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1.
Cell ; 138(3): 476-88, 2009 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-19646743

RESUMEN

The nuclear receptor PPARalpha is activated by drugs to treat human disorders of lipid metabolism. Its endogenous ligand is unknown. PPARalpha-dependent gene expression is impaired with inactivation of fatty acid synthase (FAS), suggesting that FAS is involved in generation of a PPARalpha ligand. Here we demonstrate the FAS-dependent presence of a phospholipid bound to PPARalpha isolated from mouse liver. Binding was increased under conditions that induce FAS activity and displaced by systemic injection of a PPARalpha agonist. Mass spectrometry identified the species as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-GPC). Knockdown of Cept1, required for phosphatidylcholine synthesis, suppressed PPARalpha-dependent gene expression. Interaction of 16:0/18:1-GPC with the PPARalpha ligand-binding domain and coactivator peptide motifs was comparable to PPARalpha agonists, but interactions with PPARdelta were weak and none were detected with PPARgamma. Portal vein infusion of 16:0/18:1-GPC induced PPARalpha-dependent gene expression and decreased hepatic steatosis. These data suggest that 16:0/18:1-GPC is a physiologically relevant endogenous PPARalpha ligand.


Asunto(s)
Hígado/metabolismo , PPAR alfa/metabolismo , Fosfolípidos/aislamiento & purificación , Animales , Humanos , Ligandos , Ratones , Fosfolípidos/metabolismo , Receptor fas/genética
2.
J Biol Chem ; 298(11): 102522, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36162499

RESUMEN

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs-). Unexpectedly and unlike fungi where IPCS is essential, Δipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs-, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs- parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis.


Asunto(s)
Hexosiltransferasas , Leishmania major , Leishmaniasis Cutánea , Esfingomielinas , Animales , Ratones , Leishmania major/enzimología , Leishmania major/genética , Esfingomielinas/metabolismo , Virulencia , Glicoesfingolípidos/metabolismo , Proteínas Protozoarias/genética , Hexosiltransferasas/genética , Leishmaniasis Cutánea/parasitología , Eliminación de Secuencia
3.
J Lipid Res ; 58(4): 772-782, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28154204

RESUMEN

1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H - H2O - (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.


Asunto(s)
Ceramidas/química , Ceramidas/aislamiento & purificación , Epidermis/química , Lípidos/aislamiento & purificación , Animales , Isomerismo , Lípidos/química , Ratones , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Esfingosina/química , Esfingosina/aislamiento & purificación
4.
J Biol Chem ; 291(25): 13028-39, 2016 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-27129247

RESUMEN

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of ß-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.


Asunto(s)
Acetilcoenzima A/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Osteoblastos/fisiología , Vía de Señalización Wnt , Proteína Wnt3A/fisiología , Acetilación , Animales , Línea Celular , Ácido Cítrico/metabolismo , Ciclo del Ácido Cítrico , Expresión Génica , Silenciador del Gen , Glucosa/metabolismo , Histonas/metabolismo , Ratones , Procesamiento Proteico-Postraduccional
5.
J Biol Chem ; 291(44): 23268-23281, 2016 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-27650501

RESUMEN

Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A2 (iPLA2ß) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA2ß in peritoneal macrophage immune function by comparing wild type (WT) and iPLA2ß-/- mouse macrophages. Compared with WT, iPLA2ß-/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA2ß-/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA2ß-/- macrophages compared with WT. The effects of inhibitors of iPLA2ß, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E2) recapitulated M1 phenotype in iPLA2ß-/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA2ß and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA2ß promotes macrophage M2 polarization. Reducing macrophage iPLA2ß activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu.


Asunto(s)
Polaridad Celular , Fosfolipasas A2 Grupo VI/inmunología , Inflamación/enzimología , Macrófagos/citología , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Femenino , Fosfolipasas A2 Grupo VI/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Macrófagos/enzimología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología
6.
J Biol Chem ; 291(6): 3076-89, 2016 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-26655718

RESUMEN

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2ß and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2ß is critical for spontaneous AR, whereas both iPLA2ß and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 µm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2ß, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 µm) but not at higher P4 concentrations (~10 µm).


Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Acrosoma/enzimología , Exocitosis/efectos de los fármacos , Fosfolipasas A2 Grupo VI/metabolismo , Fosfolipasas A2 Grupo X/metabolismo , Progesterona/farmacología , Animales , Fosfolipasas A2 Grupo VI/genética , Fosfolipasas A2 Grupo X/genética , Masculino , Ratones , Ratones Noqueados , Progesterona/metabolismo
7.
J Lipid Res ; 57(1): 142-55, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26574042

RESUMEN

Both phthiocerol/phthiodiolone dimycocerosate (PDIM) and phenolic glycolipids are abundant virulent lipids in the cell wall of various pathogenic mycobacteria, which can synthesize a wide range of complex high-molecular-mass lipids. In this article, we describe linear ion-trap MS(n) mass spectrometric approach for structural study of PDIMs, which were desorbed as the [M + Li](+) and [M + NH(4)](+) ions by ESI. We also applied charge-switch strategy to convert the mycocerosic acid substituents to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives and analyzed them as M (+) ions, following alkaline hydrolysis of the PDIM to release mycocerosic acids. The structural information from MS(n) on the [M + Li](+) and [M + NH(4)](+) molecular species and on the M (+) ions of the mycocerosic acid-AMPP derivative affords realization of the complex structures of PDIMs in Mycobacterium tuberculosis biofilm, differentiation of phthiocerol and phthiodiolone lipid families and complete structure identification, including the phthiocerol and phthiodiolone backbones, and the mycocerosic acid substituents, including the locations of their multiple methyl side chains, can be achieved.


Asunto(s)
Ésteres/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Lípidos/química , Mycobacterium tuberculosis/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Biopelículas , Ésteres/análisis , Ácidos Grasos/análisis , Ácidos Grasos/química , Glucolípidos/química , Lípidos/análisis , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiología
8.
Biochim Biophys Acta ; 1851(12): 1530-8, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26361872

RESUMEN

Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.


Asunto(s)
Calcio/metabolismo , Lipogénesis , Distrofias Musculares/metabolismo , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Retículo Sarcoplasmático/metabolismo , Animales , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Masculino , Ratones , Ratones Endogámicos mdx , Distrofias Musculares/genética , Distrofias Musculares/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatasa/genética , Fosfatidato Fosfatasa/metabolismo , Fosfatidilcolinas/genética , Fosfatidiletanolaminas/genética , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo
9.
Infect Immun ; 84(4): 1137-1142, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26857573

RESUMEN

Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A2ß (iPLA2ß) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA2ß may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA2ß-knockout (iPLA2ß-KO) mice infected withT. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA2ß-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA2ß-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA2ß-KO mice produced significantly less nitric oxide (NO) and caused lessT. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA2ß activity does not influence myocardial inflammation, but iPLA2ß is essential forT. cruzi clearance.


Asunto(s)
Cardiomiopatía Chagásica/metabolismo , Cardiomiopatía Chagásica/parasitología , Regulación Enzimológica de la Expresión Génica/fisiología , Fosfolipasas A2 Grupo VI/metabolismo , Macrófagos/fisiología , Animales , Línea Celular , Eliminación de Gen , Fosfolipasas A2 Grupo VI/genética , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitritos , Carga de Parásitos
10.
J Biol Chem ; 289(20): 14194-210, 2014 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-24648512

RESUMEN

Palmitate (C16:0) induces apoptosis of insulin-secreting ß-cells by processes that involve generation of reactive oxygen species, and chronically elevated blood long chain free fatty acid levels are thought to contribute to ß-cell lipotoxicity and the development of diabetes mellitus. Group VIA phospholipase A2 (iPLA2ß) affects ß-cell sensitivity to apoptosis, and here we examined iPLA2ß effects on events that occur in ß-cells incubated with C16:0. Such events in INS-1 insulinoma cells were found to include activation of caspase-3, expression of stress response genes (C/EBP homologous protein and activating transcription factor 4), accumulation of ceramide, loss of mitochondrial membrane potential, and apoptosis. All of these responses were blunted in INS-1 cells that overexpress iPLA2ß, which has been proposed to facilitate repair of oxidized mitochondrial phospholipids, e.g. cardiolipin (CL), by excising oxidized polyunsaturated fatty acid residues, e.g. linoleate (C18:2), to yield lysophospholipids, e.g. monolysocardiolipin (MLCL), that can be reacylated to regenerate the native phospholipid structures. Here the MLCL content of mouse pancreatic islets was found to rise with increasing iPLA2ß expression, and recombinant iPLA2ß hydrolyzed CL to MLCL and released oxygenated C18:2 residues from oxidized CL in preference to native C18:2. C16:0 induced accumulation of oxidized CL species and of the oxidized phospholipid (C18:0/hydroxyeicosatetraenoic acid)-glycerophosphoethanolamine, and these effects were blunted in INS-1 cells that overexpress iPLA2ß, consistent with iPLA2ß-mediated removal of oxidized phospholipids. C16:0 also induced iPLA2ß association with INS-1 cell mitochondria, consistent with a role in mitochondrial repair. These findings indicate that iPLA2ß confers significant protection of ß-cells against C16:0-induced injury.


Asunto(s)
Apoptosis/efectos de los fármacos , Fosfolipasas A2 Grupo VI/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Palmitatos/efectos adversos , Animales , Cardiolipinas/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lisofosfolípidos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Oxidación-Reducción/efectos de los fármacos , Ratas
11.
Arterioscler Thromb Vasc Biol ; 34(4): 768-78, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24482376

RESUMEN

OBJECTIVE: We previously demonstrated that nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) mediates increased monocyte priming and chemotaxis under conditions of diabetic metabolic stress, and emerging data indicate that group VIA phospholipase A2 (iPLA2ß) also participates in regulating monocyte chemotaxis. Here, we examined relationships between iPLA2ß expression and Nox4 action in mouse peritoneal macrophages subjected to diabetic metabolic stress. APPROACH AND RESULTS: Increased iPLA2ß expression and activity were observed in macrophages from low-density lipoprotein receptor knockout mice that were fed a high-fat diet, and this was associated with time-dependent increases in atherosclerotic lesion size and macrophage content. Incubating macrophages with 30 mmol/L D-glucose, 100 µg/mL low-density lipoprotein, or both (D-glucose+low-density lipoprotein) induced a robust increase in iPLA2ß expression and activity and in cell migration in response to monocyte chemoattractant protein-1. The increases in iPLA2ß activity and cell migration were prevented by a bromoenol lactone iPLA2ß suicide inhibitor or an iPLA2ß antisense oligonucleotide. Incubating macrophages under conditions that mimic diabetic metabolic stress ex vivo resulted in increased Nox4 expression and activity and hydrogen peroxide generation compared with controls. Bromoenol lactone prevented those effects without affecting Nox2 expression. Nox4 inhibition eliminated diabetic metabolic stress-induced acceleration of macrophage migration. Lysophosphatidic acid restored Nox4 expression, hydrogen peroxide generation, and migration to bromoenol lactone-treated cells, and a lysophosphatidic acid receptor antagonist abrogated iPLA2ß-mediated increases in Nox4 expression. CONCLUSIONS: Taken together, these observations identify iPLA2ß and lysophosphatidic acid derived from its action as critical in regulating macrophage Nox4 activity and migration in the diabetic state in vivo and under similar conditions ex vivo.


Asunto(s)
Aterosclerosis/enzimología , Movimiento Celular , Diabetes Mellitus/enzimología , Fosfolipasas A2 Grupo VI/metabolismo , Macrófagos Peritoneales/enzimología , NADPH Oxidasas/metabolismo , Transducción de Señal , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Diabetes Mellitus/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo VI/genética , Peróxido de Hidrógeno/metabolismo , Lisofosfolípidos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Oligonucleótidos Antisentido/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Estrés Fisiológico , Factores de Tiempo , Transfección , Regulación hacia Arriba
12.
Anal Bioanal Chem ; 407(9): 2519-28, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25656850

RESUMEN

Listeria monocytogenes (L. monocytogenes) is a facultative, Gram-positive, food-borne bacterium, which causes serious infections. Although it is known that lipids play important roles in the survival of Listeria, the detailed structures of these lipids have not been established. In this contribution, we described linear ion-trap multiple-stage mass spectrometric approaches with high-resolution mass spectrometry toward complete structural analysis including the identities of the fatty acid substituents and their position on the glycerol backbone of the polar lipids, mainly phosphatidylglycerol, cardiolipin (CL), and lysyl-CL from L. monocytogenes. The location of the methyl side group along the fatty acid chain in each lipid family was characterized by a charge-switch strategy. This is achieved by first alkaline hydrolysis to release the fatty acid substituents, followed by tandem mass spectrometry on their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives as the M+ ions. Several findings in this study are unique: (1) we confirm the presence of a plasmalogen PG family that has not been previous reported; (2) an ion arising from a rare internal loss of lysylglycerol residue was observed in the MS(2) spectrum of lysyl-CL, permitting its distinction from other CL subfamilies.


Asunto(s)
Lípidos/química , Listeria monocytogenes/química , Espectrometría de Masas en Tándem/métodos , Metabolismo de los Lípidos , Listeria monocytogenes/metabolismo , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/instrumentación
13.
J Biol Chem ; 288(12): 8028-8042, 2013 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-23355467

RESUMEN

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.


Asunto(s)
Drosophila melanogaster/metabolismo , Cuerpo Adiposo/metabolismo , Lipogénesis , Animales , Proteínas de Ciclo Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Ingestión de Energía , Metabolismo Energético , Cuerpo Adiposo/fisiología , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucólisis , Hemolinfa/metabolismo , Hiperglucemia/metabolismo , Cetonas/metabolismo , Larva/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfolípidos/metabolismo , Transcriptoma
14.
J Biol Chem ; 288(8): 5268-77, 2013 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-23300084

RESUMEN

Type 1 phosphotidylinosotol-4 phosphate 5 kinase γ (PIP5KIγ) is central to generation of phosphotidylinosotol (4,5)P(2) (PI(4,5)P(2)). PIP5KIγ also participates in cytoskeletal organization by delivering talin to integrins, thereby enhancing their ligand binding capacity. As the cytoskeleton is pivotal to osteoclast function, we hypothesized that absence of PIP5KIγ would compromise their resorptive capacity. Absence of the kinase diminishes PI(4,5) abundance and desensitizes precursors to RANK ligand-stimulated differentiation. Thus, PIP5KIγ(-/-) osteoclasts are reduced in number in vitro and confirm physiological relevance in vivo. Despite reduced numbers, PIP5KIγ(-/-) osteoclasts surprisingly have normal cytoskeletons and effectively resorb bone. PIP5KIγ overexpression, which increases PI(4,5)P(2), also delays osteoclast differentiation and reduces cell number but in contrast to cells lacking the kinase, its excess disrupts the cytoskeleton. The cytoskeleton-disruptive effects of excess PIP5KIγ reflect its kinase activity and are independent of talin recognition. The combined arrested differentiation and disorganized cytoskeleton of PIP5KIγ-transduced osteoclasts compromises bone resorption. Thus, optimal PIP5KIγ and PI(4,5)P(2) expression, by osteoclasts, are essential for skeletal homeostasis.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Osteoclastos/citología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Animales , Transporte Biológico , Resorción Ósea , Calcio/metabolismo , Diferenciación Celular , Ligandos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoclastos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Plásmidos/metabolismo , Ligando RANK/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos
15.
Hepatology ; 57(6): 2202-12, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23401290

RESUMEN

UNLABELLED: Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes, also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP(-/-) mice contain fewer LDs than wild-type (WT) HSCs, and exhibit up-regulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP(-/-) mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP(-/-) and WT mice a high-fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP(-/-) mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP(-/-) mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. CONCLUSION: L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. (Hepatology 2013).


Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Hígado Graso/metabolismo , Células Estrelladas Hepáticas/fisiología , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Animales , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Hígado Graso/etiología , Femenino , Fibrosis , Fructosa/efectos adversos , Técnicas de Transferencia de Gen , Lipogénesis , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Perilipina-5 , Proteínas/metabolismo , Triglicéridos/metabolismo
16.
Hepatology ; 57(6): 2213-23, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23359250

RESUMEN

UNLABELLED: Earlier reports suggest a link between mitochondrial dysfunction and development of hepatic insulin resistance. Here we used a murine model heterozygous (HET) for a mitochondrial trifunctional protein (MTP) gene defect to determine if a primary defect in mitochondrial long-chain fatty acid oxidation disrupts hepatic insulin action. Hyperinsulinemic-euglycemic clamps and signaling studies were performed for assessment of whole-body and hepatic insulin resistance/signaling. In addition, hepatic fatty acid oxidation and hepatic insulin action were assessed in vitro using primary hepatocytes isolated from HET and wildtype (WT) mice. In both hepatic mitochondria and isolated primary hepatocytes, heterozygosity of MTP caused an ∼50% reduction in mitochondrial fatty acid oxidation, a significantly impaired glucose disposal during the insulin clamp, and a markedly lower insulin-stimulated suppression of hepatic glucose production. HET mice also exhibited impaired insulin signaling, with increased hepatic phosphorylation of IRS2 (ser731) and reduced Akt phosphorylation (ser473) in both hepatic tissue and isolated primary hepatocytes. Assessment of insulin-stimulated FOXO1/phospho-FOXO1 protein content and PEPCK/G6Pase messenger RNA (mRNA) expression did not reveal differences between HET and WT mice. However, insulin-induced phosphorylation of GSK3ß was significantly blunted in HET mice. Hepatic insulin resistance was associated with an increased methylation status of the catalytic subunit of protein phosphatase 2A (PP2A-C), but was not associated with differences in hepatic diacylglycerol content, activated protein kinase C-ϵ (PKC-ϵ), inhibitor κB kinase ß (IKK-ß), c-Jun N-terminal kinase (JNK), or phospho-JNK protein contents. Surprisingly, hepatic ceramides were significantly lower in the HET mice compared with WT. CONCLUSION: A primary defect in mitochondrial fatty acid ß-oxidation causes hepatic insulin resistance selective to hepatic glycogen metabolism that is associated with elevated methylated PP2A-C, but independent of other mechanisms commonly considered responsible for insulin resistance. (HEPATOLOGY 2013;).


Asunto(s)
Hígado Graso/metabolismo , Resistencia a la Insulina , Metabolismo de los Lípidos , Complejos Multienzimáticos/genética , Animales , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Hígado Graso/genética , Técnica de Clampeo de la Glucosa , Heterocigoto , Insulina/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Proteína Trifuncional Mitocondrial , Oxidación-Reducción
17.
Neurochem Res ; 39(8): 1522-32, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24919816

RESUMEN

Calcium-independent phospholipase A2 group VIa (iPLA2ß) preferentially releases docosahexaenoic acid (DHA) from the sn-2 position of phospholipids. Mutations of its gene, PLA2G6, are found in patients with several progressive motor disorders, including Parkinson disease. At 4 months, PLA2G6 knockout mice (iPLA2ß(-/-)) show minimal neuropathology but altered brain DHA metabolism. By 1 year, they develop motor disturbances, cerebellar neuronal loss, and striatal α-synuclein accumulation. We hypothesized that older iPLA2ß(-/-) mice also would exhibit inflammatory and other neuropathological changes. Real-time polymerase chain reaction and Western blotting were performed on whole brain homogenate from 15 to 20-month old male iPLA2ß(-/-) or wild-type (WT) mice. These older iPLA2ß(-/-) mice compared with WT showed molecular evidence of microglial (CD-11b, iNOS) and astrocytic (glial fibrillary acidic protein) activation, disturbed expression of enzymes involved in arachidonic acid metabolism, loss of neuroprotective brain derived neurotrophic factor, and accumulation of cytokine TNF-α messenger ribonucleic acid, consistent with neuroinflammatory pathology. There was no evidence of synaptic loss, of reduced expression of dopamine active reuptake transporter, or of accumulation of the Parkinson disease markers Parkin or Pink1. iPLA2γ expression was unchanged. iPLA2ß deficient mice show evidence of neuroinflammation and associated neuropathology with motor dysfunction in later life. These pathological biomarkers could be used to assess efficacy of dietary intervention, antioxidants or other therapies on disease progression in this mouse model of progressive human motor diseases associated with a PLA2G6 mutation.


Asunto(s)
Envejecimiento/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fosfolipasas A2 Grupo VI/deficiencia , Trastornos de la Destreza Motora/metabolismo , Envejecimiento/genética , Animales , Fosfolipasas A2 Grupo VI/genética , Masculino , Ratones , Ratones Noqueados , Trastornos de la Destreza Motora/genética , Trastornos de la Destreza Motora/patología
18.
J Biol Chem ; 287(8): 5528-41, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22194610

RESUMEN

Group VIA phospholipase A(2) (iPLA(2)ß) in pancreatic islet ß-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)ß activity and amplified by iPLA(2)ß overexpression. While exploring signaling events that occur downstream of iPLA(2)ß activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)ß expression level, and that it is stimulated by the iPLA(2)ß reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates ß-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)ß inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)ß in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both ß-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)ß products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced ß-cell p38 MAPK phosphorylation. Thapsigargin-induced ß-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)ß in ß-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)ß activation, and that p38 MAPK is involved in the ß-cell functional responses of insulin secretion and apoptosis in which iPLA(2)ß participates.


Asunto(s)
Fosfolipasas A2 Grupo VI/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ácido Araquidónico/farmacología , Línea Celular Tumoral , Ceramidas/metabolismo , Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Fosfolipasas A2 Grupo VI/antagonistas & inhibidores , Imidazoles/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/enzimología , Masculino , Ratones , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , Pironas/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismo
19.
J Biol Chem ; 287(29): 24739-53, 2012 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-22637477

RESUMEN

Whether group VIA phospholipase A(2) (iPLA(2)ß) is involved in vascular inflammation and neointima formation is largely unknown. Here, we report that iPLA(2)ß expression increases in the vascular tunica media upon carotid artery ligation and that neointima formation is suppressed by genetic deletion of iPLA(2)ß or by inhibiting its activity or expression via perivascular delivery of bromoenol lactone or of antisense oligonucleotides, respectively. To investigate whether smooth muscle-specific iPLA(2)ß is involved in neointima formation, we generated transgenic mice in which iPLA(2)ß is expressed specifically in smooth muscle cells and demonstrate that smooth muscle-specific expression of iPLA(2)ß exacerbates ligation-induced neointima formation and enhanced both production of proinflammatory cytokines and vascular infiltration by macrophages. With cultured vascular smooth muscle cell, angiotensin II, arachidonic acid, and TNF-α markedly induce increased expression of IL-6 and TNF-α mRNAs, all of which were suppressed by inhibiting iPLA(2)ß activity or expression with bromoenol lactone, antisense oligonucleotides, and genetic deletion, respectively. Similar suppression also results from genetic deletion of 12/15-lipoxygenase or inhibiting its activity with nordihydroguaiaretic acid or luteolin. Expression of iPLA(2)ß protein in cultured vascular smooth muscle cells was found to depend on the phenotypic state and to rise upon incubation with TNF-α. Our studies thus illustrate that smooth muscle cell-specific iPLA(2)ß participates in the initiation and early progression of vascular inflammation and neointima formation and suggest that iPLA(2)ß may represent a novel therapeutic target for preventing cardiovascular diseases.


Asunto(s)
Calcio/metabolismo , Inflamación/metabolismo , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Neointima/inmunología , Neointima/metabolismo , Fosfolipasas A2 Calcio-Independiente/metabolismo , Angiotensina II , Animales , Western Blotting , Arterias Carótidas/inmunología , Arterias Carótidas/metabolismo , Células Cultivadas , Inmunohistoquímica , Inflamación/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Oligonucleótidos Antisentido , Fosfolipasas A2 Calcio-Independiente/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Conejos , Ratas , Ratas Sprague-Dawley
20.
Biochim Biophys Acta ; 1821(9): 1278-86, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22349267

RESUMEN

Calcium-independent phospholipase A(2) group VIA (iPLA(2)ß) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA(2)ß gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA(2)ß in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA(2)ß(+/+)) and knockout (iPLA(2)ß(-/-)) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA(2), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA(2)ß(+/+) mice, iPLA(2)ß(-/-) mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA(2)ß(-/-) mice, brain levels of iPLA(2)ß mRNA, protein, and activity were decreased, as was the iPLA(2)γ (Group VIB PLA(2)) mRNA level, while levels of secretory sPLA(2)-V mRNA, protein, and activity and cytosolic cPLA(2)-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA(2)ß deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.


Asunto(s)
Encéfalo/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Fosfolipasas A2 Grupo VI , Metabolismo de los Lípidos , Proteínas del Tejido Nervioso/metabolismo , Fosfolípidos/metabolismo , Animales , Encéfalo/patología , Química Encefálica/genética , Ciclooxigenasa 1/biosíntesis , Ciclooxigenasa 1/genética , Ácidos Docosahexaenoicos/genética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Lipooxigenasa/biosíntesis , Lipooxigenasa/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/genética , Fosfolipasas A2 Secretoras/biosíntesis , Fosfolipasas A2 Secretoras/genética , Fosfolípidos/genética
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