RESUMEN
Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.
Asunto(s)
Mitocondrias/metabolismo , Fosfolípidos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transporte Biológico , Carboxiliasas/genética , Carboxiliasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.
Asunto(s)
Retículo Endoplásmico , Toxoplasma , Toxoplasma/enzimología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/enzimología , Humanos , Fosfatidilserinas/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , CarboxiliasasRESUMEN
Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.
Asunto(s)
Aciltransferasas/metabolismo , Glicerilfosforilcolina/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acilación , Aciltransferasas/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
In the vertebrate nervous system, myelination of axons for rapid impulse propagation requires the synthesis of large amounts of lipids and proteins by oligodendrocytes and Schwann cells. Myelin membranes are thought to be cell-autonomously assembled by these axon-associated glial cells. Here, we report the surprising finding that in normal brain development, a substantial fraction of the lipids incorporated into central nervous system (CNS) myelin are contributed by astrocytes. The oligodendrocyte-specific inactivation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), an essential coactivator of the transcription factor SREBP and thus of lipid biosynthesis, resulted in significantly retarded CNS myelination; however, myelin appeared normal at 3 months of age. Importantly, embryonic deletion of the same gene in astrocytes, or in astrocytes and oligodendrocytes, caused a persistent hypomyelination, as did deletion from astrocytes during postnatal development. Moreover, when astroglial lipid synthesis was inhibited, oligodendrocytes began incorporating circulating lipids into myelin membranes. Indeed, a lipid-enriched diet was sufficient to rescue hypomyelination in these conditional mouse mutants. We conclude that lipid synthesis by oligodendrocytes is heavily supplemented by astrocytes in vivo and that horizontal lipid flux is a major feature of normal brain development and myelination.
Asunto(s)
Astrocitos/metabolismo , Enfermedades Desmielinizantes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Animales , Astrocitos/patología , Astrocitos/ultraestructura , Biomarcadores/metabolismo , Cruzamientos Genéticos , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/prevención & control , Dieta Alta en Grasa , Acido Graso Sintasa Tipo I/metabolismo , Eliminación de Gen , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mutación , Vaina de Mielina/patología , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/patología , Oligodendroglía/ultraestructura , Especificidad de Órganos , Procesamiento Proteico-Postraduccional , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genéticaRESUMEN
BACKGROUND: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. RESULTS: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. CONCLUSIONS: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.
Asunto(s)
Proteínas Bacterianas/genética , Melibiosa/metabolismo , Salmonella typhimurium/genética , Simportadores/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cardiolipinas/química , Cardiolipinas/metabolismo , Melibiosa/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Salmonella typhimurium/metabolismo , Simportadores/química , Simportadores/metabolismoRESUMEN
Toxoplasma gondii is among the most prevalent protozoan parasites, which infects a wide range of organisms, including one-third of the human population. Its rapid intracellular replication within a vacuole requires efficient synthesis of glycerophospholipids. Cytidine diphosphate-diacylglycerol (CDP-DAG) serves as a major precursor for phospholipid synthesis. Given the peculiarities of lipid biogenesis, understanding the mechanism and physiological importance of CDP-DAG synthesis is particularly relevant in T. gondii Here, we report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in the parasite. The eukaryotic-type TgCDS1 and the prokaryotic-type TgCDS2 reside in the endoplasmic reticulum and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuated the parasite growth and resulted in a nearly complete loss of virulence in a mouse model. Moreover, mice infected with the TgCDS1 mutant became fully resistant to challenge infection with a hyper-virulent strain of T. gondii The residual growth of the TgCDS1 mutant was abolished by consecutive deletion of TgCDS2. Lipidomic analyses of the two mutants revealed significant and specific declines in phosphatidylinositol and phosphatidylglycerol levels upon repression of TgCDS1 and after deletion of TgCDS2, respectively. Our data suggest a "division of labor" model of lipid biogenesis in T. gondii in which two discrete CDP-DAG pools produced in the endoplasmic reticulum and apicoplast are subsequently used for the synthesis of phosphatidylinositol in the Golgi bodies and phosphatidylglycerol in the mitochondria. The essential and divergent nature of CDP-DAG synthesis in the parasite apicoplast offers a potential drug target to inhibit the asexual reproduction of T. gondii.
Asunto(s)
Diacilglicerol Colinafosfotransferasa/genética , Glicerofosfolípidos/biosíntesis , Proteínas Protozoarias/genética , Toxoplasma/enzimología , Animales , Animales Modificados Genéticamente , Apicoplastos/enzimología , Diacilglicerol Colinafosfotransferasa/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Aparato de Golgi/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Mutación , Fosfatidilgliceroles/química , Fosfatidilinositoles/química , Filogenia , Dominios Proteicos , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , VirulenciaRESUMEN
Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Gotas Lipídicas/metabolismo , Lisosomas/metabolismo , Esterol Esterasa/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Células Estrelladas Hepáticas/efectos de los fármacos , Gotas Lipídicas/efectos de los fármacos , Lisosomas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Wistar , Esterol Esterasa/antagonistas & inhibidores , Esterol Esterasa/deficiencia , Relación Estructura-ActividadRESUMEN
The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.
Asunto(s)
Retículo Endoplásmico/enzimología , Glicerofosfolípidos/metabolismo , Proteínas Protozoarias/metabolismo , Treonina/análogos & derivados , Toxoplasma/fisiología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/parasitología , Encéfalo/patología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente/inmunología , Organismos Modificados Genéticamente/metabolismo , Enquistamiento de Parásito , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Piel/inmunología , Piel/metabolismo , Piel/parasitología , Treonina/metabolismo , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Toxoplasmosis/prevención & control , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Vacunas Atenuadas/uso terapéutico , VirulenciaRESUMEN
The hydrophobic molecules of the metabolome - also named the lipidome - constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host-pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Hongos/fisiología , Interacciones Huésped-Patógeno/fisiología , Metabolismo de los Lípidos , Metaboloma , Fenómenos Fisiológicos de los Virus , Fenómenos Fisiológicos Bacterianos/genética , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Hongos/genética , Interacciones Huésped-Patógeno/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunidad Innata , Metabolismo de los Lípidos/fisiología , Metaboloma/fisiología , Fenómenos Fisiológicos de los Virus/genéticaRESUMEN
The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682.
Asunto(s)
Astrocitos/metabolismo , Potenciales Postsinápticos Excitadores/genética , Regulación de la Expresión Génica/genética , Metabolismo de los Lípidos/fisiología , Sinapsis/fisiología , Animales , Astrocitos/ultraestructura , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Acido Graso Sintasa Tipo I/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/ultraestructura , Tinción con Nitrato de Plata , Sinapsis/ultraestructura , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC-MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT-/- HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT-/- HSCs (1080nm) is significantly smaller than in wild type HSCs (1618nm). This is a consequence of an altered lipid droplet size distribution with 50.5±9.0% small (≤700nm) lipid droplets in LRAT-/- HSCs and 25.6±1.4% large (1400-2100nm) lipid droplets in wild type HSC cells. Upon prolonged (24h) incubation, the amounts of small (≤700nm) lipid droplets strongly increased both in wild type and in LRAT-/- HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.
Asunto(s)
Aciltransferasas/metabolismo , Ésteres/metabolismo , Células Estrelladas Hepáticas/metabolismo , Gotas Lipídicas/metabolismo , Lípidos/fisiología , Animales , Línea Celular , Diacilglicerol O-Acetiltransferasa/metabolismo , Humanos , Hepatopatías/metabolismo , Ratones , Espectrometría de Masas en Tándem/métodosRESUMEN
Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.
Asunto(s)
Células del Cúmulo/enzimología , Ácidos Grasos/toxicidad , Oocitos/fisiología , Estearoil-CoA Desaturasa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Bovinos , Células del Cúmulo/efectos de los fármacos , Técnicas de Cultivo de Embriones , Inhibidores Enzimáticos/farmacología , Femenino , Fertilización In Vitro , Metabolismo de los Lípidos/genética , Necrosis , Ácido Oléico/metabolismo , Ácido Oléico/farmacología , Oocitos/efectos de los fármacos , Ovario/citología , Ácidos Esteáricos/metabolismo , Ácidos Esteáricos/farmacología , Estearoil-CoA Desaturasa/antagonistas & inhibidoresRESUMEN
Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Células Estrelladas Hepáticas/metabolismo , Lipasa/genética , Triglicéridos/biosíntesis , Animales , Ésteres del Colesterol/genética , Ésteres del Colesterol/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Ácidos Grasos Insaturados/biosíntesis , Células Estrelladas Hepáticas/patología , Gotas Lipídicas/metabolismo , Lipogénesis/genética , Lipólisis/genética , Ratones , Ratones Noqueados , Compuestos de Fenilurea/administración & dosificación , Ratas , Triglicéridos/genéticaRESUMEN
Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.
Asunto(s)
Coenzima A Ligasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Células Estrelladas Hepáticas/enzimología , Triglicéridos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular , Coenzima A Ligasas/antagonistas & inhibidores , Coenzima A Ligasas/genética , Inhibidores Enzimáticos/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Masculino , Fosfatidilcolinas/metabolismo , Interferencia de ARN , Ratas Wistar , Rosiglitazona , Tiazolidinedionas/farmacología , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
The deregulation of brain cholesterol metabolism is typical in acute neuronal injury (such as stroke, brain trauma and epileptic seizures) and chronic neurodegenerative diseases (Alzheimer's disease). Since both conditions are characterized by excessive stimulation of glutamate receptors, we have here investigated to which extent excitatory neurotransmission plays a role in brain cholesterol homeostasis. We show that a short (30 min) stimulation of glutamatergic neurotransmission induces a small but significant loss of membrane cholesterol, which is paralleled by release to the extracellular milieu of the metabolite 24S-hydroxycholesterol. Consistent with a cause-effect relationship, knockdown of the enzyme cholesterol 24-hydroxylase (CYP46A1) prevented glutamate-mediated cholesterol loss. Functionally, the loss of cholesterol modulates the magnitude of the depolarization-evoked calcium response. Mechanistically, glutamate-induced cholesterol loss requires high levels of intracellular Ca(2+), a functional stromal interaction molecule 2 (STIM2) and mobilization of CYP46A1 towards the plasma membrane. This study underscores the key role of excitatory neurotransmission in the control of membrane lipid composition, and consequently in neuronal membrane organization and function.
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Colesterol/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Transmisión Sináptica , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol 24-Hidroxilasa , Técnicas de Silenciamiento del Gen , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Hidroxicolesteroles/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Molécula de Interacción Estromal 2RESUMEN
Lipid rafts are micro-domains of ordered lipids (Lo phase) in biological membranes. The Lo phase of cellular membranes can be isolated from disordered lipids (Ld phase) after treatment with 1 % Triton X-100 at 4 °C in which the Lo phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and porcine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with <5 mM methyl-ß-cyclodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein-cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the Lo phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.
Asunto(s)
Colesterol/análisis , Células Epiteliales/citología , Microdominios de Membrana/química , Fosfolípidos/análisis , Espermatozoides/citología , Esfingolípidos/análisis , Esfingomielinas/análisis , Animales , Detergentes/química , Perros , Células Epiteliales/química , Células Epiteliales/ultraestructura , Masculino , Microdominios de Membrana/ultraestructura , Espermatozoides/química , Espermatozoides/ultraestructura , PorcinosRESUMEN
Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.
Asunto(s)
Células del Cúmulo/fisiología , Ácidos Grasos/efectos adversos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Oogénesis , Animales , Apoptosis/efectos de los fármacos , Bovinos , Células Cultivadas , Citoprotección , Femenino , Técnicas de Maduración In Vitro de los Oocitos , Lípidos/efectos adversos , Lípidos/análisis , Oogénesis/efectos de los fármacos , Oogénesis/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The fertility of high-yielding dairy cows has declined during the last 3 decades, in association with a more profound negative energy balance (NEB) during the early weeks postpartum. One feature of this NEB is a marked elevation in circulating free fatty acid (FFA) concentrations. During the early postpartum period (≤ d 42), circulatory FFA levels were measured weekly, and progesterone concentrations and the diameter of the dominant follicles were determined thrice weekly. Retrospectively, cows that ovulated within 35 d postpartum were grouped as "normal ovulating" cows (n = 5), and the others were grouped as "delayed ovulating" cows (n = 5). In both groups, high total FFA levels (>500 µM) were evident immediately postpartum. Interestingly, cows with delayed ovulation had higher plasma FFA concentrations in the first weeks postpartum compared with normal ovulating cows. In both cow groups, FFA decreased to control levels of non-NEB cows within 3 wk postpartum. The FFA compositions and concentrations in fluids from the dominant follicles of postpartum cows were not different between the normal and delayed ovulating cows when measured at potential insemination points: d 55, 80, and 105 postpartum. Interestingly, the concentration of monounsaturated oleic acid was higher and that of saturated stearic acid lower in follicular fluids of both groups compared with that in blood. The level of FFA in follicular fluid was correlated with the ratio of 17ß-estradiol (E2) to progesterone (P4) in follicular fluid, with a relatively high level of unsaturated FFA in follicles with a low E2:P4 ratio. Taken together, these results indicate that a more severe NEB early postpartum is related to a delay in the first postpartum ovulation and does not affect FFA composition in follicular fluid at the preferred insemination time. The high FFA level in dominant follicles with a low E2:P4 ratio may be due to a different FFA metabolism in these follicles. The diagnostic value of this observation for selective screening of dominant follicles needs further investigation.
Asunto(s)
Ácidos Grasos no Esterificados/sangre , Líquido Folicular/química , Inseminación/fisiología , Periodo Posparto , Animales , Bovinos , Metabolismo Energético , Estradiol/sangre , Femenino , Ácido Oléico/sangre , Ovulación , Análisis de Componente Principal , Progesterona/sangre , Estudios Retrospectivos , Ácidos Esteáricos/sangre , Estrés FisiológicoRESUMEN
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.
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Proteínas Portadoras/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lisofosfolipasa/metabolismo , Proteínas de la Membrana/metabolismo , Palmitatos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipasas A2/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.