BBA - Molecular and Cell Biology of Lipids (v.1861, #11)

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro by Krishna Chaithanya Batchu; Satu Hänninen; Sawan Kumar Jha; Michael Jeltsch; Pentti Somerharju (1597-1604).
Cytosolic phospholipase A2 alpha (cPLA2α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA2α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA2α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA2α. Such promiscuous binding would explain the previous finding that cPLA2α has both PLA1 and PLA2 activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA2α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA2α.
Keywords: Arachidonic acid; Bilayer; Catalytic site; Micelle; Phospholipase A; Mass spectrometry;

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C 4 synthase activity and evaluation of inhibitors by Stefanie Liening; Gerhard K. Scriba; Silke Rummler; Christina Weinigel; Thea K. Kleinschmidt; Jesper Z. Haeggström; Oliver Werz; Ulrike Garscha (1605-1613).
Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~ 1.3 μg/200 μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2 +-ionophore A23187 and 20 μM AA resulted in strong LTC4 formation (~ 250 ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50  = 3.1–3.5 μM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50  = 17 and 300 nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.
Keywords: Leukotriene C 4; LTC4 synthase; Inhibitor; Assay; HEK293 cells;

Brown adipose tissue in obesity: Fractalkine-receptor dependent immune cell recruitment affects metabolic-related gene expression by Ágnes Polyák; Zsuzsanna Winkler; Dániel Kuti; Szilamér Ferenczi; Krisztina J. Kovács (1614-1622).
Brown adipose tissue (BAT) plays essential role in metabolic- and thermoregulation and displays morphological and functional plasticity in response to environmental and metabolic challenges. BAT is a heterogeneous tissue containing adipocytes and various immune-related cells, however, their interaction in regulation of BAT function is not fully elucidated. Fractalkine is a chemokine synthesized by adipocytes, which recruits fractalkine receptor (CX3CR1)-expressing leukocytes into the adipose tissue. Using transgenic mice, in which the fractalkine receptor, Cx3cr1 gene was replaced by Gfp, we evaluated whether deficiency in fractalkine signaling affects BAT remodeling and function in high-fat-diet - induced obesity. Homo- and heterozygote male CX3CR1-GFP mice were fed with normal or fat enriched (FatED) diet for 10 weeks. Interscapular BAT was collected for molecular biological analysis. Heterozygous animals in which fractalkine signaling remains intact, gain more weight during FatED than CX3CR1 deficient gfp/gfp homozygotes. FatED in controls resulted in macrophage recruitment to the BAT with increased expression of proinflammatory mediators (Il1a, b, Tnfa and Ccl2). Local BAT inflammation was accompanied by increased expression of lipogenic enzymes and resulted in BAT “whitening”. By contrast, fractalkine receptor deficiency prevented accumulation of tissue macrophages, selectively attenuated the expression of Tnfa, Il1a and Ccl2, increased BAT expression of lipolytic enzymes (Atgl, Hsl and Mgtl) and upregulated genes involved thermo-metabolism (Ucp1, Pparg Pgc1a) in response to FatED. These results highlight the importance of fractalkine-CX3CR1 interaction in recruitment of macrophages into the BAT of obese mice which might contribute to local tissue inflammation, adipose tissue remodeling and regulation of metabolic-related genes.Display Omitted
Keywords: Fractalkine; Macrophage; Inflammation; Triglyceride metabolism; Thermogenesis; Obesity; BAT;

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.
Keywords: Microsomal triglyceride transfer protein; Low density; Lipoprotein; Apolipoprotein B; Abetalipoproteinemia; Hypobetalipoproteinemia;

Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures by Tomáš Řezanka; Irena Kolouchová; Karel Sigler (1634-1642).
Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4–28 °C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10 °C. On temperature drop to 4 °C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice.A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7 °C and the difference between PO-PE and OP-PE is some 10 °C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions.
Keywords: Psychrophilic yeast; Lipidomic analysis; Phospholipids; Phosphatidylcholine; Phosphatidylethanolamine; Regioisomers;

ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism by Aditi Chaudhari; Liliana Håversen; Reza Mobini; Linda Andersson; Marcus Ståhlman; Emma Lu; Mikael Rutberg; Per Fogelstrand; Kim Ekroos; Adil Mardinoglu; Malin Levin; Rosie Perkins; Jan Borén (1643-1651).
Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.
Keywords: ARAP2; lipid droplets; triglyceride synthesis; GLUT1; Glucosylceramide synthase;

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling by Walter Boiten; Samira Absalah; Rob Vreeken; Joke Bouwstra; Jeroen van Smeden (1652-1661).
In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.Display Omitted
Keywords: Stratum corneum; Skin; Tape-stripping; Extraction efficiency; Mass spectrometry; Stereoisomers;

SCD1 deficiency protects mice against ethanol-induced liver injury by Mohamed A. Lounis; Quentin Escoula; Cathy Veillette; Karl-F. Bergeron; James M. Ntambi; Catherine Mounier (1662-1670).
Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.
Keywords: Alcoholic fatty liver disease; SCD1; De novo lipogenesis; Inflammation;

Genes and miRNA expression signatures in peripheral blood mononuclear cells in healthy subjects and patients with metabolic syndrome after acute intake of extra virgin olive oil by Simona D'Amore; Michele Vacca; Marica Cariello; Giusi Graziano; Andria D'Orazio; Roberto Salvia; Rosa Cinzia Sasso; Carlo Sabbà; Giuseppe Palasciano; Antonio Moschetta (1671-1680).
Extra virgin olive oil (EVOO) consumption has been associated with reduced cardiovascular risk but molecular mechanisms underlying its beneficial effects are not fully understood. Here we aimed to identify genes and miRNAs expression changes mediated by acute high- and low-polyphenols EVOO intake. Pre- and post-challenge gene and miRNAs expression analysis was performed on the peripheral blood mononuclear cells (PBMCs) of 12 healthy subjects and 12 patients with metabolic syndrome (MS) by using microarray and RT-qPCR. In healthy subjects, acute intake of EVOO rich in polyphenols was able to ameliorate glycaemia and insulin sensitivity, and to modulate the transcription of genes and miRNAs involved in metabolism, inflammation and cancer, switching PBMCs to a less deleterious inflammatory phenotype; weaker effects were observed in patients with MS as well as in healthy subjects following low-polyphenol EVOO challenge. Concluding, our study shows that acute high-polyphenols EVOO intake is able to modify the transcriptome of PBMCs through the modulation of different pathways associated with the pathophysiology of cardio-metabolic disease and cancer. These beneficial effects are maximized in healthy subjects, and by the use of EVOO cultivars rich in polyphenols. Nutrigenomic changes induced by EVOO thus legitimate the well-known beneficial effects of EVOO in promoting human health and, potentially, preventing the onset of cardiovascular disease and cancer.
Keywords: Extra virgin olive oil; Gene expression; miRNAs; PBMCs;

Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa by Swathi Banthiya; Jacqueline Kalms; Etienne Galemou Yoga; Igor Ivanov; Xavi Carpena; Mats Hamberg; Hartmut Kuhn; Patrick Scheerer (1681-1692).
Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48 Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system.
Keywords: Eicosanoids; Bacteria; Biomembranes; Protein X-ray crystallography; Protein structure; Inflammation; Infection;

Circulating levels of endocannabinoids and oxylipins altered by dietary lipids in older women are likely associated with previously identified gene targets by Bruce A. Watkins; Jeffrey Kim; Anne Kenny; Theresa L. Pedersen; Kirk L. Pappan; John W. Newman (1693-1704).
Postmenopausal women (PMW) report marginal n − 3 PUFA intakes and are at risk of chronic diseases associated with the skeletal, muscular, neuroendocrine, and cardiovascular systems. How n − 3 PUFA affect the amounts of endocannabinoids (ECs) and oxylipins (OLs) of metabolic and physiologic importance in PMW is not clear. Based on our recent findings that dietary n − 3 PUFA alter gene targets of the EC system and lower pro-inflammatory OL we proceeded to characterize these actions in blood of PMW. Our aim was to determine levels of the ECs, OLs, and global metabolites (GM) in white PMW (75 ± 7 y), randomized in a double-masked manner, from baseline to 6 mo after receiving a fish oil supplement of n − 3 PUFA (720 mg 20:5n3 + 480 mg 22:6n3/d, n = 20) or placebo (1.8 g oleic acid/d, n = 20). ECs and OLs in serum were determined by UPLC-MS/MS and GM by GC–MS and LC-MS/MS. Plasma 20:5n3 and 22:6n3 levels increased in PMW given fish oil. EC n − 6 acyl-ethanolamides, arachidonate-derived diols were decreased and 20:5n3 and 22:6n3 diols, epoxides, and alcohols were increased in PMW given fish oil. GM analysis revealed that n − 3 PUFA supplementation increased renal steroid hormone and proteolytic metabolite levels in PMW. Herein, we confirm that gene targets of the EC system, previously found as modifiable by n − 3 PUFA result in changes in the levels of ECs and OLs in PMW. This study shows phenotypic responses (in levels) to n − 3 PUFA supplementation in PMW and increases of n − 3 acyl-ethanolamide and n − 3-derived OL of clinical considerations in aging.
Keywords: Postmenopausal women; Endocannabinoids; Oxylipins; Global metabolites;

The endoplasmic reticulum (ER) has numerous biological functions including protein synthesis, protein folding, and lipid synthesis. The CAX4 gene encodes dolichyl pyrophosphate (Dol-PP) phosphatase, which is involved in protein N-glycosylation. In cax4Δ cells, the N-glycosylation of the vacuolar carboxypeptidase (CPY) was severely affected, and expression of the ER chaperone Kar2p was elevated, which resulted in UPR activation as an adaptive response. The cax4Δ cell growth was reduced, and this could be attributed to the formation of clumped aggregates, high vesiculation of the intracellular membrane, and plasma membrane alterations were depicted using DiOC6 fluorescence. In the cax4 deletion strain, the transcription factors INO2 and INO4 were upregulated, and the negative regulator OPI1 was concomitantly down regulated, which led to the derepression of the phospholipid genes CHO2, OPI3, PSD1, and PSD2 and resulted in increased phospholipid levels. However, the TAG, SE, and LD levels were significantly reduced, and FFA, sterol, and DAG levels were increased. These findings could be attributed to the derepression of the TAG and SE lipases TGL3, TGL4, TGL5, YEH1, and YEH2 and the repression of LRO1, DGA1, ARE1, and ARE2 in cax4Δ cells. Interestingly, the overexpression of SEC59 or CAX4 in cax4Δ cells prevented the ER stress and growth defect, and restored normal level of phospholipids, neutral lipids, and LDs. The current study revealed the disruption of N-glycosylation-induced ER stress, altered lipid homeostasis accounts for pleiotropic phenotype. Thus, CAX4 regulates membrane biogenesis by coordinating lipid homeostasis with protein quality control.
Keywords: ER stress; Protein glycosylation; Phospholipids; Lipid droplets; Triacylglycerol lipase;

Stearoyl-CoA desaturase-1 and adaptive stress signaling by Andreas Koeberle; Konstantin Löser; Maria Thürmer (1719-1726).
Stearoyl-CoA desaturase (SCD), the central enzyme in the biosynthesis of monounsaturated fatty acids, introduces a cis-Δ9 double bond into saturated fatty acids. SCD-1 has been proposed as promising target for the treatment of cancer, skin disorders and metabolic diseases, and strong efforts have been made during the last decade to develop clinical drug candidates. While the regulation and biological implications of SCD-1 have been extensively reviewed, the molecular mechanisms through which SCD-1 mediates cellular responses remained a mystery. An important aspect seems to be that SCD-1 induces adaptive stress signaling that maintains cellular persistence and fosters survival and cellular functionality under distinct pathological conditions. Here, we will first provide an overview about the function, regulation, structure and mechanism of SCD-1 and then focus on mitogenic and stress-related signal transduction pathways orchestrated by SCD-1. Moreover, we will discuss molecular mechanisms and potential lipid factors that link SCD-1 activity with initial signal transduction.Display Omitted
Keywords: Stearoyl-CoA desaturase; Monounsaturated fatty acid; Lipotoxicity; Stress adaption; Endoplasmic reticulum; Lipidomics;

The total and mitochondrial lipidome of Artemia franciscana encysted embryos by Emily Chen; Michael A. Kiebish; Justice McDaniel; Fei Gao; Niven R. Narain; Rangaprasad Sarangarajan; Gergely Kacso; Dora Ravasz; Thomas N. Seyfried; Vera Adam-Vizi; Christos Chinopoulos (1727-1735).
Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the ‘late embryogenesis abundant’ proteins, which are known to interact with lipid membranes.
Keywords: Extremophilia; Cardiolipin; Phosphatidylethanolamine; Ceramide; Phosphatidylglycerol; Phosphatidylserine;

Uncovering the benefits of fluctuating thermal regimes on cold tolerance of drosophila flies by combined metabolomic and lipidomic approach by Hervé Colinet; David Renault; Marion Javal; Petra Berková; Petr Šimek; Vladimír Koštál (1736-1745).
When exposed to constant low temperatures (CLTs), insects often suffer from cumulative physiological injuries that can severely compromise their fitness and survival. Yet, mortality can be considerably lowered when the cold stress period is interrupted by periodic warm interruption(s), referred to as fluctuating thermal regimes, FTRs. In this study, we have shown that FTRs strongly promoted cold tolerance of Drosophila melanogaster adults. We then assessed whether this marked phenotypic shift was associated with detectable physiological changes, such as synthesis of cryoprotectants and/or membrane remodeling. To test these hypotheses, we conducted two different time-series Omics analyzes in adult flies submitted to CLTs vs. FTRs: metabolomics (GC/MS) and lipidomics (LC/ESI/MS) targeting membrane phospholipids. We observed increasing levels in several polyhydric alcohols (arabitol, erythritol, sorbitol, mannitol, glycerol), sugars (fructose, mannose) and amino acids (serine, alanine, glutamine) in flies under CLT. Prolonged exposure to low temperature was also associated with a marked deviation of metabolic homeostasis and warm interruptions as short as 2 h were sufficient to periodically return the metabolic system to functionality. Lipidomics revealed an increased relative proportion of phosphatidylethanolamines and a shortening of fatty acyl chains in flies exposed to cold, likely to compensate for the ordering effect of low temperature on membranes. We found a remarkable correspondence in the time-course of changes between the metabolic and phospholipids networks, both suggesting a fast homeostatic regeneration during warm intervals under FTRs. In consequence, we suggest that periodic opportunities to restore system-wide homeostasis contribute to promote cold tolerance under FTRs.
Keywords: Cold stress; fluctuating thermal regimes; recovery; Omics; Drosophila;

Omega-3 fatty acid supplementation influences the whole blood transcriptome in women with obesity, associated with pro-resolving lipid mediator production by Anna Polus; Barbara Zapala; Urszula Razny; Anna Gielicz; Beata Kiec-Wilk; Malgorzata Malczewska-Malec; Marek Sanak; Caroline E. Childs; Philip C. Calder; Aldona Dembinska-Kiec (1746-1755).
The n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce low-grade inflammation associated with obesity. The relationship between therapeutic response to n-3 PUFAs and modification of the transcriptome in obesity or metabolic syndrome remains to be explored.Blood samples were obtained from women with obesity before and after three-months supplementation with a moderate dose of n-3 PUFAs (1.8 g EPA + DHA per day) or from controls. n-3 PUFAs (GC) and plasma concentrations of lipoxins, resolvins, protectin X (GC–MS/MS) and inflammatory markers (ELISA) were measured. Whole blood transcriptome was assayed using microarray.Women supplemented with n-3 PUFAs for 3 months had significantly higher levels of EPA and DHA in plasma phosphatidylcholine. n-3 PUFA supplementation, in contrast to placebo, significantly decreased the concentrations of several inflammatory markers (SELE, MCP-1, sVCAM-1, sPECAM-1, and hsCRP), fasting triglycerides and insulin and increased the concentrations of pro-resolving DHA derivatives in plasma. The microarray data demonstrated effects of n-3 PUFAs on PPAR-α, NRF2 and NF-κB target genes.N-3 PUFAs increased DHA-derived pro-resolving mediators in women with obesity. Elevated resolvins and up-regulation of the resolvin receptor occurred in parallel with activation of PPAR-α target genes related to lipid metabolism and of NRF2 up-regulated antioxidant enzymes.
Keywords: DHA and EPA supplementation; Human obesity; Inflammation; Microarray; Pro-resolving mediators;

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction by Shivantika Bisen; Olga Seleverstov; Jitendra Belani; Scott Rychnovsky; Alex M. Dopico; Anna N. Bukiya (1756-1766).
Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30–80 mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory β1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50 mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK β1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK β1 (KCNMB1) knockout mice. Thus, BK β1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.
Keywords: MaxiK channel; Cerebral artery; ent-Cholesterol; KCNMB1 knockout; Lipid-protein interaction;

Ceramide and polyunsaturated phospholipids are strongly reduced in human hepatocellular carcinoma by Sabrina Krautbauer; Elisabeth M. Meier; Lisa Rein-Fischboeck; Rebekka Pohl; Thomas S. Weiss; Alexander Sigruener; Charalampos Aslanidis; Gerhard Liebisch; Christa Buechler (1767-1774).
Lipid composition affects membrane function, cell proliferation and cell death and is changed in cancer tissues. Hepatocellular carcinoma (HCC) is an aggressive cancer and this study aimed at a comprehensive characterization of hepatic and serum lipids in human HCC. Cholesteryl ester were higher in tumorous tissues (TT) compared to adjacent non-tumorous tissues (NT). Free cholesterol exerting cytotoxic effects was not changed. Phosphatidylethanolamine, -serine (PS) and -inositol but not phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) were reduced in HCC tissues. Saturated species mostly increased and polyunsaturated species were diminished in all of these phospholipids. Ceramide (Cer) was markedly reduced in HCC tissues and higher levels of sphingomyelin suggest impaired sphingomyelinase activity as one of the underlying mechanisms. Importantly, ceramide in NT increased in HCC stage T3. Ceramide released from hepatocytes attracts immune cells and a positive association of the macrophage specific receptor CD163 with NT ceramide was identified. HCC associated lipid changes did not differ in patients suffering from type 2 diabetes. Protein levels of p53 were induced in TT and negatively correlated with Cer d18:1/16:0 and PS 36:1. Of the lipid species changed in HCC tissues only TT Cer d18:1/16:0, Cer d18:1/24:1, PC 38:6 and LPC 22:6 correlated with the respective serum levels. Our study demonstrates a considerably altered hepatic lipidome in HCC tissues. Ceramide was markedly reduced in HCC tissues, and therefore, raising ceramide levels specifically in the tumor represents a reasonable therapeutic approach for the treatment of this malignancy.
Keywords: Saturated phospholipids; Cholesterol; Sphingomyelin; CD163; p53;

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane by Paweł Mystek; Przemysław Dutka; Magdalena Tworzydło; Marta Dziedzicka-Wasylewska; Agnieszka Polit (1775-1786).
G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein–lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi3 prefer different types of membrane domains they are both co-localized with the D1 receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi3 and their heterotrimers, as well as the D1 receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-β-cyclodextrin or Fumonisin B1 led to the disruption of cholesterol–sphingolipids containing domains and changed the diffusion of Gαi3 and the D1 receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi3 prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D1 receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.
Keywords: G proteins; Dopamine D1 receptor; Sphingolipids; Cholesterol; Lipids binding motifs;

Acylation of lysine residues in human plasma high density lipoprotein increases stability and plasma clearance in vivo by Yaliu Yang; Corina Rosales; Baiba K. Gillard; Antonio M. Gotto; Henry J. Pownall (1787-1795).
Although human plasma high density lipoproteins (HDL) concentrations negatively correlate with atherosclerotic cardiovascular disease, underlying mechanisms are unknown. Thus, there is continued interest in HDL structure and functionality. Numerous plasma factors disrupt HDL structure while inducing the release of lipid free apolipoprotein (apo) AI. Given that HDL is an unstable particle residing in a kinetic trap, we tested whether HDL could be stabilized by acylation with acetyl and hexanoyl anhydrides, giving AcHDL and HexHDL respectively. Lysine analysis with fluorescamine showed that AcHDL and HexHDL respectively contained 11 acetyl and 19 hexanoyl groups. Tests with biological and physicochemical perturbants showed that HexHDL was more stable than HDL to perturbant-induced lipid free apo AI formation. Like the reaction of streptococcal serum opacity factor against HDL, the interaction of HDL with its receptor, scavenger receptor class B member 1 (SR-B1), removes CE from HDL. Thus, we tested and validated the hypothesis that selective uptake of HexHDL-[3H]CE by Chinese Hamster Ovary cells expressing SR-B1 is less than that of HDL-[3H]CE; thus, selective SR-B1 uptake of HDL-CE depends on HDL instability. However, in mice, plasma clearance, hepatic uptake and sterol secretion into bile were faster from HexHDL-[3H]CE than from HDL-[3H]CE. Collectively, our data show that acylation increases HDL stability and that the reaction of plasma factors with HDL and SR-B1-mediated uptake are reduced by increased HDL stability. In vivo data suggest that HexHDL promotes charge-dependent reverse cholesterol transport, by a mechanism that increases hepatic sterol uptake via non SR-B1 receptors, thereby increasing bile acid output.
Keywords: Reverse cholesterol transport; High density lipoprotein stability; Scavenger receptor class B member 1; High density lipoprotein acylation; Atherogenesis;

Recent research considerably changed our knowledge how cellular metabolism affects the immune system. We appreciate that metabolism not only provides energy to immune cells, but also actively influences diverse immune cell phenotypes. Fatty acid metabolism, particularly mitochondrial fatty acid oxidation (FAO) emerges as an important regulator of innate and adaptive immunity. Catabolism of fatty acids also modulates the progression of disease, such as the development of obesity-driven insulin resistance and type II diabetes. Here, we summarize (i) recent developments in research how FAO modulates inflammatory signatures in macrophages in response to saturated fatty acids, and (ii) the role of FAO in regulating anti-inflammatory macrophage polarization. In addition, we define the contribution of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptors (PPARs), in controlling macrophage biology towards fatty acid metabolism and inflammation.
Keywords: Macrophages; Fatty acids; Fatty acid oxidation; Metabolism; Inflammation; AMP-activated protein kinase;

d-3-Deoxy-dioctanoylphosphatidylinositol induces cytotoxicity in human MCF-7 breast cancer cells via a mechanism that involves downregulation of the D-type cyclin-retinoblastoma pathway by Cheryl S. Gradziel; Peter A. Jordan; Delilah Jewel; Fay J. Dufort; Scott J. Miller; Thomas C. Chiles; Mary F. Roberts (1808-1815).
Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. d-3-Deoxy-dioctanoylphosphatidylinositol (d-3-deoxy-diC8PI), but not compounds with altered inositol stereochemistry (e.g., l-3-deoxy-diC8PI and l-3,5-dideoxy-diC8PI), is cytotoxic. However, high resolution NMR field cycling relaxometry shows that both cytotoxic and non-toxic PIAs bind to the Akt1 PH domain at the site occupied by the cytotoxic alkylphospholipid perifosine. This suggests that another mechanism for cytotoxicity must account for the difference in efficacy of the synthetic short-chain PIAs. In MCF-7 breast cancer cells, with little constitutively active Akt, d-3-deoxy-diC8PI (but not l-compounds) decreases viability concomitant with increased cleavage of PARP and caspase 9, indicative of apoptosis. d-3-Deoxy-diC8PI also induces a decrease in endogenous levels of cyclins D1 and D3 and blocks downstream retinoblastoma protein phosphorylation. siRNA-mediated depletion of cyclin D1, but not cyclin D3, reduces MCF-7 cell proliferation. Thus, growth arrest and cytotoxicity induced by the soluble d-3-deoxy-diC8PI occur by a mechanism that involves downregulation of the D-type cyclin-pRb pathway independent of its interaction with Akt. This ability to downregulate D-type cyclins contributes, at least in part, to the anti-proliferative activity of d-3-deoxy-diC8PI and may be a common feature of other cytotoxic phospholipids.Display Omitted
Keywords: 3-Deoxy-phosphatidylinositol; Field-cycling NMR relaxometry; MCF-7 cells; D-type cyclin-retinoblastoma protein pathway;

Antagonizing effect of CLPABP on the function of HuR as a regulator of ARE-containing leptin mRNA stability and the effect of its depletion on obesity in old male mouse by Tasuku Nishino; Ryota Matsunaga; Hiroshi Jikihara; Moe Uchida; Akane Maeda; Guangying Qi; Takaya Abe; Hiroshi Kiyonari; Satoshi Tashiro; Kyoko Inagaki-Ohara; Fumio Shimamoto; Hiroaki Konishi (1816-1827).
Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein–RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3′UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.
Keywords: Cardiolipin; Mitochondria; Obesity; RNA granule; mRNA stability; Knockout mouse;

Regulation of glucose homeostasis and insulin action by ceramide acyl-chain length: A beneficial role for very long-chain sphingolipid species by Magdalene K. Montgomery; Simon H.J. Brown; Xin Y. Lim; Corrine E. Fiveash; Brenna Osborne; Nicholas L. Bentley; Jeremy P. Braude; Todd W. Mitchell; Adelle C.F. Coster; Anthony S. Don; Gregory J. Cooney; Carsten Schmitz-Peiffer; Nigel Turner (1828-1839).
In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N mice all developed glucose intolerance, while BALB/c mice displayed minimal deterioration in glucose tolerance and insulin action. Lipidomic analysis of livers across these five strains has revealed marked strain-specific differences in ceramide (Cer) and sphingomyelin (SM) species with high-fat feeding; with increases in C16-C22 (long-chain) and reductions in C > 22 (very long-chain) Cer and SM species observed in the four strains that developed HFD-induced glucose intolerance. Intriguingly, the opposite pattern was observed in sphingolipid species in BALB/c mice. These strain-specific changes in sphingolipid acylation closely correlated with ceramide synthase 2 (CerS2) protein content and activity, with reduced CerS2 levels/activity observed in glucose intolerant strains and increased content in BALB/c mice. Overexpression of CerS2 in primary mouse hepatocytes induced a specific elevation in very long-chain Cer, but despite the overall increase in ceramide abundance, there was a substantial improvement in insulin signal transduction, as well as decreased ER stress and gluconeogenic markers. Overall our findings suggest that very long-chain sphingolipid species exhibit a protective role against the development of glucose intolerance and hepatic insulin resistance.
Keywords: Insulin sensitivity and resistance; Ceramide species; Obesity; Lipid metabolism; Lipidomics; Endoplasmic reticulum stress;

Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation by Iuliia Filipenko; Stephanie Schwalm; Luca Reali; Josef Pfeilschifter; Doriano Fabbro; Andrea Huwiler; Uwe Zangemeister-Wittke (1840-1851).
Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P3 receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca2 +]i), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E2 synthase expression, and consequently with increased PGE2 synthesis. PGE2 synthesis was decreased by antagonists and siRNA against S1P3 and S1P2. Moreover, in parental MDA-MB-231 cells overexpression of S1P3 by cDNA transfection also increased PGE2 synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P3 antagonism, COX-2 inhibition or PGE2 receptor 2 (EP2) and 4 (EP4) antagonism, but not by S1P2 antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P3 and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE2 signaling via EP2 and EP4.
Keywords: Breast cancer metastasis; Sphingosine 1-phosphate; Sphingosine 1-phosphate receptor 3; Cyclooxygenase; Prostaglandin E2; Prostaglandin E2 receptor;

The effect of regulating molecules on the structure of the PPAR-RXR complex by Ortal Amber-Vitos; Navaneet Chaturvedi; Esther Nachliel; Menachem Gutman; Yossi Tsfadia (1852-1863).
The PPAR-RXR complex is one of the most significant and prevalent regulatory systems, controlling lipid metabolism by gene expression. Both proteins are members of the nuclear hormone receptor family, consisting of a ligand-binding domain (LBD), a hinge and a DNA binding domain (DBD). The two proteins form a heterodimer in the nucleus. The ligand-free complex interacts with corepressor proteins and blocks the expression of the genes. With the activating ligands and coactivator segments of regulating proteins, the heterodimer becomes active and allows translation of the genes under its control. We implemented model-independent all-atom molecular dynamics simulations for clarifying the structure changes that the activating ligand and the regulatory peptides impose on the PPAR-RXR system, starting with an LBD up to the PPAR-RXR-DNA complex. The simulations were carried out first with an active state of the protein. Once the relaxed state was attained, it was transformed into the inactive-state, the resulting structure was simulated. As the complex alternates between the active-inactive conformations, most of the changes are noticed at the junction area between the two subunits, located on the surface of a long fused helical structure made of H10–H11 of the proteins. The significant differences between the states included enhanced rigidity of the inactive complex, enhancement of tight contacts. The main drive for the transformation is the relocation of the tip of H12 of the PPAR that drives the carboxylate of the C-terminal towards the junction between H10–H11 of the RXR, leading to rearrangement of the main contact zone of the proteins.
Keywords: PPAR; RXR; Regulation; Heterodimer-complex; All-atom molecular dynamics;