BBA - Molecular and Cell Biology of Lipids (v.1862, #2)

Abscisic acid enhances glucose disposal and induces brown fat activity in adipocytes in vitro and in vivo by Laura Sturla; Elena Mannino; Sonia Scarfì; Santina Bruzzone; Mirko Magnone; Giovanna Sociali; Valeria Booz; Lucrezia Guida; Tiziana Vigliarolo; Chiara Fresia; Laura Emionite; Ambra Buschiazzo; Cecilia Marini; Gianmario Sambuceti; Antonio De Flora; Elena Zocchi (131-144).
Abscisic acid (ABA) is a plant hormone also present in animals, where it is involved in the regulation of innate immune cell function and of glucose disposal, through its receptor LANCL2. ABA stimulates glucose uptake by myocytes and pre-adipocytes in vitro and oral ABA improves glycemic control in rats and in healthy subjects. Here we investigated the role of the ABA/LANCL2 system in the regulation of glucose uptake and metabolism in adipocytes.Silencing of LANCL2 abrogated both the ABA- and insulin-induced increase of glucose transporter-4 expression and of glucose uptake in differentiated 3T3-L1 murine adipocytes; conversely, overexpression of LANCL2 enhanced basal, ABA- and insulin-stimulated glucose uptake. As compared with insulin, ABA treatment of adipocytes induced lower triglyceride accumulation, CO2 production and glucose-derived fatty acid synthesis. ABA per se did not induce pre-adipocyte differentiation in vitro, but stimulated adipocyte remodeling in terminally differentiated cells, with a reduction in cell size, increased mitochondrial content, enhanced O2 consumption, increased transcription of adiponectin and of brown adipose tissue (BAT) genes. A single dose of oral ABA (1 μg/kg body weight) increased BAT glucose uptake 2-fold in treated rats compared with untreated controls. One-month-long ABA treatment at the same daily dose significantly upregulated expression of BAT markers in the WAT and in WAT-derived preadipocytes from treated mice compared with untreated controls. These results indicate a hitherto unknown role of LANCL2 in adipocyte sensitivity to insulin-stimulated glucose uptake and suggest a role for ABA in the induction and maintenance of BAT activity.
Keywords: LANCL2; Insulin; WAT; BAT; MicroPET; Glycemic control;

Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice by Sandra F. Gallego; Richard R. Sprenger; Ditte Neess; Josch K. Pauling; Nils J. Færgeman; Christer S. Ejsing (145-155).
The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.
Keywords: Acyl-CoA binding protein; Weaning; Lipidomics; Lipid metabolism;

Reorganization of plasma membrane lipid domains during conidial germination by Filipa C. Santos; Andreia S. Fernandes; Catarina A.C. Antunes; Filipe P. Moreira; Arnaldo Videira; H. Susana Marinho; Rodrigo F.M. de Almeida (156-166).
Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted.Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30 °C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.Display Omitted
Keywords: Neurospora crassa; Saccharomyces cerevisiae; Fluorescence spectroscopy; Lipid domains/rafts; Fungal sphingolipids; Ergosterol;

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae by Yana Y. Toporkova; Valeria S. Fatykhova; Yuri V. Gogolev; Bulat I. Khairutdinov; Lucia S. Mukhtarova; Alexander N. Grechkin (167-175).
Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [18O2]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.
Keywords: Oxylipins; Cytochrome P450; CYP74 clan; Epoxyalcohol synthase; Brown alga Ectocarpus siliculosus;

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT by Mokrish Ajat; Martijn Molenaar; Jos F.H.M. Brouwers; Arie B. Vaandrager; Martin Houweling; J. Bernd Helms (176-187).
Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.
Keywords: Hepatic stellate cells; LRAT; DGAT1; Retinyl esters; Lipid droplets; Lipidomics;

Poor glycemic control in type 2 diabetes enhances functional and compositional alterations of small, dense HDL3c by Leonardo Gomez Rosso; Marie Lhomme; Tomas Meroño; Ana Dellepiane; Patricia Sorroche; Lyamine Hedjazi; Emile Zakiev; Vasily Sukhorukov; Alexander Orekhov; Julieta Gasparri; M. John Chapman; Fernando Brites; Anatol Kontush (188-195).
High-density lipoprotein (HDL) possesses multiple biological activities; small, dense HDL3c particles displaying distinct lipidomic composition exert potent antiatherogenic activities which can be compromised in dyslipidemic, hyperglycemic insulin-resistant states. However, it remains indeterminate (i) whether such functional HDL deficiency is related to altered HDL composition, and (ii) whether it originates from atherogenic dyslipidemia, dysglycemia, or both.In the present work we analyzed compositional characteristics of HDL subpopulations and functional activity of small, dense HDL3c particles in treatment-naïve patients with well-controlled (n = 10) and poorly-controlled (n = 8) type 2 diabetes (T2D) and in normolipidemic age- and sex-matched controls (n = 11).Our data reveal that patients with both well- and poorly-controlled T2D displayed dyslipidemia and low-grade inflammation associated with altered HDL composition. Such compositional alterations in small, dense HDL subfractions were specifically correlated with plasma HbA1c levels. Further analysis using a lipidomic approach revealed that small, dense HDL3c particles from T2D patients with poor glycemic control displayed additional modifications of their chemical composition. In parallel, antioxidative activity of HDL3c towards oxidation of low-density lipoprotein was diminished.These findings indicate that defective functionality of small, dense HDL particles in patients with T2D is not only affected by the presence of atherogenic dyslipidemia, but also by the level of glycemic control, reflecting compositional alterations of HDL.
Keywords: Type 2 diabetes; HDL; Antioxidative activity; Lipidomics; Functionality;

This review focuses on the chemistry and structures of (Brady)rhizobium lipids A, indispensable parts of lipopolysaccharides. These lipids contain unusual (ω-1) hydroxylated very long chain fatty acids, which are synthesized by a very limited group of bacteria, besides rhizobia. The significance and requirement of the very long chain fatty acids for outer membrane stability as well as the genetics of the synthesis pathway are discussed. The biological role of these fatty acids for bacterial life in extremely different environments (soil and intracellular space within nodules) is also considered.
Keywords: Lipid A; Nodule-forming bacteria; Rhizobium; Very long chain fatty acids (VLCFA);

Cholesteryl hemiesters alter lysosome structure and function and induce proinflammatory cytokine production in macrophages by Neuza Domingues; Luís M.B.B. Estronca; João Silva; Marisa R. Encarnação; Rita Mateus; Diogo Silva; Inês B. Santarino; Margarida Saraiva; Maria I.L. Soares; Teresa M.V.D. Pinho e Melo; António Jacinto; Winchil L.C. Vaz; Otília V. Vieira (210-220).
Cholesteryl hemiesters are oxidation products of polyunsaturated fatty acid esters of cholesterol. Their oxo-ester precursors have been identified as important components of the “core aldehydes” of human atheromata and in oxidized lipoproteins (Ox-LDL). We had previously shown, for the first time, that a single compound of this family, cholesteryl hemisuccinate (ChS), is sufficient to cause irreversible lysosomal lipid accumulation (lipidosis), and is toxic to macrophages. These features, coupled to others such as inflammation, are typically seen in atherosclerosis.To obtain insights into the mechanism of cholesteryl hemiester-induced pathological changes in lysosome function and induction of inflammation in vitro and assess their impact in vivo.We have examined the effects of ChS on macrophages (murine cell lines and primary cultures) in detail. Specifically, lysosomal morphology, pH, and proteolytic capacity were examined. Exposure of macrophages to sub-toxic ChS concentrations caused enlargement of the lysosomes, changes in their luminal pH, and accumulation of cargo in them. In primary mouse bone marrow-derived macrophages (BMDM), ChS-exposure increased the secretion of IL-1β, TNF-α and IL-6. In zebrafish larvae (wild-type AB and PU.1:EGFP), fed with a ChS-enriched diet, we observed lipid accumulation, myeloid cell-infiltration in their vasculature and decrease in larval survival. Under the same conditions the effects of ChS were more profound than the effects of free cholesterol (FC).Our data strongly suggest that cholesteryl hemiesters are pro-atherogenic lipids able to mimic features of Ox-LDL both in vitro and in vivo.
Keywords: Atherosclerosis; Cholesteryl hemiesters; Lysosome malfunction; Inflammation; Oxidized lipids; Zebrafish larvae;

Apolipoprotein A-I (apoA-I) is a prominent member of the exchangeable apolipoprotein class of proteins, capable of transitioning between lipid-bound and lipid-free states. It is the primary structural and functional protein of high density lipoprotein (HDL). Lipid-free apoA-I is critical to de novo HDL formation as it is the preferred substrate of the lipid transporter, ATP Binding Cassette Transporter A1 (ABCA1) Remaley et al. (2001) . Lipid-free apoA-I is an important element in reverse cholesterol transport and comprehension of its structure is a core issue in our understanding of cholesterol metabolism. However, lipid-free apoA-I is highly conformationally dynamic making it a challenging subject for structural analysis. Over the past 20 years there have been significant advances in overcoming the dynamic nature of lipid-free apoA-I, which have resulted in a multitude of proposed conformational models.
Keywords: Apolipoprotein A-I; High density lipoprotein cholesterol; Protein structure;

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts by Marina Sokolova; Leif Erik Vinge; Katrine Alfsnes; Maria Belland Olsen; Lars Eide; Ole Jørgen Kaasbøll; Håvard Attramadal; May-Kristin Torp; Linn E. Fosshaug; Azita Rashidi; Egil Lien; Alexandra Vanessa Finsen; Øystein Sandanger; Pål Aukrust; Trine Ranheim; Arne Yndestad (234-245).
Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3−/− and ASC−/−  mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1β, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated β-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.Display Omitted
Keywords: Inflammation; Palmitate; Cardiac fibroblast; TLR4; NLRP3; Cellular senescence;

Schistosomal-derived lysophosphatidylcholine triggers M2 polarization of macrophages through PPARγ dependent mechanisms by Leonardo Santos Assunção; Kelly G. Magalhães; Alan Brito Carneiro; Raphael Molinaro; Patrícia E. Almeida; Georgia C. Atella; Hugo C. Castro-Faria-Neto; Patrícia T. Bozza (246-254).
Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFβ and production of IL-10 and PGE2 24 h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis.
Keywords: Lysophosphatidylcholine; Macrophage; PPARgamma; Parasites; S. mansoni;

Epilepsy is a highly common chronic neurological disorder, manifested in many different types, affecting ~ 1% of the worldwide human population. The molecular mechanisms of epileptogenesis have not yet been clarified, and pharmacoresistance exhibited by 30–40% of epilepsy patients remains a major obstacle in medical care. Growing evidence indicates a role of lipid signalling pathways in epileptogenesis, thus lipid signals emerge as potential biomarkers for the onset and evolving course of the epileptic disorder, as well as potential therapeutic agents and targets. For this purpose, we applied a lipidomic strategy to unravel lipid alterations in brain regions, periphery tissues and plasma that are specific for acute epileptic seizures in mice at 1 h after seizure induction by systemic kainic acid injection as compared to vehicle controls. Specifically, levels of (i) selected phospholipids and sphingomyelins, (ii) the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), and the endocannabinoid-related compounds oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), (iii) arachidonic acid (AA), (iv) selected eicosanoids, and (v) fatty acyl content of lipidome were determined in pulverized tissues from six brain regions of kainic acid induced epileptic seizure models and vehicle controls: hypothalamus, hippocampus, thalamus, striatum, cerebellum and cerebral cortex, and from peripheral organs, such as heart and lungs, and in plasma. Alterations in lipid levels after acute epileptic seizures as compared to non-seizure controls were found to be brain region- and periphery tissue-specific, including specific plasma lipid correlates, highlighting their value as marker candidates in translational research studies, and/or drug discovery and response monitoring.Display Omitted
Keywords: Epilepsy; Lipid biomarkers; Phospholipids; Endocannabinoids; Eicosanoids; LC/MRM;

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500 × 300 μm and weigh ~ 20 μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2–1, fad3–2, fad6–1, and fae1–1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective.Display Omitted
Keywords: 3D; MALDI-MSI; Lipids; Arabidopsis; Phosphatidylcholine; Phosphatidylinositol;