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BBA - Molecular and Cell Biology of Lipids (v.1811, #2)
PPARβ/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells by Alvarez-Guardia David Álvarez-Guardia; Xavier Palomer; Teresa Coll; Lucía Serrano; Rodriguez-Calvo Ricardo Rodríguez-Calvo; Mercy M. Davidson; Manuel Merlos; Ilhem El Kochairi; Liliane Michalik; Walter Wahli; Vazquez-Carrera Manuel Vázquez-Carrera (pp. 59-67).
Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARβ/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARβ/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARβ/δ activation by GW501516 enhanced the physical interaction between PPARβ/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARβ/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARβ/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.► GW501516-induced PPARbeta/delta activation can attenuate the inflammatory. ► Response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed an HFD. ► PPARβ/δ may serve as a therapeutic target to prevent cardiac hypertrophy and heart failure in metabolic disorders.
Keywords: Abbreviations; AMPK; AMP-activated protein kinase; HFD; high-fat diet; IL-6; interleukin-6; MCP-1; monocyte chemoattractant protein-1; NF-κB; nuclear factor-κB; PPAR; peroxisome proliferator-activated receptor; SOCS; suppressor of cytokine signaling; STAT; signal transducer and activator of transcription; TNF-α; tumor necrosis factor-αHigh-fat diet; Inflammation; Heart; NF-κB; Nuclear factor-κB; Peroxisome proliferator-activated receptor
Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry by Max Scherer; Bottcher Alfred Böttcher; Gerd Schmitz; Gerhard Liebisch (pp. 68-75).
Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids.► Quantification of free sphingoid bases, sphingosylphosphorylcholine, hexosyl-, lactosylceramide, ceramide-1-phosphate. ► Besides sphingosine d18:1, d16:1 and d18:2 species were found in most lipid classes. ► Hexosyl- and lactosylceramide showed pronounced differences in their species pattern. ► More than 90% of Hexosyl- and lactosylceramides were found in LDL and HDL.
Keywords: Abbreviations; CE; collision energy; Cer1P; ceramide-1-phosphate; CV; coefficient of variation; DimetSPH; dimethyl-sphingosine; ESI-MS/MS; electrospray ionization tandem mass spectrometry; FPLC; fast performance liquid chromatography; HexCer; hexosylceramide; HILIC; hydrophilic interaction chromatography; IS; internal standard; LacCer; lactosylceramide; LOD; limit of detection; LPDS; lipoprotein deficient serum; MRM; multiple reaction monitoring; S.D.; standard deviation; S1P; sphingosine-1-phosphate; SPC; sphingosylphosphorylcholine; SPH; free sphingoid base; TrimetSPH; trimethyl-sphingosine; var.; variableLactosylceramide; Sphingoid base; Glycosylceramide; High throughput; Lipidomics; Sphingolipidomics
Differential regulation of human apolipoprotein AI and high-density lipoprotein by fenofibrate in hapoAI and hapoAI–CIII–AIV transgenic mice by Rai Ajit K. Srivastava; Shirley He; Roger S. Newton (pp. 76-83).
Fenofibrate, a PPAR- α agonist, lowers triglycerides (TG) and raises high-density lipoproteins (HDL-C) in humans. While fenofibrate is very effective in lowering TG, it does not raise HDL-C in humans to the same extent as seen in human apoAI transgenic (hAI-Tg) mice. We studied the mechanism of this discordance using the following compounds as tools: cholic acid that down-regulates human apoAI, and fenofibrate, that elevates hapoAI and HDL-C in hAI-Tg mice. We hypothesized that additional sequences, including apoCIII and AIV genes on chromosome 11, not present in the hapoAI transgene may be responsible for the dampened effect of fibrates on HDL-C seen in humans. For this, hAI-Tg mice with 11kb DNA segment and hapoAI–CIII–AIV-Tg mice with 33kb DNA segment harboring apoCIII and AIV genes were employed. These mice were treated with fenofibrate and cholic acid. Fenofibrate increased apoAI and HDL-C levels, and HDL size in the apoAI-Tg mice via up-regulation of the hapoAI mRNA and increased activity and mRNA of PLTP, respectively. Consistent with earlier findings, cholic acid showed similar effects of lowering HDL-C, and elevating LDL-C in hAI-Tg mice as well as in the hAI–CIII–AIV-Tg mice. Fenofibrate decreased TG and increased HDL size in hAI–CIII–AIV-Tg mice as well, but surprisingly, did not elevate serum levels of hapoAI or hepatic AI mRNA, suggesting that additional sequences not present in the hapoAI transgene (11kb) may be partly responsible for the dampened effect on HDL-C seen in hAI–CIII–AIV-Tg mice. Since hAI–CIII–AIV-Tg mouse mimics fenofibrate effects seen in humans, this transgenic mouse could serve as a better predictive model for screening HDL-C raising compounds.►Using apoAI- and apoAI–CIII–AIV-transgenic mice we have investigated regulation of fenofibrate-induced regulation of apoAI gene expression in AI and AI-CIII-AIV transgenic mouse lines. ► Fenofibrate up-regulates apoAI gene in apoAI transgenic mice, but not in apoAI–CIII–AIV transgenic mice. ► Cholic acid down-regulates apoAI gene expression both in apoAI and apoAI–CIII–AIV transgenic mice. ► Fenofibrate and cholic acid regulate SR-BI gene expression in opposite direction, which may be partly responsible for circulating levels of HDL-C. ► Fenofibrate up-regulated sterol career protein 2, suggesting increased delivery of cholesterol to the liver. ► Fenofibrate increased HDL size both in the apoAI and AI–CIII–AIV transgenic mice via up-regulation of the PLTP gene.
Keywords: Abbreviations; TG; triglycerides; HDL-C; high-density lipoprotein cholesterol; apo; apolipoprotein; PLTP; phospholipid transfer protein; SCP2; sterol career protein-2; SR-BI; scavenger receptor class B type I; PPAR; peroxisome proliferator activated receptor; ACC; acetyl CoA carboxylase; ACO; acetyl CoA oxidase; FAS; fatty acid synthase; FPLC; fast protein liquid chromatography; hAI; human apolipoprotein AIApolipoprotein A1; Fenofibrate; Cholic acid; HDL; Phospholipid transfer protein; SR-BI
Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity by Olga M. Chaim; Rafael B. da Silveira; Dilza Trevisan-Silva; Valéria P. Ferrer; Youssef B. Sade; Boia-Ferreira Mariana Bóia-Ferreira; Luiza H. Gremski; Waldemiro Gremski; Andrea Senff-Ribeiro; Hélio K. Takahashi; Marcos S. Toledo; Helena B. Nader; Silvio S. Veiga (pp. 84-96).
Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin — LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.► Brown spider recombinant toxin hydrolyzes sphingomyelin. ► Catalytic site-directed mutagenesis H12A reduces drastically substrate hydrolysis. ► Dermonecrosis and inflammation induced by toxin depend on phospholipase activity. ► Both recombinant forms bound to lipids in blot and endothelial cell membrane. ► Phospholipase-D activity induces endothelial citotoxicity. ► Lysophospholipids and glycerophospholipids are also substrates for LiRecDT1.
Keywords: Venom; Loxosceles intermedia; Dermonecrotic toxin; Phospholipase-D; Endothelial cell; Inflammatory response
Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells by Alma M. Astudillo; Perez-Chacon Gema Pérez-Chacón; David Balgoma; Gil-de-Gomez Luis Gil-de-Gómez; Ruiperez Violeta Ruipérez; Carlos Guijas; María A. Balboa; Jesús Balsinde (pp. 97-103).
The availability of free arachidonic acid (AA) constitutes a limiting step in the synthesis of biologically active eicosanoids. Free AA levels in cells are regulated by a deacylation/reacylation cycle of membrane phospholipids, the so-called Lands cycle, as well as by further remodeling reactions catalyzed by CoA-independent transacylase. In this work, we have comparatively investigated the process of AA incorporation into and remodeling between the various phospholipid classes of human monocytes and monocyte-like U937 cells. AA incorporation into phospholipids was similar in both cell types, but a marked difference in the rate of remodeling was appreciated. U937 cells remodeled AA at a much faster rate than human monocytes. This difference was found not to be related to the differentiation state of the U937 cells, but rather to the low levels of esterified arachidonate found in U937 cells compared to human monocytes. Incubating the U937 cells in AA-rich media increased the cellular content of this fatty acid and led to a substantial decrease of the rate of phospholipid AA remodeling, which was due to reduced CoA-independent transacylase activity. Collectively, these findings provide the first evidence that cellular AA levels determine the amount of CoA-independent transacylase activity expressed by cells and provide support to the notion that CoA-IT is a major regulator of AA metabolism in human monocytes.► Phospholipid arachidonate remodeling in U937 cells is faster than in human monocytes. ► Arachidonate remodeling does not depend on the differentiation stage of the cells. ► The intracellular content of arachidonate determines the rate of remodeling. ► Arachidonate‐loaded U937 cells exhibit a reduced CoA-transacylase activity.
Keywords: Abbreviations; AA; arachidonic acid; CoA-IT; coenzyme A-independent transacylase; PC; choline glycerophospholipids; PE; ethanolamine glycerophospholipids; PI; phosphatidylinositol; PMA; phorbol myristate acetate; TAG; triacylglycerolArachidonic acid; Phospholipase A; 2; Free fatty acid; CoA-independent transacylase; Phospholipid remodeling
Differential effects of gemfibrozil and fenofibrate on reverse cholesterol transport from macrophages to feces in vivo by Noemí Rotllan; Llaverias Gemma Llaverías; Josep Julve; Matti Jauhiainen; Laura Calpe-Berdiel; Hernandez Cristina Hernández; Simo Rafael Simó; Francisco Blanco–Vaca; Escola-Gil Joan Carles Escolà-Gil (pp. 104-110).
Gemfibrozil and fenofibrate, two of the fibrates most used in clinical practice, raise HDL cholesterol (HDLc) and are thought to reduce the risk of atherosclerotic cardiovascular disease. These drugs act as PPARα agonists and upregulate the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, we determined the effects of these two fibrates on RCT from macrophages to feces in vivo in human apoA-I transgenic (hApoA-ITg) mice. [3H]cholesterol-labeled mouse macrophages were injected intraperitoneally into hApoA-ITg mice treated with intragastric doses of fenofibrate, gemfibrozil or a vehicle solution for 17days, and radioactivity was determined in plasma, liver and feces. Fenofibrate, but not gemfibrozil, enhanced [3H]cholesterol flux to plasma and feces of female hApoA-ITg mice. Fenofibrate significantly increased plasma HDLc, HDL phospholipids, hApoA-I levels and phospholipid transfer protein activity, whereas these parameters were not altered by gemfibrozil treatment. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, together with higher potential to generate preβ-HDL formation and caused a significant increase in [3H]cholesterol efflux to plasma. Our findings demonstrate that fenofibrate promotes RCT from macrophages to feces in vivo and, thus, highlight a differential action of this fibrate on HDL.► Fenofibrate increases plasma HDLc and human (h) apoA-I in hapoA-I transgenic mice. ► Fenofibrate induces the generation of larger HDL particles and preβ-HDL formation. ► Fenofibrate promotes reverse cholesterol transport from macrophages to feces in vivo. ► Our data would be consistent with a protective clinical effect of fenofibrate.
Keywords: Fibrate; PPAR-alpha; HDL; apoA-I; Atherosclerosis; Cardiovascular disease
Dietary n-6 PUFA deprivation downregulates arachidonate but upregulates docosahexaenoate metabolizing enzymes in rat brain by Hyung-Wook Kim; Jagadeesh S. Rao; Stanley I. Rapoport; Miki Igarashi (pp. 111-117).
Dietary n-3 polyunsaturated fatty acid (PUFA) deprivation increases expression of arachidonic acid (AA 20:4n-6)-selective cytosolic phospholipase A2 (cPLA2) IVA and cyclooxygenase (COX)-2 in rat brain, while decreasing expression of docosahexaenoic acid (DHA 22:6n-3)-selective calcium-independent iPLA2 VIA. Assuming that these enzyme changes represent brain homeostatic responses to deprivation, we hypothesized that dietary n-6 PUFA deprivation would produce changes in the opposite directions.Brain expression of PUFA-metabolizing enzymes and their transcription factors was quantified in male rats fed an n-6 PUFA adequate or deficient diet for 15weeks post-weaning.The deficient compared with adequate diet increased brain mRNA, protein and activity of iPLA2 VIA and 15-lipoxygenase (LOX), but decreased cPLA2 IVA and COX-2 expression. The brain protein level of the iPLA2 transcription factor SREBP-1 was elevated, while protein levels were decreased for AP-2α and NF-κB p65, cPLA2 and COX-2 transcription factors, respectively.With dietary n-6 PUFA deprivation, rat brain PUFA metabolizing enzymes and some of their transcription factors change in a way that would homeostatically dampen reductions in brain n-6 PUFA concentrations and metabolism, while n-3 PUFA metabolizing enzyme expression is increased. The changes correspond to reported in vitro enzyme selectivities for AA compared with DHA.► Dietary n-6 PUFA deprivation downregulated cPLA2 and COX-2 in rat brain. ► Dietary n-6 PUFA deprivation upregulated iPLA2 and 15-LOX in rat brain. ► These changes are opposite to n-3 PUFA deprivation in rat brain. ► These changes would tend to maintain homeostasis of brain AA and DHA function.
Keywords: Abbreviations; AA; arachidonic acid; DHA; docosahexaenoic acid; DPA; docosapentaenoic acid; PUFA; polyunsaturated fatty acid; cPLA; 2; cytosolic phospholipase A; 2; sPLA; 2; secretory PLA; 2; iPLA; 2; Ca; 2+; -independent PLA; 2; COX; cyclooxygenase; SREBP; sterol regulatory element binding protein; LOX; lipoxygenase; NF-κB; nuclear factor-κB; AP; activator protein; sn; stereospecifically numberedArachidonic; Brain; PUFA; Phospholipase; Lipoxygenase; SREBP