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BBA - Molecular and Cell Biology of Lipids (v.1831, #6)

Editorial Board (pp. i).

Regulation of mevalonate metabolism in cancer and immune cells by Martin Thurnher; Georg Gruenbacher; Oliver Nussbaumer (pp. 1009-1015).

The mevalonate pathway is a highly conserved metabolic cascade and provides isoprenoid building blocks for the biosynthesis of vital cellular products such as cholesterol or prenyl pyrophosphates that serve as substrates for the posttranslational prenylation of numerous proteins. The pathway, which is frequently hyperactive in cancer cells, is considered an important target in cancer therapy, since prenylated members of the Ras superfamily are crucially involved in the control of proliferation, survival, invasion and metastasis of tumour cells. Upstream accumulation and downstream depletion of mevalonate pathway intermediates as induced for instance by aminobisphosphonates translate into different effects in cancer and immune cells. Thus, mevalonate pathway regulation can affect tumour biology either directly or exhibit indirect antitumour effects through stimulating cancer immune surveillance. The present review summarizes major effects of pharmacologic mevalonate pathway regulation in cancer and immune cells that may collaboratively contribute to the efficacy of cancer therapy.•p53 inactivation by mutation can enhance mevalonate metabolism in cancer cells.•Mevalonate pathway inhibitors may be able to reverse the malignant phenotype.•Mevalonate pathway inhibitors also have multifaceted immunostimulatory effects.•The mevalonate pathway is an important target in cancer therapy.

Keywords: Mevalonate; Statins; Bisphosphonates; Prenylation; Caspase-1; Innate immunity

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia by Ji-Sun Jung; Kyong-Oh Shin; Yong-Moon Lee; Jin A. Shin; Eun-Mi Park; Jinju Jeong; Dong-Hyun Kim; Ji Woong Choi; Hee-Sun Kim (pp. 1016-1026).

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.► Short chain ceramides inhibited iNOS and proinflammatory cytokines in microglia. ► C2 ceramide suppressed microglial activation in the brain of septic mice. ► C2 ceramide inhibited ROS, MAPKs, PI3K/Akt and Jak/STAT pathways. ► C2 ceramide upregulated PKA and hemeoxygenase-1 expressions. ► C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions.

Keywords: Abbreviations; ARE; antioxidant response element; C1P; ceramide-1-phosphate; CRE; cAMP responsive element; HO-1; heme oxygenase-1; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; MMP; matrix metalloproteinase; Nrf2; nuclear factor E2-related factor-2; ROS; reactive oxygen species; aSMase; acidic sphingomyelinase; nSMase; neutral sphingomyelinase; S1P; sphingosine-1-phosphate; TLR4; toll like receptor4Microglia; C2 ceramide; Short chain ceramide; Neuroinflammation; TLR4; Molecular mechanism

Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes by Daniela Bonofiglio; Antonella Santoro; Emanuela Martello; Donatella Vizza; Daniela Rovito; Anna Rita Cappello; Ines Barone; Cinzia Giordano; Salvatore Panza; Stefania Catalano; Vito Iacobazzi; Vincenza Dolce; Ando Sebastiano Andò (pp. 1027-1036).

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.Display Omitted► PPARγ regulates CIC expression during fibroblast differentiation into fat cells. ► Activated PPARγ up-regulates CIC expression in fibroblasts, but not in adipocytes. ► In fibroblasts PPARγ ligand enhances the recruitment of PPARγ/Sp1/PGC1α、ARA70 coactivators to CIC promoter. ► In adipocytes PPARγ/Sp1/SMRT corepressor are recruited on CIC promoter. ► Divergent effects of PPARγ on CIC expression during adipogenesis are proposed.

Keywords: Citrate carrier; Mitochondrion; PPARgamma; Adipocyte; Sp1

Ubiquitous transcription factor YY1 promotes zebrafish liver steatosis and lipotoxicity by inhibiting CHOP-10 expression by Guor Mour Her; Wan-Yu Pai; Chi-Yu Lai; Yang-Wen Hsieh; Hsi-Wen Pang (pp. 1037-1051).

The ubiquitous transcription factor Yin Yang 1 (YY1) is known to have diverse and complex cellular functions. Although relevant literature has reported that YY1 expression can induce the down-regulation of C/EBP homologous protein 10 (CHOP-10) and then allow the transactivation of certain transcription factors required for lipogenesis, similar properties of YY1 are poorly understood in animal model systems. In this study, we demonstrate hepatic lipid accumulation in YY1 transgenic zebrafish (GY). Oil-red staining cells were predominantly increased in the livers of both GY larvae and adults, indicating that YY1 functionally promoted lipid accumulation in GY livers. Molecular analysis revealed that YY1 over-expression contributed to the accumulation of hepatic triglycerides (TGs) by inhibiting CHOP-10 expression in the juvenile GY and 3 other fish cell lines; the decreased CHOP-10 expression then induced the transactivation of C/EBP-α and PPAR-γ expression. CHOP-10 morpholino (MO)-injected and rosiglitazone-treated G-liver larvae showed liver steatosis by transactivating PPAR-γ. PPAR-γ MO-injected, and GW9662- and astaxanthin-treated GY larvae showed no liver steatosis by inhibiting PPAR-γ. Moreover, a fatty acid (FA) accumulation and a TG decrease were found in the liver of aged GY, leading to the induction of FA-oxidizing systems that increased hepatic oxidative stress and liver damage. This study is the first to examine YY1 as a potential stimulator for GY liver steatosis and lipotoxicity.► Overexpression of YY1 induces hepatic steatosis in zebrafish. ► YY1 alters the expression of 16 hepatic genes associated with lipid metabolism. ► YY1 induces C/EBP-α and PPAR-γ expression by suppressing CHOP-10 expression. ► Overexpression of YY1 increases the gene expression of de novo FA synthesis. ► Overexpression of YY1 increases hepatic lipotoxicity and liver injury.

Keywords: Hepatic steatosis; Lipid metabolism; Liver hypoplasia; Lipotoxicity; YY1

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles by Leah E. Ross; Xunjun Xiao; Mark E. Lowe (pp. 1052-1059).

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile–salt–oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I₃₃₀/I₃₅₀ ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.•Tyr 55 of colipase inserts into mixed micelles.•Phe 84 is critical for colipase structure.•Phe 84 does not interact with lipid or mixed micelle interfaces.•Amino acids 31–36 influence colipase adsorption to lipid interfaces.•The second and third loops have a major role colipase adsorption to interfaces.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; PTL; pancreatic triglyceride lipase; NaTDC; sodium taurodeoxycholateColipase; Dietary lipid; Mixed micelle; Pancreatic lipase; Site-directed mutagenesis

New insights on the role of ceramide 1-phosphate in inflammation by Gomez-Munoz Antonio Gomez-Muñoz; Patricia Gangoiti; Lide Arana; Alberto Ouro; Io-Guané Rivera; Ordonez Marta Ordoñez; Miguel Trueba (pp. 1060-1066).

Inflammation is a complex biological process involving a variety of locally produced molecules, as well as different types of white blood cells. Some of the so-called inflammatory mediators include cytokines, chemokines, interleukins, prostaglandins, or bioactive lipids, all of which provide protection from infection and foreign substances, such as bacteria, yeast, viruses or some chemicals. Under some circumstances, however, the organism inappropriately activates the immune system triggering an inflammatory response in the absence of foreign insults thereby leading to the establishment of autoimmune diseases. Therefore, inflammation must be tightly regulated in order to ensure sufficient protection to the organism in the absence of unwanted, and at times dangerous, side effects. Increasing experimental evidence implicates sphingolipids as major inducers of inflammatory responses and regulators of immune cell functions. In particular, ceramides and sphingosine 1-phosphate have been extensively implicated in inflammation, and ceramide 1-phosphate has also been shown to participate in these processes. The present review highlights novel aspects on the regulation of inflammation by sphingolipids, with special emphasis to the role played by ceramide 1-phoshate and ceramide kinase, the enzyme responsible for its biosynthesis, in inflammatory responses.► Ceramide 1-phosphate (C1P) is mitogenic and antiapoptotic for different cell types. ► C1P induces macrophage or stem cell migration through a G_i protein-coupled receptor. ► C1P can act both as pro-inflammatory or anti-inflammatory agent.

Keywords: Cell survival; Cell migration; Ceramide; Ceramide 1-phosphate; Inflammation; Sphingolipid

Characterization of palmitoylation of ATP binding cassette transporter G1: Effect on protein trafficking and function by Hong-mei Gu; Ge Li; Xia Gao; Luc G. Berthiaume; Da-wei Zhang (pp. 1067-1078).

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol efflux onto lipidated apolipoprotein A-I and HDL and plays a role in various important physiological functions. However, the mechanism by which ABCG1 mediates cholesterol translocation is unclear. Protein palmitoylation regulates many functions of proteins such as ABCA1. Here we investigated if ABCG1 is palmitoylated and the subsequent effects on ABCG1-mediated cholesterol efflux. We demonstrated that ABCG1 is palmitoylated in both human embryonic kidney 293 cells and in mouse macrophage, J774. Five cysteine residues located at positions 26, 150, 311, 390 and 402 in the NH₂-terminal cytoplasmic region of ABCG1 were palmitoylated. Removal of palmitoylation at Cys311 by mutating the residue to Ala (C311A) or Ser significantly decreased ABCG1-mediated cholesterol efflux. On the other hand, removal of palmitoylation at sites 26, 150, 390 and 402 had no significant effect. We further demonstrated that mutations of Cys311 affected ABCG1 trafficking from the endoplasmic reticulum. Therefore, our data suggest that palmitoylation plays a critical role in ABCG1-mediated cholesterol efflux through the regulation of trafficking.► ABCG1 is palmitoylated at Cys 26, Cys150, Cys311, Cys390 and Cys402. ► Removal of palmitoylation at Cys311 decreased ABCG1-mediated cholesterol efflux. ► Palmitoylation regulates ABCG1 trafficking. ► ABCG1 is palmitoylated in macrophages.

Keywords: Abbreviations; 15-HDYA; 15-hexadecynoic acid; 17-ODYA; 17-octadecynoic acid; ABC; ATP-binding cassette transporter; apo; apolipoprotein; BK channels; voltage- and calcium-activated potassium channels; BSA; bovine serum albumin; DAPI; 4′,6-diamidino-2-phenylindole; ECL; enhanced chemiluminescence; HDL; high density lipoprotein; HEK; human embryonic kidney; HRP; horseradish peroxidase; ldlr; low density lipoprotein receptor; LXR; the liver X receptor; Na; +/K+-ATPase; sodium potassium ATPase; PBS; phosphate buffer saline; PVDF; polyvinylidene fluoride; RCT; reverse cholesterol transport; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisATP-binding cassette transport; ABCG1; Cholesterol efflux; Protein palmitoylation; Cysteine residue; Atherosclerosis

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase by Almerinda Di Venere; Thomas Horn; Sabine Stehling; Giampiero Mei; Laura Masgrau; Gonzalez-Lafont Àngels González-Lafont; Kuhn Hartmut Kühn; Igor Ivanov (pp. 1079-1088).

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme–substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.Display Omitted► Arg403 is important for structural integrity of 12/15-LOX. ► Side-chain of Arg403 contributes to stability of LOX's tertiary structure. ► Substrate binding to Arg403 might be associated with α2-helix rearrangement. ► Mutation of Arg403 leads to distortion of catalytic properties of LOX enzyme.

Keywords: Abbreviations; LOX(s); lipoxygenase(s); LA; linoleic acid; AA; arachidonic acid; Me-AA; methyl arachidonate; 15-HETE; (5Z,8Z,11Z,13E)-15-hydroxyeicosa-5,8,11,13-tetraenoic acid; 12-HETE; (5Z,8Z,10E,14Z)-12-hydroxyeicosa-5,8,10,14-tetraenoic acid; 5,15-DiHETE; (6E,8Z,11Z,13E)-5,15-dihydroxyeicosa-6,8,11,13-tetraenoic acid; 8,15-DiHETE; (5Z,9E,11Z,13E)-8,15-dihydroxyeicosa-5,9,11,13-tetraenoic acid; IPTG; isopropyl-β-; d; -1-thiogalactopyranosideLipoxygenases; Lipid peroxidation; Fluorescence studies; Motional flexibility; Thermal stability; Molecular dynamics

Lipid-coated gold nanoparticles promote lamellar body formation in A549 cells by Meijing Wang; Nils O. Petersen (pp. 1089-1097).

Gold nanoparticles (GNPs) have been applied as diagnostic and therapeutic agents because they can be targeted, localized, and be heated to cause cell death. However, their use has been limited by their relatively low biocompatibility. In this work, we coated the GNPs' surface by a biocompatible phospholipid bilayer composed of 1-stearoyl-2-oleoyl- sn-glycero-3-phospho-(1′- rac-glycerol) (SOPG). We tested their interaction with A549 cells to investigate their uptake and intracellular fate as well as the response of the cells to the presence of the GNPs. We used flow cytometry and confocal microscopy to show that the SOPG coated GNPs were readily taken up by the A549 cells. Transmission electron microscopy (TEM) images and fluorescence images further showed that the number of granular structures in the cells was increased following exposure to the lipid coated GNPs. Co-localization experiments demonstrated that SOPG coated GNPs localize in acidic compartments in a time dependent manner and that the number of these increase as the cells are exposed to the GNPs suggesting that they induce formation of lamellar bodies (LBs) which in A549 cells in turn can serve as a means of exporting the GNPs.Display Omitted► Lipid coated gold nanoparticles are readily taken up by A549 cells. ► The lipid coated gold nanoparticles exclusively locate in the acidic compartments of A549 cells. ► The lipid coated gold nanoparticles promote the formation of lamellar bodies (LBs) in A549 cells.

Keywords: Phospholipid; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol) (SOPG); Gold nanoparticle; Endocytosis; A549 cell; Lamellar bodies (LBs)

Characterisation of the prostaglandin E₂-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells by Kirsten L. Brown; Jillian Davidson; Dino Rotondo (pp. 1098-1107).

Prostaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E₂ has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E₂ ethanolamide (PGE₂-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE₂.Whole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE₂-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE₂-EA and directly compared to those of PGE₂. The effect of PGE₂-EA on the binding of radiolabelled PGE₂ to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE₂-EA.PGE₂-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE₂-EA.This study reports that PGE₂-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP₂ receptor.•PGE₂-ethanolamide (PGE₂-EA) potently suppressed TNF-α production.•TNF-α suppression by PGE₂-EA occurred via cyclic AMP pathways in human monocytes.•PGE₂-EA increased intracellular cyclic AMP levels in human monocytes.•PGE₂-ethanolamide displaced specific binding of radiolabelled PGE₂ on human monocytes.•Dual-labelled PGE₂-EA was produced from labelled arachidonic acid and ethanolamine.

Keywords: Prostaglandin–ethanolamide; TNF-α production; Human immune cells; Monocytes; Inflammatory response

The polar lipids of Clostridium psychrophilum, an anaerobic psychrophile by Ziqiang Guan; Bing Tian; Amedea Perfumo; Howard Goldfine (pp. 1108-1112).

We have examined the polar lipids of Clostridium psychrophilum, a recently characterized psychrophilic Clostridium isolated from an Antarctic microbial mat. Lipids were extracted from cells grown near the optimal growth temperature (+5°C) and at −5°C, and analyzed by two-dimensional thin layer chromatography and liquid chromatography coupled with mass spectrometry. The major phospholipids of this species are: cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol. Phosphatidylserine and lyso-phosphatidylethanolamine were found as minor components. The most abundant glycolipids are a monoglycosyldiradylglycerol (MGDRG) and a diglycosyldiradylglycerol (DGDRG). The latter was only seen in cells grown at −5°C. An ethanolamine-phosphate derivative of N-acetylglucosaminyldiradylglycerol was seen in cells grown at −5°C and an ethanolamine-phosphate derivative of MGDRG was found in cells grown at +5°C. All lipids were present in both the all acyl and plasmalogen (alk-1′-enyl acyl) forms with the exception of PS and MGDRG, which were predominantly in the diacyl form. The significance of lipid changes at the two growth temperatures is discussed.► Lipids of Clostridium psychrophilum were analyzed. ► All major lipids contained plasmalogens (alk-1’-enyl ether lipids). ► Differences were observed in lipids at two growth temperatures. ► Lipid composition at low temperatures is discussed.

Keywords: Abbreviations; DGDG; diglucosyldiacylglycerol; DGDRG; diglycosyldiradylglycerol (diradyl indicates the presence of diacyl and alk-1′-enyl, acyl glycerolipids); EtnP-GlcNAcDRG; ethanolaminephosphate-N-acetylglucosaminyldiradylglycerol; EtnPMGDRG; ethanolaminephosphate monoglycosyldiradylglycerol; LC-ESI/MS; liquid chromatography-electrospray ionization mass spectrometry; MGDG; monoglucosyldiacylglycerol; MGDRG; monoglycosyldiradylglycerol; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PS; phosphatidylserine; TLC; thin layer chromatographyPhospholipid; Glycolipid; Phosphoglycolipid; Plasmalogen; Mass spectrometry

Endoplasmic reticulum-localized hepatic lipase decreases triacylglycerol storage and VLDL secretion by Bruce Erickson; Senthamil Paramadayalan Selvan; Kerry W.S. Ko; Karen Kelly; Ariel D. Quiroga; Lena Li; Randy Nelson; Kirst King-Jones; René L. Jacobs; Richard Lehner (pp. 1113-1123).

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.► Secretion-destined hepatic lipase becomes active in the endoplasmic reticulum. ► Intracellular hepatic lipase activity hydrolyzes newly synthesized lipids. ► Intracellular hepatic lipase activity decreases the assembly of VLDL. ► Fatty acids released by intracellular hepatic lipase are oxidized.

Keywords: Abbreviations; apoB; apolipoprotein B; ATGL; adipose triglyceride lipase; CE; cholesteryl ester; DG; diacylglycerol; ER; endoplasmic reticulum; E600; diethyl-; p; -nitrophenylphosphate; FA; fatty acid; HL; hepatic lipase; HL-R; ER-retained hepatic lipase; McA; McArdle-RH7777; PBS; phosphate-buffered saline; MTP; microsomal triglyceride transfer protein; OA; oleic acid; PC; phosphatidylcholine; PDI; protein disulfide isomerase; PL; phospholipids; Ppia; cyclophilin; R12; McArdle cells expressing HL-R; TG; triacylglycerol; VLDL; very-low density lipoproteinHepatic lipase; Triacylglycerol; VLDL; apoB; Fatty acid oxidation; Endoplasmic reticulum

Autophagy in obesity and atherosclerosis: Interrelationships between cholesterol homeostasis, lipoprotein metabolism and autophagy in macrophages and other systems by Mireille Ouimet (pp. 1124-1133).

The incidence of diseases characterized by a dysregulation of lipid metabolism such as obesity, diabetes and atherosclerosis is rising at alarming rates, driving research to uncover new therapies to manage dyslipidemias and resolve the metabolic syndrome conundrum. Autophagy and lipid homeostasis – both ancient cellular pathways – have seemingly co-evolved to share common regulatory elements, and autophagy has emerged as a prominent mechanism involved in the regulation of lipid metabolism. This review highlights recent findings on the role of autophagy in the regulation of cellular cholesterol homeostasis and lipoprotein metabolism, with special emphasis on macrophages. From modulation of inflammation to regulation of cellular cholesterol levels, a protective role for autophagy in atherosclerosis is emerging. The manipulation of autophagic activity represents a new possible therapeutic approach for the treatment complex metabolic disorders such as obesity and the metabolic syndrome.•The autophagy and lipid metabolism pathways share common regulatory elements.•Autophagy is emerging as a prominent mechanism for lipid metabolism regulation.•This occurs via lipophagy or through other autophagy-mediated regulatory functions.•Autophagy's impact on lipid metabolism and atherosclerosis is discussed.

Keywords: Autophagy; Lipid metabolism; Atherosclerosis; Macrophage; Lipoprotein

DNA topoisomerase II inhibitors induce macrophage ABCA1 expression and cholesterol efflux—An LXR-dependent mechanism by Ling Zhang; Meixiu Jiang; Yongsheng Shui; Yuanli Chen; Qixue Wang; Wenquan Hu; Xingzhe Ma; Xiaoju Li; Xin Liu; Xingyue Cao; Mengyang Liu; Yajun Duan; Jihong Han (pp. 1134-1145).

ATP-binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux and thereby inhibits lipid-laden macrophage/foam cell formation and atherosclerosis. ABCA1 expression is transcriptionally regulated by activation of liver X receptor (LXR). Both etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors and are chemotherapeutic medications used in the treatment of various cancers. Interestingly, etoposide inhibits atherosclerosis in rabbits by unclear mechanisms. Herein, we report the effects of etoposide and teniposide on macrophage ABCA1 expression and cholesterol efflux. Both etoposide and teniposide increased macrophage free cholesterol efflux. This increase was associated with increased ABCA1 mRNA and protein expression. Etoposide and teniposide also increased ABCA1 promoter activity in an LXR-dependent manner and formation of the LXRE-LXR/RXR complex indicating that transcriptional induction had occurred. Expression of ABCG1 and fatty acid synthase (FAS), another two LXR-targeted genes, was also induced by etoposide and teniposide. In vivo, administration of mice with either etoposide or teniposide induced macrophage ABCA1 expression and enhanced reverse cholesterol transport from macrophages to feces. Taken together, our study indicates that etoposide and teniposide increase macrophage ABCA1 expression and cholesterol efflux that may be attributed to the anti-atherogenic properties of etoposide. Our study also describes a new function for Topo II inhibitors in addition to their role in anti-tumorigenesis.•Topo II inhibitor, etoposide, inhibits lesions in rabbits with unclear mechanisms;•Both etoposide and teniposide induced Mϕ ABCA1 expression and cholesterol efflux;•The induction of ABCA1 expression is LXR-dependent;•We disclose a new function of etoposide and teniposide and the involved mechanism.

Keywords: Abbreviations; ABCA1; ATP-binding cassette transporter A1; ABCG1; ATP-binding cassette transporter G1; apoAI; apolipoprotein AI; FAS; fatty acid synthase; HDL; high-density lipoprotein; LXR; liver X receptor; LXRE; LXR responsive element; RIP140; receptor-interacting protein 140; RXR; retinoid X receptor; Topo II; DNA topoisomerase IIABCA1; Atherosclerosis; Topo II inhibitor; LXR; Macrophage

Cytosolic phospholipase A₂α sustains pAKT, pERK and AR levels in PTEN-null/mutated prostate cancer cells by Sheng Hua; Mu Yao; Soma Vignarajan; Paul Witting; Leila Hejazi; Zhen Gong; Ying Teng; Marzieh Niknami; Stephen Assinder; Des Richardson; Qihan Dong (pp. 1146-1157).

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog ( PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 ( PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.•cPLA2α role in oncogenic pathway in tumor suppressor null-cancer cells is examined.•Increase or decrease in cPLA2α expression enhances or reduces AKT signaling.•Silencing of cPLA2α causes a decrease in ERK signaling and AR levels.•Inhibitory effect of cPLA2α siRNA is reduced by addition of arachidonic acid.•Stimulatory effect of arachidonic acid is decreased by blocking 5-HETE production.

Keywords: cPLA; ₂; α; AKT; ERK; AR; PTEN; Prostate cancer

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris by Vasyl A. Ivashov; Guenther Zellnig; Karlheinz Grillitsch; Guenther Daum (pp. 1158-1166).

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1∆lro1∆ double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.•We identified acyltransferases involved in non-polar lipid synthesis of P. pastoris.•Dga1p is an acyl-CoA dependent, Lro1p an acyl-CoA independent triglyceride synthase.•Both enzymes have limited substrate specificity; Lro1p has higher activity than Dga1p.•Are2p is the only acyl CoA:sterol acyltransferase of P. pastoris.•A triplet mutant deleted of all three acyltransferases lacks lipid droplets.

Keywords: Acyltransferase; Triacylglycerol; Steryl ester; Lipid droplet; Pichia pastoris

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p by Cedric H. De Smet; Ruud Cox; Jos F. Brouwers; Anton I.P.M. de Kroon (pp. 1167-1176).

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.•Sct1p overexpression in yeast increases the extent of acyl chain remodeling of PC.•Overexpression of Sct1p is a unique new tool for studying acyl chain exchange.•PC is remodeled at both the sn-1 and sn-2 positions of the glycerol backbone.•Plb1p is required for efficient remodeling of newly synthesized PC.

Keywords: Abbreviations; ESI-MS/MS; electrospray ionization tandem mass spectrometry; GPC; glycerophosphocholine; PE; phosphatidylethanolamine; PC; phosphatidylcholine; SL; synthetic lactate medium; SGR; synthetic galactose–raffinose mediumAcyl chain exchange; Fatty acid metabolism; Glycerophospholipid metabolism; Lipid remodeling; Phosphatidylcholine; Yeast

Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance by Nagatoshi Fujiwara; Steven A. Porcelli; Takashi Naka; Ikuya Yano; Shinji Maeda; Hirotaka Kuwata; Shizuo Akira; Satoshi Uematsu; Takemasa Takii; Hisashi Ogura; Kazuo Kobayashi (pp. 1177-1184).

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.• Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs).•These bacterial SPLs activated murine bone marrow macrophages via TLR4.•A non-hydroxy fatty acid in ceramide was necessary for the SPLs activation via TLR4.•The SPLs activation induced an innate immune response and contributed to bacterial clearance.

Keywords: Abbreviations; SPL; sphingophospholipid; GSL; glycosphingolipid; BMM; bone marrow macrophageSphingophospholipid; Toll-like receptor (TLR) 4; Sphingobacterium spiritivorum; Ceramide

An ethanolamine-phosphate modified glycolipid in Clostridium acetobutylicum that responds to membrane stress by Bing Tian; Ziqiang Guan; Howard Goldfine (pp. 1185-1190).

Two phosphorus-containing glycolipids have previously been observed in Clostridium acetobutylicum. We had shown that the concentration of one of them increases in response to increased unsaturation of the membrane lipid hydrocarbon chains, suggesting a potential role in the regulation of lipid polymorphism in this organism. Mass spectrometry shows that these glycolipids are ethanolamine phosphate (Etn-P)-containing derivatives of a mono- and di-glycosyldiradylglycerol. The content of both diglycosyldiradylglycerol and the Etn-P-monoglycosyldiradylglycerol, which increases upon increased unsaturation of the membrane, also increases upon addition of octanol to the medium. Thus, it appears that the Etn-P-monoglycosyldiradylglycerol along with the diglycosyldiradylglycerol may serve to stabilize the membrane bilayer during membrane stress caused by the presence of the solvents produced during fermentation.• Clostridium acetobutylicum is an important producer of industrial solvents.•Novel ethanolamine-phosphate glycosyldiradylglycerols are characterized•Lipids respond to membrane stress.•Potential involvement in solvent resistance

Keywords: Abbreviations; DGDRG; diglycosyldiradylglycerol; Etn-P; ethanolamine-phosphate; MGDG; monoglycosyldiacylglycerol; MGDRG; monoglycosyldiradylglycerolGlycolipid; Plasmalogen; Phosphoglycolipid; Solvent

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BBA - Molecular and Cell Biology of Lipids (v.1831, #6)