Regulación de la formación de gotas lipídicas por ácido araquidónico en monocitos humanos: importancia del ácido graso

Las gotas lipídicas son orgánulos compuestos por un núcleo de lípidos neutros rodeado de una monocapa de fosfolípidos. Su formación está implicada en numerosas enfermedades, como la aterosclerosis. Se ha descrito que la formación de gotas lipídicas en monocitos circulantes es un factor de riesgo en el desarrollo temprano de esta enfermedad. En este trabajo se muestra que el ácido araquidónico induce una rápida formación de gotas lipídicas en monocitos humanos, dependiente de la síntesis de novo de ácidos grasos, así como de la activación de la fosfolipasa A2 de grupo IVA. Entre los ácidos grasos que se incorporan a las gotas lipídicas destaca el ácido graso 16:1n-9, que podría constituir un buen marcador de la formación de estos orgánulos. En el trabajo se demuestra que este ácido graso se sintetiza a partir del ácido oleico y presenta un robusto comportamiento antiinflamatorio en modelos de ratón.Departamento de Bioquímica y Biología Molecular y Fisiologí

Lipin-1 integrates lipid synthesis with proinflammatory responses during TLR activation in macrophages

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase involved in the de novo synthesis of phospholipids and triglycerides. Using macrophages from lipin-1-deficient animals and human macrophages deficient in the enzyme, we show in this work that this phosphatase acts as a proinflammatory mediator during TLR signaling and during the development of in vivo inflammatory processes. After TLR4 stimulation lipin-1-deficient macrophages showed a decreased production of diacylglycerol and activation of MAPKs and AP-1. Consequently, the generation of proinflammatory cytokines like IL-6, IL-12, IL-23, or enzymes like inducible NO synthase and cyclooxygenase 2, was reduced. In addition, animals lacking lipin-1 had a faster recovery from endotoxin administration concomitant with a reduced production of harmful molecules in spleen and liver. These findings demonstrate an unanticipated role for lipin-1 as a mediator of macrophage proinflammatory activation and support a critical link between lipid biosynthesis and systemic inflammatory responses.This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055, SAF2010-18831, and BFU2010-18826) and the Regional Government of Castile and Leon (Grants BIO39/VA04/10 and CSI168A12-1). L.P. and G.L. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation (Plan de Formación de Personal Investigador and Plan de Formación de Profesorado Universitario programs). M.V. was supported by a predoctoral fellowship from the Regional Government of Castile and Leon. E.E. was supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). C.G. was supported by a predoctoral fellowship from the University of Valladolid.Peer Reviewe

Sequestration of 9-hydroxystearic acid in FAHFA (Fatty Acid Esters of Hydroxy Fatty Acids) as a protective mechanism for colon carcinoma cells to avoid apoptotic cell death

Hydroxy fatty acids are known to cause cell cycle arrest and apoptosis. The best studied of them, 9-hydroxystearic acid (9-HSA), induces apoptosis in cell lines by acting through mechanisms involving different targets. Using mass spectrometry-based lipidomic approaches, we show in this study that 9-HSA levels in human colorectal tumors are diminished when compared with normal adjacent tissue. Since this decrease could be compatible with an escape mechanism of tumors from 9-HSA-induced apoptosis, we investigated different features of the utilization of this hydroxyfatty acid in colon. We show that in colorectal tumors and related cell lines such as HT-29 and HCT-116, 9-HSA is the only hydroxyfatty acid constituent of branched fatty acid esters of hydroxyfatty acids (FAHFA), a novel family of lipids with anti-inflammatory properties. Importantly, FAHFA levels in tumors are elevated compared with normal tissue and, unlike 9-HSA, they do not induce apoptosis of colorectal cell lines over a wide range of concentrations. Further, the addition of 9-HSA to colon cancer cell lines augments the synthesis of different FAHFA before the cells commit to apoptosis, suggesting that FAHFA formation may function as a buffer system that sequesters the hydroxyacid into an inactive form, thereby restricting apoptosis.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers SAF2016-80883-R and SAF2015-73000-EXP. C.G. was funded by a predoctoral fellowship from the University of Valladolid (Plan de Formación de Personal Investigador). CIBERDEM is an initiative of Instituto de Salud Carlos IIIWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Regulación de la formación de gotas lipídicas por ácido araquidónico en monocitos humanos: importancia del ácido graso 16:1n-9

Tesis Doctorla presentada por Carlos Guijas Maté para optar al grado de doctor por la Universidad de Valladolid, Facultad de Medicina e Instituto de Biología y Genética Molecular: Dpto. de Bioquímica y Biología Molecular y Fisiología.Las gotas lipídicas son orgánulos compuestos por un núcleo de lípidos neutros rodeado de una monocapa de fosfolípidos. Su formación está implicada en numerosas enfermedades, como la aterosclerosis. Se ha descrito que la formación de gotas lipídicas en monocitos circulantes es un factor de riesgo en el desarrollo temprano de esta enfermedad. En este trabajo se muestra que el ácido araquidónico induce una rápida formación de gotas lipídicas en monocitos humanos, dependiente de la síntesis de novo de ácidos grasos, así como de la activación de la fosfolipasa A2 de grupo IVA. Entre los ácidos grasos que se incorporan a las gotas lipídicas destaca el ácido graso 16:1n-9, que podría constituir un buen marcador de la formación de estos orgánulos. En el trabajo se demuestra que este ácido graso se sintetiza a partir del ácido oleico y presenta un robusto comportamiento antiinflamatorio en modelos de ratón.Esta tesis doctoral ha sido realizada gracias a la concesión de una beca de Formación de Personal Investigador de la Universidad de Valladolid, en colaboración con el Banco Santander (Convocatoria del año 2012), y financiada por los siguientes proyectos de investigación: Rutas anti-inflamatorias mediadas por lípidos que regulan la activación del inflamasoma: Papel de los ácidos grasos omega-3 (SAF2013-48201-R1). Estudio lipidómico de los mecanismos que gobiernan la disponibilidad y metabolismo oxidativo de ácido araquidónico en macrófagos humanos (CSI007U13). Estudio por espectrometría de masas del perfil lipidómico de macrófagos humanos polarizados (BFU2010-18826). Una aproximación de lipidómica al estudio de la respuesta inmune innata: mecanismos que gobiernan la disponibilidad y metabolismo oxidativo de ácido araquidónico en macrófagos (BFU2007-67154/BMC). CIBERDEM - Diabetes and Associated Metabolic Disorders (CB07/08/0004).Peer Reviewe

Opposite cross-talk by oleate and palmitate on insulin signaling in hepatocytes through macrophage activation

Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B4 (LTB4) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB4 suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB4 decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB4 receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB4.This work was supported by Ministerio de Economía y Competitividad, Spain, Grants SAF2012-33283 and SAF2013-48201-R; Comunidad de Madrid Grant S2010/BMD-2423 (Spain); an European Foundation for the Studies of Diabetes and Amylin Paul Langerhans grant; and the Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, Instituto de Salud Carlos III, Spain).Peer reviewe

Phospholipase A2 regulation of lipid droplet formation

The classical regard of lipid droplets as mere static energy-storage organelles has evolved dramatically. Nowadays these organelles are known to participate in key processes of cell homeostasis, and their abnormal regulation is linked to several disorders including metabolic diseases (diabetes, obesity, atherosclerosis or hepatic steatosis), inflammatory responses in leukocytes, cancer development and neurodegenerative diseases. Hence, the importance of unraveling the cell mechanisms controlling lipid droplet biosynthesis, homeostasis and degradation seems evident. Phospholipase A2s, a family of enzymes whose common feature is to hydrolyze the fatty acid present at the sn-2 position of phospholipids, play pivotal roles in cell signaling and inflammation. These enzymes have recently emerged as key regulators of lipid droplet homeostasis, regulating their formation at different levels. This review summarizes recent results on the roles that various phospholipase A2 forms play in the regulation of lipid droplet biogenesis under different conditions. These roles expand the already wide range of functions that these enzymes play in cell physiology and pathophysiology.Work in our laboratories was supported by the Spanish Ministry of Science and Innovation (Grants BFU2010-18826, SAF2010-18831, and SAF2013-48201-R), and the Education Department of the Autonomous Government of Castile and Leon (Grant CSI007U13). C. G. was supported by a predoctoral fellowship from the University of Valladolid. CIBERDEM is an initiative of Instituto de Salud Carlos III.Peer Reviewe

Stimulated occurrence and biological activity of palmitoleic acid and its isomers in metabolic diseases

Resumen del trabajo presentado presentado al CIBERDEM Annual Meeting, celebrado en Cerdanyola del Vallès, Barcelona (España) del 11 al 13 de mayo de 2016.Atherosclerosis, a major cause for cardiovascular disease, can be initiated by the increased activation of endothelial cells lining the inside of the blood vessels. This activation may obey to multiple causes, such as diabetes or hyperlipidemia, and results in the endothelial cells releasing a variety of products with inflammatory potential that may attract monocytes and favor their infiltration into the subendothelial space. We have recently shown that human monocytes respond to free arachidonic acid, a well-defined secretory product of activated endothelial cells, by activating the de novo pathway of fatty acid biosynthesis, resulting in the formation of neutral lipids and the acquisition of a foamy phenotye due to the accumulation of cytoplasmic lipid droplets. In this work we describe that the neutral lipids of foamy monocytes are selectively enriched in a rather uncommon fatty acid, cis-7-hexadecenoic acid (16:n-9), a positional isomer of the more frequently described palmitoleic acid, which is strikingly absent from lipid droplets. Experiments addressing the biological role for 16:1n-9 indicate that this fatty acid prevents the inflammatory response of human monocytes and murine macrophages to bacterial lipopolysaccharide both in vitro and in vivo. Collectively, our results provide evidence that a previously unrecognized fatty acid, 16:1n-9, possesses anti-inflammatory activity that is comparable to that of omega-3 fatty acids and is clearly distinguishable from the effects of palmitoleic acid. Moreover, the selective accumulation in the neutral lipids of phagocytic cells of a rather uncommon fatty acid, reveals an early phenotypic change that may provide a biomarker of proatherogenicity, and a potential target for intervention in the early stages of cardiovascular disease.Peer reviewe

Phospholipid sources for adrenic acid mobilization in RAW 264.7 macrophages. Comparison with arachidonic acid

8 páginas, 6 figuras.Cells metabolize arachidonic acid (AA) to adrenic acid (AdA) via 2-carbon elongation reactions. Like AA, AdA can be converted into multiple oxygenated metabolites, with important roles in various physiological and pathophysiological processes. However, in contrast to AA, there is virtually no information on how the cells regulate the availability of free AdA for conversion into bioactive products. We have used a comparative lipidomic approach with both gas chromatography and liquid chromatography coupled to mass spectrometry to characterize changes in the levels of AA- and AdA-containing phospholipid species in RAW 264.7 macrophage-like cells. Incubation of the cells with AA results in an extensive conversion to AdA but both fatty acids do not compete with each other for esterification into phospholipids. AdA but not AA, shows preference for incorporation into phospholipids containing stearic acid at the sn-1 position. After stimulation of the cells with zymosan, both AA and AdA are released in large quantities, albeit AA is released to a greater extent. Finally, a variety of phosphatidylcholine and phosphatidylinositol molecular species contribute to AA; however, AdA is liberated exclusively from phosphatidylcholine species. Collectively, these results identify significant differences in the cellular utilization of AA and AdA by the macrophages, suggesting non-redundant biological actions for these two fatty acids.This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2010-18826, and SAF2010-18831).Peer reviewe

Cytosolic group IVA and calcium-independent group VIA phospholipase A2s act on distinct phospholipid pools in zymosan-stimulated mouse peritoneal macrophages

Phospholipase A2s generate lipid mediators that constitute an important component of the integrated response of macrophages to stimuli of the innate immune response. Because these cells contain multiple phospholipase A2 forms, the challenge is to elucidate the roles that each of these forms plays in regulating normal cellular processes and in disease pathogenesis. A major issue is to precisely determine the phospholipid substrates that these enzymes use for generating lipid mediators. There is compelling evidence that group IVA cytosolic phospholipase A2 (cPLA2α) targets arachidonic acid-containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca2+-independent group VIA phospholipase A2 (iPLA2β) has not been clearly defined. We applied mass spectrometry-based lipid profiling to study the substrate specificities of these two enzymes during inflammatory activation of macrophages with zymosan. Using selective inhibitors, we find that, contrary to cPLA2α, iPLA2β spares arachidonate-containing phospholipids and hydrolyzes only those that do not contain arachidonate. Analyses of the lysophospholipids generated during activation reveal that one of the major species produced, palmitoyl-glycerophosphocholine, is generated by iPLA2β, with minimal or no involvement of cPLA 2α. The other major species produced, stearoyl- glycerophosphocholine, is generated primarily by cPLA2α. Collectively, these findings suggest that cPLA2α and iPLA 2β act on different phospholipids during zymosan stimulation of macrophages and that iPLA2β shows a hitherto unrecognized preference for choline phospholipids containing palmitic acid at the sn-1 position that could be exploited for the design of selective inhibitors of this enzyme with therapeutic potential. Copyright © 2014 by The American Association of Immunologists, Inc.This work was supported by Spanish Ministry of Science and Innovation Grants BFU2010-18826 and SAF2010-18831. L.G.-d.-G. and C.G. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation and the University of Valladolid, respectively (Plan de Formación de Personal Investigador Program).Peer Reviewe