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BBA - Molecular and Cell Biology of Lipids (v.1821, #4)

Editorial Board (pp. i).

Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages by Makoto Ayaori; Emi Yakushiji; Masatsune Ogura; Kazuhiro Nakaya; Tetsuya Hisada; Harumi Uto-Kondo; Shunichi Takiguchi; Yoshio Terao; Makoto Sasaki; Tomohiro Komatsu; Maki Iizuka; Makiko Yogo; Yoshinari Uehara; Hiroyuki Kagechika; Tsuyoshi Nakanishi; Katsunori Ikewaki (pp. 561-572).

ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All- trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms—RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.► Ligands for RARs promoted cholesterol efflux from macrophages by increasing ABCA1/G1. ► RARs regulated ABCG1 transcription driven by dual promoters. ► Its transcription involved LXR-responsive element-dependent pathways. ► RARα activated ABCG1 promoter by binding to LXR-responsive element.

Keywords: Abbreviations; ABCA1; ATP-binding cassette transporter A1; ABCG1; ATP-binding cassette transporter G1; AcLDL; acetylated low-density lipoprotein; apoA-I; apolipoprotein A-I; ATRA; all-; trans; retinoic acid; BSA; bovine serum albumin; DMEM; Dulbecco's modified Eagle's medium; DR; direct repeat; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HDL; high-density lipoprotein; LXR; liver X receptor; LXRE; LXR-responsive element; RAR; retinoic acid receptor; RXR; retinoid X receptor; Sult2b1; sulfotransferase family cytosolic 2B member 1; TTNPB; 4-[E-2-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl] benzoic acid; 5CPPSS-50; 5-chloro-N-2′-n-pentylphenyl-1,3-dithiophthalimide; 8-Br-cAMP; 8-bromoadenocine 3′,5′-cyclic monophosphate; 9cRA; 9-; cis; -retinoic acidRAR; ABCA1; ABCG1; HDL; Cholesterol efflux; LXR

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti by Solis-Oviedo Rosa L. Solís-Oviedo; Martinez-Morales Fernando Martínez-Morales; Otto Geiger; Christian Sohlenkamp (pp. 573-581).

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.► Alanine scanning mutagenesis of 55 residues identifies 9 essential amino acids. ► Four conserved aspartate residues are essential for Pcs activity. ► Sinorhizobial Pcs has eight transmembrane helices.

Keywords: Abbreviations; AAPT; aminoalcohol phosphotransferase; CL; cardiolipin; CPT; choline phosphotransferase; DAG; diacylglycerol; LacZ; β-galactosidase; PC; phosphatidylcholine; Pcs; phosphatidylcholine synthase; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PhoA; alkaline phosphatase; TLC; thin-layer chromatographyMembrane lipid biosynthesis; Phosphatidylcholine; Symbiosis; Topology analysis; Alanine scanning; CDP-alcohol phosphotransferase

Minimally modified LDL upregulates endothelin type B receptors in rat coronary artery via ERK1/2 MAPK and NF-κB pathways by Jie Li; Yong-Xiao Cao; Zu-Yi Yuan; Cang-Bao Xu (pp. 582-589).

Minimally modified low density lipoprotein (mmLDL) is a well-known risk factor for coronary artery disease. Upregulation of vascular endothelin type B (ET_B) receptors on the vascular smooth muscle cells is predicted to be the molecular mechanism that leads to cardiovascular pathogenesis. The objective of the present study was to examine the hypothesis that mmLDL upregulates ET_B receptors in rat coronary artery. The contractile responses to sarafotoxin 6c (ET_B receptor agonist) were studied using a sensitive myograph. ET_B receptor mRNA and protein expression was determined using real-time PCR and Western blot analysis. The results showed that organ culture increased the contractile responses induced by sarafotoxin 6c and the levels of ET_B receptor mRNA and protein. This increase was further enhanced by the addition of mmLDL (10μg/mL). Specific ERK1/2 inhibitors (SB386023 and U0126) and an NF-κB inhibitor (wedelolactone) attenuated the mmLDL-increased ET_B receptor-mediated contraction and ET_B receptor mRNA and protein levels. Wedelolactone significantly attenuated the mmLDL-decreased IκB_α protein expression. Consistent with this result, IκB_α protein expression was significantly decreased by culture with mmLDL compared to the level of expression in the organ culture group. However, the JNK inhibitor, SP600125 or p38 pathway inhibitor, SB203580 did not inhibit mmLDL-enhanced effects. The PKC inhibitor, staurosporine attenuated only culture-alone-increased effects. In conclusion, mmLDL upregulates the ET_B receptors in rat coronary arterial smooth muscle cells, mainly via activation of the ERK1/2 MAPK and the downstream transcriptional factor NF-κB.► Rat coronary artery was cultured in the present study. ► Minimally modified LDL upregulates ETB receptors in rat coronary artery. ► ERK and NF-κB inhibitors attenuate the upregulation. ► The upregulation occurs via ERK1/2 MAPK and NF-κB signal pathways.

Keywords: Minimally modified low density lipoprotein; Coronary artery; Endothelin type B receptor; Extracellular signal related kinases 1/2; Nuclear factor-κB

Minimally modified LDL upregulates endothelin type B receptors in rat coronary artery via ERK1/2 MAPK and NF-κB pathways by Jie Li; Yong-Xiao Cao; Zu-Yi Yuan; Cang-Bao Xu (pp. 582-589).

Keywords: Minimally modified low density lipoprotein; Coronary artery; Endothelin type B receptor; Extracellular signal related kinases 1/2; Nuclear factor-κB

Role of β-adrenergic receptors in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotien (ZAG) by Steven Thomas Russell; Michael John Tisdale (pp. 590-599).

Objectives: The goal of the current study is to determine whether the β-adrenoreceptor (β-AR) plays a role in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotein (ZAG). Material and methods: This has been investigated in CHO-K1 cells transfected with the human β1-, β2-, β3-AR and in ob/ob mice. Cyclic AMP assays were carried out along with binding studies. Ob/ob mice were treated with ZAG and glucose transportation and insulin were examined in the presence or absence of propranolol. Results: ZAG bound to the β3-AR with higher affinity (Kd 46±1nM) than the β2-AR (Kd 71±3nM) while there was no binding to the β1-AR, and this correlated with the increases in cyclic AMP in CHO-K1 cells transfected with the various β-AR and treated with ZAG. Treatment of ob/ob mice with ZAG increased protein expression of β3-AR in gastrocnemius muscle, and in white and brown adipose tissues, but had no effect on expression of β1- and β2-AR. A reduction of body weight was seen and urinary glucose excretion, increase in body temperature, reduction in maximal plasma glucose and insulin levels in the oral glucose tolerance test, and stimulation of glucose transport into skeletal muscle and adipose tissue, were completely attenuated by the non-specific β-AR antagonist propranolol. Conclusion: The results suggest that the effects of ZAG on body weight and insulin sensitivity in ob/ob mice are manifested through a β-3AR, or possibly a β2-AR.► Zinc-a2-glycoprotein (ZAG) binds and activates b3- and b2-adrenergic receptors, but not b1. ► ZAG through binding to these adrenergic receptors effects on body weight and insulin sensitivity. ► These interactions are shown for the first time using a recombinant form of ZAG.

Keywords: Abbreviations; AUC; area under the curve; AMPK; AMPkinase; β-AR; beta adrenergic receptor; BAT; brown adipose tissue; CHOK1; Chinese hamster ovary cells; DMEM; Dulbecco's modified Eagle's medium; ECL; enhanced chemiluminescence; FCS; foetal calf serum; LMF; lipid mobilizing factor; PBS; phosphate buffered saline; NEFA; non-esterified fatty acids; TG; triglycerides; UCP; uncoupling protein; WAT; white adipose tissue; ZAG; zinc-alpha2-glycoproteinZinc-α2-glycoprotein; β-Adrenoreceptor; Obesity; Diabetes

Role of β-adrenergic receptors in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotien (ZAG) by Steven Thomas Russell; Michael John Tisdale (pp. 590-599).

Lymphopenia induced by a novel selective S1P₁ antagonist structurally unrelated to S1P by Yasuyuki Fujii; Hidenori Ohtake; Naoya Ono; Toshifumi Hara; Takanobu Sakurai; Syuya Takahashi; Tetsuo Takayama; Yoshiki Fukasawa; Fumiyasu Shiozawa; Narutoshi Tsukahara; Takehiro Hirayama; Yasuyuki Igarashi; Ryo Goitsuka (pp. 600-606).

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P₁) and participates in many pathological conditions. We developed a novel type S1P₁-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P₁ resulting in reduced signaling downstream of S1P₁, including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P₁-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P₁ antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P₁ agonists on lymphocyte sequestration results from their functional antagonism.► Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P1). ► We developed a novel type S1P1-selective antagonist, TASP0251078. ► TASP0251078 induces lymphocyte sequestration from circulation and suppresses immune responses. ► TASP0251078 will be highly beneficial for the development of immunosuppressive drugs.

Keywords: Abbreviations; S1P; sphingosine 1-phosphate; S1P; ₁; type 1 sphingosine 1-phosphate receptor; CHS; contact hypersensitivity response; HEK293; human embryonic kidney cell line; CHO-K1; Chinese hamster ovary cell line; HTS; high-throughput screening; GTP; guanosine-5′-triphosphate; GDP; guanosine-5′-diphosphate; HEPES; N-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid; BSA; bovine serum albumin; cAMP; cyclic adenosine monophosphate; TNCB; 2,4,6-trinitrochlorobenzene; IC; ₅₀; half maximal (50%) inhibitory concentration; TC; ₅₀; half maximal (50%) toxic concentration; EIA; Enzyme-Linked ImmunoSorbent AssaySphingosine 1-phosphate (S1P); Type-1 S1P receptor (S1P; ₁; ); S1P; ₁; -selective antagonist; Lymphopenia; Immunosuppressive agent; Chemotaxis

Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia by Armen Khatchadourian; Simon D. Bourque; Vincent R. Richard; Vladimir I. Titorenko; Dusica Maysinger (pp. 607-617).

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/β (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A₂ (cPLA₂-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/β in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.► LPS treatment induces triglyceride synthesis and LD formation in microglia. ► LPS treatment leads to activation of p38 and JNK MAPKs. ► Perilipin-2 expression is increased following LPS treatment. ► Inhibition of p38 blocks LD accumulation and perilipin-2 expression. ► Cytosolic phospholipase A2-α colocalizes with LPS-induced LDs.

Keywords: Abbreviations; LD; lipid droplet; TG; triglyceride; TLR4; Toll-like receptor 4; LPS; lipopolysaccharides; ADRP; adipocyte differentiation-related protein; CNS; central nervous system; NO; nitric oxide; MAPK; mitogen-activated protein kinases; JNK; c-Jun N-terminal kinase; OA; oleic acid; BODIPY 493/503; 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; PI3K; phosphatidylinositol 3-kinase; MKK; MAPK kinase; AP-1; activator protein-1; FFA; free fatty acid; cPLA; ₂; -α; cytosolic phospholipase A; ₂; alphaLipid droplets; Microglia activation; Lipopolysaccharides; Perilipin-2; JNK phosphorylation; p38 phosphorylation

Diacylglycerol-containing oleic acid induces increases in [Ca²⁺]_i via TRPC3/6 channels in human T-cells by Carrillo Perez Celia Carrillo; Aziz Hichami; Pierre Andreoletti; Mustapha Cherkaoui-Malki; María del Mar Cavia; Souleymane Abdoul-Azize; Sara R. Alonso-Torre; Naim Akhtar Khan (pp. 618-626).

Though most of the studies have focused on the effects of free fatty acids on T-cell activation, fatty acids incorporated into plasma membrane phospholipids may also affect cell signaling via diacylglycerol (DAG), generally produced by phospholipid hydrolysis. In the present study, we have synthesized a DAG-containing oleic acid and studied its implication in the modulation of calcium signaling in human Jurkat T-cells. 1-palmitoyl-2-oleoyl- sn-glycerol (POG) induced a dose-dependent increase in [Ca²⁺]_i. This effect was due to the presence of oleic acid at the sn-2 position as no differences were observed between POG and 1-stearoly-2-oleoyl- sn-glycerol (SOG). However, the substitution of oleic acid with arachidonic acid at the sn-2 position of the DAG moiety exerted a different response on the increases in [Ca²⁺]_i in these cells. POG-evoked increases in [Ca²⁺]_i were not due to its metabolites. Furthermore, POG-induced increases in [Ca²⁺]_i were due to the opening of TRPC3/TRPC6 channels as silencing of TRPC3 and TRPC6 genes by shRNA abolished calcium entry. Moreover, disruption of lipid rafts with methyl-β-cyclodextrin completely abolished POG-evoked increases in [Ca²⁺]_i. In conclusion, our results demonstrate that oleic acid can influence T-lymphocyte functions, in the conjugated form of DAG, via opening TRPC3/6 channels.► DAG-containing OA induces increases in [Ca²⁺]_i in human T-cells. ► Such increases are via opening of TRPC3 and TRPC6 channels. ► Oleic acid at the sn-2 position confers the molecular property to the diacylglycerol. ► Disruption of lipid rafts completely abolished POG-evoked increases in [Ca²⁺]_i.

Keywords: Diacylglycerols; T-cells; Fatty acids; Lipids; Calcium channels; Calcium signaling; TRPC3; TRPC6; shRNA

Cardiac lipoprotein lipase activity in the hypertrophied heart may be regulated by fatty acid flux by David Hauton; Germaine M. Caldwell (pp. 627-636).

Cardiac hypertrophy is characterised by an imbalance between lipid uptake and fatty acid β-oxidation leading to an accumulation of lipids, particularly triacylglycerol (TAG). It is unclear whether uptake mechanisms such as lipoprotein lipase (LPL) can be attenuated to diminish this uptake. Rats were cold acclimated to induce cardiac hypertrophy and increase cardiac LPL. Lipid uptake and metabolism were altered by feeding a ‘Western-style’ high fat diet (WSD) or feeding oxfenicine (2g/L) in the drinking water. Diastolic stiffness (increased volume change/unit pressure change) was induced in hypertrophied hearts for rats fed WSD (P<0.05) or WSD+oxfenicine (P<0.01), although absolute performance of cardiac muscle, estimated from stress–strain calculations was unchanged. Cold acclimation increased cardiac endothelial LPL (P<0.05) but this was diminished following oxfenicine. Following WSD LPL was further decreased below WSD-fed control hearts (P<0.05) with no further decrease by oxfenicine supplementation. A negative correlation was noted between plasma TAG and endothelial LPL (correlation coefficient=−0.654; P<0.001) but not cardiac TAG concentration. Transcript levels of angiopoietin-like protein-4 (ANGPTL4) were increased 6-fold by WSD (P<0.05) and increased 15-fold following WSD+oxfenicine (P<0.001). For CA-hearts fed WSD or WSD+oxfenicine ANGPTL4 mRNA levels were preserved at chow-fed levels. VLDLR protein levels were increased 10-fold (P<0.01) by CA. ANGPTL4 protein levels were increased 2-fold (P<0.05) by WSD, but restored following oxfenicine. For CA-hearts WSD increased ANGPTL4 protein levels 3-fold (P<0.01) with WSD+oxfenicine increasing ANGPTL4 protein 4-fold (P<0.01). These data suggest that endothelial LPL levels in the heart are altered to maintain FA flux and may exploit ANGPTL4.► Cardiac endothelial LPL inversely correlates with plasma triacylglycerol. ► Heart TAG levels do not relate to tissue LPL levels. ► Cardiac ANGPTL4 protein levels increased in response to high plasma lipids. ► LPL in the heart may be controlled to maintain fatty acid flux.

Keywords: Lipoprotein lipase; Very-low-density lipoprotein; Cardiac hypertrophy; ANGPTL4

Cardiac lipoprotein lipase activity in the hypertrophied heart may be regulated by fatty acid flux by David Hauton; Germaine M. Caldwell (pp. 627-636).

Keywords: Lipoprotein lipase; Very-low-density lipoprotein; Cardiac hypertrophy; ANGPTL4

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization by Jiahn-Dar Huang; Juan Amaral; Jung Wha Lee; Ignacio M. Larrayoz; Ignacio R. Rodriguez (pp. 637-646).

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5–10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.► Sterculic acid is a potent inhibitor of ER-stress induced by 7KCh. ► 7KCh is a potent inhibitor of SCD expression. ► SCD inhibition by sterculic acid is unlikely related to anti-inflammatory effects. ► The laser-injury rat angiogenesis model likely to be associated with 7KCh formation. ► Mechanistic data supports 7KCh's involvement in ER-stress related inflammation.

Keywords: ER stress; 7-ketocholesterol; Inflammation; Sterculic acid; CHOP; RPE

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae by Miroslava Spanova; Dagmar Zweytick; Karl Lohner; Lisa Klug; Erich Leitner; Albin Hermetter; Günther Daum (pp. 647-653).

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127–6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.► Deletion of HEM1 causes squalene accumulation in yeast. ► Squalene accumulated in lipid droplets has a disordering effect. ► In biological membranes squalene modulates fluidity/rigidity.

Keywords: Abbreviations; LP; lipid particles; PL; phospholipids; SQ; squalene; SE; steryl ester(s); TG; triacylglycerol(s); QM; quadruple mutantSqualene; Ergosterol; Lipid droplet; Plasma membrane; Fluidity; Yeast

The role of lecithin:cholesterol acyltransferase in the modulation of cardiometabolic risks — A clinical update and emerging insights from animal models by Dominic S. Ng (pp. 654-659).

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in mediating the esterification of cholesterol on circulating lipoproteins. It has long been suggested that LCAT plays a crucial role in reverse cholesterol transport, a process depicting the removal of cellular cholesterol through efflux to high density lipoproteins (HDL) and its delivery to the liver for eventual excretion from the body. Although loss-of-function LCAT mutations invariably result in profound HDL deficiency, the role of LCAT in atherogenesis continues to be clouded with controversy. Increasing number of large scale, population-based studies failed to detect an elevated cardiac risk with reduced blood levels of LCAT, suggesting that reduced LCAT activity may not be a risk factor nor a therapeutic target. More recent studies in human LCAT gene mutation carriers tend to suggest that atherogenicity in LCAT deficiency may be dependent on the nature of the mutations, providing plausible explanations for the otherwise contradictory findings. Genetic models of LCAT excess or deficiency yielded mixed findings. Despite its known profound effects on HDL and triglyceride metabolism, the role of LCAT in metabolic disorders, including obesity and diabetes, has not received much attention. Recent studies in LCAT deficient mouse models suggest that absence of LCAT may protect against insulin resistance, diabetes and obesity. Coordinated modulation of a number of anti-obesity and insulin sensitizing pathways has been implicated. Further studies to explore the role of LCAT in the modulation of cardiometabolic disorders and the underlying mechanisms are warranted.► LCAT is a major modulator of HDL metabolism. ► Complete LCAT deficiency causes profound low HDL and modest hypertriglyceridemia. ► The role of LCAT in atherosclerosis remains controversial despite the dyslipidemia. ► Recent animal model studies revealed novel protective metabolic phenotypes. ► LCAT null mice are more insulin sensitive and resists diet induced obesity.

Keywords: Lecithin cholesterol acyltransferase (LCAT); High density lipoproteins; Atherosclerosis; Insulin resistance; ER stress; Brown adipose tissue

The role of lecithin:cholesterol acyltransferase in the modulation of cardiometabolic risks — A clinical update and emerging insights from animal models by Dominic S. Ng (pp. 654-659).

Keywords: Lecithin cholesterol acyltransferase (LCAT); High density lipoproteins; Atherosclerosis; Insulin resistance; ER stress; Brown adipose tissue

Long chain polyunsaturated fatty acid synthesis in a marine vertebrate: Ontogenetic and nutritional regulation of a fatty acyl desaturase with Δ4 activity by Sofia Morais; Filipa Castanheira; Laura Martinez-Rubio; Conceicao Luis E.C. Conceição; Douglas R. Tocher (pp. 660-671).

Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally characterized fatty acyl desaturase and elongase cDNAs corresponding to Δ4fad (with some Δ5 activity for the n−3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to higher or lower dietary n−3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Δ4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Δ4fad was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway.► We functionally characterized a fatty acyl desaturase and elongase in sole. ► Fad has Δ4-activity and Elovl has substrate specificity typical of fish Elovl5. ► Sole appears able to synthesize DHA from EPA via a Sprecher-independent pathway. ► Δ4fad and elovl5 showed a marked pattern of regulation during early ontogenesis. ► Δ4fad shows nutritional regulation, up-regulated in response to LC-PUFA-poor diets.

Keywords: Abbreviations; ALA; α-linolenic acid; ARA; arachidonic acid; DAH; days after hatching; DHA; docosahexaenoic acid; DPA; docosapentaenoic acid; EFA; essential fatty acid; EPA; eicosapentaenoic acid; FA; fatty acid; FAME; Fatty acid methyl esters; LC-PUFA; long-chain polyunsaturated fatty acids; LOA; linoleic acid; ORF; open reading frame; PCR; polymerase chain reaction; RACE; rapid amplification of cDNA endsΔ4 desaturase; Polyunsaturated fatty acid synthesis; Ontogenesis; Transcriptional regulation; Solea senegalensis; Marine teleost

Retinoic acid increases hypoxia-inducible factor-1α through intracrine prostaglandin E₂ signaling in human renal proximal tubular cells HK-2 by Fernandez-Martinez Ana B. Fernández-Martínez; Arenas Jimenez María I. Arenas Jiménez; Lucio Cazana Francisco J. Lucio Cazaña (pp. 672-683).

We have previously shown in HK-2 cells that ATRA (all- trans-retinoic acid) up-regulates HIF-1α (hypoxia-inducible factor-1α) in normoxia, which results in increased production of renal protector VEGF-A (vascular endothelial growth factor-A). Here we investigated the role of COXs (cyclooxygenases) in these effects and we found that, i) ATRA increased the expression of COX-1 and COX-2 mRNA and protein and the intracellular levels (but not the extracellular ones) of PGE₂. Furthermore, inhibitors of COX isoenzymes blocked ATRA-induced increase in intracellular PGE₂, HIF-1α up-regulation and increased VEGF-A production. Immunofluorescence analysis found intracellular staining for EP1-4 receptors (PGE₂ receptors). These results indicated that COX activity is critical for ATRA-induced HIF-1α up-regulation and suggested that intracellular PGE₂ could mediate the effects of ATRA; ii) Treatment with PGE₂ analog 16,16-dimethyl-PGE₂ resulted in up-regulation of HIF-1α and antagonists of EP1-4 receptors inhibited 16,16-dimethyl-PGE₂- and ATRA-induced HIF-1α up-regulation. These results confirmed that PGE₂ mediates the effects of ATRA on HIF-1α expression; iii) Prostaglandin uptake transporter inhibitor bromocresol green blocked the increase in HIF-1α expression induced by PGE₂ or by PGE₂-increasing cytokine interleukin-1β, but not by ATRA. Therefore only intracellular PGE₂ is able to increase HIF-1α expression. In conclusion, intracellular PGE₂ increases HIF-1α expression and mediates ATRA-induced HIF-1α up-regulation.Display Omitted

Keywords: Hypoxia inducible factor-1α; All trans retinoic acid; Cyclooxigenases; Prostaglandin E; ₂; Human proximal tubular cells

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis by Elodie Lafont; Romain Dupont; Nathalie Andrieu-Abadie; Toshiro Okazaki; Klaus Schulze-Osthoff; Thierry Levade; Hervé Benoist; Segui Bruno Ségui (pp. 684-693).

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.► Caspase-9 is dispensable for ceramide generation triggered by CD95L. ► Caspase-9 activation is enhanced by endogenous ceramide. ► Ceramide induces apoptosis in a caspase-9-dependent manner.

Keywords: Abbreviations; Bcl-2/xL/x(s); B-cell lymphoma-2/xL/x(s); Bid; Bcl-2 interacting domain; DISC; death-inducing signaling complex; GlcCer; glucosylceramide; GCS; glucosylceramide synthase; SM; sphingomyelin; SMS; sphingomyelin synthaseSphingolipid; Sphingomyelin; Sphingomyelin synthase 1; Death receptor; Mitochondria; Cytochrome c

Lysophosphatidylinositol signalling: New wine from an old bottle by Pineiro Roberto Piñeiro; Marco Falasca (pp. 694-705).

Lysophosphatidylinositol (LPI) is a bioactive lipid generated by phospholipase A2 which is believed to play an important role in several diseases. Indeed LPI can affect various functions such as cell growth, differentiation and motility, in a number of cell-types, including cancer cells, endothelial cells and nervous cells. Despite the fact that LPI-induced cellular functions had been known for more than twenty years, the recent discovery that in several cell-types the orphan G protein-coupled receptor GPR55 acts as the specific receptor for LPI has fuelled novel interest in this lysolipid. Different research groups, including our own, have recently suggested that LPI may be the specific and functional ligand for GPR55, triggering signalling cascades that are relevant to cell proliferation, migration, survival and tumourigenesis. Recently published data suggest that the LPI/GPR55 axis plays an important role in different physiological and pathological contexts. Here we review the available data supporting the role of LPI in cell signalling and the pharmacology of its putative receptor GPR55.► LPI regulates diverse physiological and pathophysiological processes. ► GPR55 is a specific and a functional receptor of LPI. ► LPI/GPR55 axis plays a role in physiological and pathological contexts. ► LPI and GPR55 are relevant for the proliferation and migration of cancer cells. ► LPI/GPR55 axis could be a possible therapeutic target.

Keywords: Lysophospolipid; Lysophosphatidylinositol; GPCR; GPR55

Lysophosphatidylinositol signalling: New wine from an old bottle by Pineiro Roberto Piñeiro; Marco Falasca (pp. 694-705).

Keywords: Lysophospolipid; Lysophosphatidylinositol; GPCR; GPR55

Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7 macrophages by U. Stemmer; C. Ramprecht; E. Zenzmaier; Stojcic B. Stojčić; G. Rechberger; M. Kollroser; A. Hermetter (pp. 706-718).

The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl- sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid–protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid.► Identification of primary protein targets of truncated phospholipids. ► Covalent modification of proteins by aldehydophospholipids. ► Exchange of fluorescent aldehydophospholipids between cells, lipoproteins and albumin. ► Imaging of fluorescent oxidized phospholipids in live cells.

Keywords: Abbreviations; aSMase; acid sphingomyelinase; BSA; bovine serum albumin; BY; BODIPY™; DMEM; Dulbecco's modified Eagle's medium; HAEC; human aortic endothelial cells; IL; interleukin; LDL; low density lipoprotein; MAPK; mitogen-activated protein kinase; mmLDL; minimally modified LDL; oxLDL; oxidized LDL; oxPL; oxidized phospholipids; PAF; platelet activating factor; PAPC; 1-palmitoyl-2-arachidonoyl-; sn; -glycero-3-phosphocholine; Paze-PC; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PGPC; 1-palmitoyl-2-glutaroyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POVPC; 1-palmitoyl-2-(5-oxovaleroyl)-; sn; -glycero-3-phosphocholine; Poxno-PC; 1-palmitoyl-2-(9-oxo-nonanoyl)-; sn; -glycero-3-phosphocholine; PS; phosphatidylserine; ROS; reactive oxygen species; TCA; trichloroacetic acid; TLC; thin layer chromatography; TLR; toll-like receptor; VDAC; voltage dependent anion channelAldehydophospholipid; Schiff base; Atherosclerosis; Phospholipid exchange; Oxidized LDL; Functional proteomic analysis

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BBA - Molecular and Cell Biology of Lipids (v.1821, #4)

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BBA - Molecular and Cell Biology of Lipids (v.1821, #4)