1 is involved in peroxisome biogenesis.

Keywords: Abbreviations; ROS; reactive oxygen species; GPx; glutathione peroxidase; PTS; peroxisome targeting signal; ER; endoplasmic reticulum; SD; synthetic dextrose; PMSF; phenylmethylsulfonyl fluoride; PAGE; polyacrylamide gel electrophoresis; 3-AT; 3-amino 1,2,4-triazole; t; -BHP; tert; -butyl hydroperoxideGlutathione peroxidase; Peroxisome; Yeast; Peroxiredoxin; Reactive oxygen species

Corrigendum to “Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica” [Biochim. Biophys. Acta 1811 (2011) 323–332] by Taketoshi Takara; Tetsuto Nakagawa; Masami Isobe; Nozomu Okino; Sachiyo Ichinose; Akira Omori; Makoto Ito (pp. 1306-1307).

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BBA - Molecular and Cell Biology of Lipids (v.1821, #9)

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BBA - Molecular and Cell Biology of Lipids (v.1821, #9)