RESUMEN
Hepatic stellate cells (HSCs) are liver-resident cells best known for their role in vitamin A storage under physiological conditions. Upon liver injury, HSCs activate into myofibroblast-like cells, a key process in the onset of liver fibrosis. Lipids play an important role during HSC activation. Here, we provide a comprehensive characterization of the lipidomes of primary rat HSCs during 17 days of activation in vitro. For lipidomic data interpretation, we expanded our previously described Lipid Ontology (LION) and associated web application (LION/Web) with the LION-PCA heatmap module, which generates heatmaps of the most typical LION-signatures in lipidomic datasets. Furthermore, we used LION to perform pathway analysis to determine the significant metabolic conversions in lipid pathways. Together, we identify two distinct stages of HSC activation. In the first stage, we observe a decrease of saturated phosphatidylcholine, sphingomyelin, and phosphatidic acid and an increase in phosphatidylserine and polyunsaturated bis(monoacylglycero)phosphate (BMP), a lipid class typically localized at endosomes and lysosomes. In the second activation stage, BMPs, hexosylceramides, and ether-linked phosphatidylcholines are elevated, resembling a lysosomal lipid storage disease profile. The presence of isomeric structures of BMP in HSCs was confirmed ex vivo in MS-imaging datasets of steatosed liver sections. Finally, treatment with pharmaceuticals targeting the lysosomal integrity led to cell death in primary HSCs but not in HeLa cells. In summary, our combined data suggest that lysosomes play a critical role during a two-stage activation process of HSCs.
Asunto(s)
Células Estrelladas Hepáticas , Lipidómica , Humanos , Ratas , Animales , Células Estrelladas Hepáticas/metabolismo , Células HeLa , Cirrosis Hepática/metabolismo , Lisosomas/metabolismo , Lípidos/fisiologíaRESUMEN
In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination.
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Cisteína , Semen , Masculino , Porcinos , Animales , Espermatozoides/metabolismo , Reacción Acrosómica , Fertilización In Vitro/veterinariaRESUMEN
The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.
Asunto(s)
Hígado Graso , Neoplasias Hepáticas , Animales , Hígado Graso/patología , Hepatocitos/metabolismo , Lípidos , Hígado/patología , Neoplasias Hepáticas/patología , Ratones , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-aktRESUMEN
BACKGROUND: Improvements in mass spectrometry (MS) technologies coupled with bioinformatics developments have allowed considerable advancement in the measurement and interpretation of lipidomics data in recent years. Since research areas employing lipidomics are rapidly increasing, there is a great need for bioinformatic tools that capture and utilize the complexity of the data. Currently, the diversity and complexity within the lipidome is often concealed by summing over or averaging individual lipids up to (sub)class-based descriptors, losing valuable information about biological function and interactions with other distinct lipids molecules, proteins and/or metabolites. AIM OF REVIEW: To address this gap in knowledge, novel bioinformatics methods are needed to improve identification, quantification, integration and interpretation of lipidomics data. The purpose of this mini-review is to summarize exemplary methods to explore the complexity of the lipidome. KEY SCIENTIFIC CONCEPTS OF REVIEW: Here we describe six approaches that capture three core focus areas for lipidomics: (1) lipidome annotation including a resolvable database identifier, (2) interpretation via pathway- and enrichment-based methods, and (3) understanding complex interactions to emphasize specific steps in the analytical process and highlight challenges in analyses associated with the complexity of lipidome data.
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Biología Computacional , Lipidómica , Bases de Datos Factuales , Lípidos , Espectrometría de MasasRESUMEN
The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases.
Asunto(s)
Proteínas Amiloidogénicas/fisiología , Agregado de Proteínas/fisiología , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Glicosaminoglicanos , Humanos , Iones , Lípidos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Metales , Ácidos Nucleicos , Dominios Proteicos/fisiologíaRESUMEN
The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat-beclin 1-derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef-is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat-beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.
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Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/uso terapéutico , Autofagia/efectos de los fármacos , Proteínas de la Membrana/química , Proteínas de la Membrana/uso terapéutico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/farmacología , Beclina-1 , Permeabilidad de la Membrana Celular , Células Cultivadas , Virus Chikungunya/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/metabolismo , VIH-1/fisiología , Células HeLa , Humanos , Macrófagos/citología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Replicación Viral/efectos de los fármacos , Virus del Nilo Occidental/efectos de los fármacos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
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
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
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.
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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
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
BACKGROUND: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. RESULTS: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. CONCLUSIONS: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.
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GTP Fosfohidrolasas/inmunología , Proteínas de Unión al GTP/inmunología , Lisosomas/inmunología , Animales , Autofagia , Línea Celular , GTP Fosfohidrolasas/análisis , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/genética , Eliminación de Gen , Inmunidad Innata , Infecciones/genética , Infecciones/inmunología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
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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.
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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.
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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
Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release.
Asunto(s)
Aciltransferasas , Células Estrelladas Hepáticas , Células Estrelladas Hepáticas/metabolismo , Aciltransferasas/metabolismo , Aciltransferasas/genética , Animales , Ésteres de Retinilo/metabolismo , Células Cultivadas , Diterpenos/metabolismo , Diterpenos/farmacología , Ratas , RatonesRESUMEN
Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed.
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Exosomas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Colesterol/metabolismo , Células Epiteliales/metabolismo , Glicerofosfolípidos/metabolismo , Humanos , Metabolismo de los Lípidos , Masculino , Próstata/citología , Próstata/metabolismo , Semen/metabolismo , Esfingomielinas/metabolismoRESUMEN
Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR-1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR-1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the previously published GAPR-1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR-1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR-1 caused a rapid and stable tethering of liposomes. [D81K]GAPR-1, a mutant predicted to stabilize the IP6-induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR-1 however, a mutant predicted to stabilize the non-rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function.