RESUMEN
Farber disease is a rare autosomal recessive disorder caused by acid ceramidase deficiency that usually presents as early-onset progressive visceral and neurologic disease. To understand the neurologic abnormality, we investigated behavioral, biochemical, and cellular abnormalities in the central nervous system of Asah1P361R/P361R mice, which serve as a model of Farber disease. Behaviorally, the mutant mice had reduced voluntary locomotion and exploration, increased thigmotaxis, abnormal spectra of basic behavioral activities, impaired muscle grip strength, and defects in motor coordination. A few mutant mice developed hydrocephalus. Mass spectrometry revealed elevations of ceramides, hydroxy-ceramides, dihydroceramides, sphingosine, dihexosylceramides, and monosialodihexosylganglioside in the brain. The highest accumulation was in hydroxy-ceramides. Storage compound distribution was analyzed by mass spectrometry imaging and morphologic analyses and revealed involvement of a wide range of central nervous system cell types (eg, neurons, endothelial cells, and choroid plexus cells), most notably microglia and/or macrophages. Coalescing and mostly perivascular granuloma-like accumulations of storage-laden CD68+ microglia and/or macrophages were seen as early as 3 weeks of age and located preferentially in white matter, periventricular zones, and meninges. Neurodegeneration was also evident in specific cerebral areas in late disease. Overall, our central nervous system studies in Asah1P361R/P361R mice substantially extend the understanding of human Farber disease and suggest that this model can be used to advance therapeutic approaches for this currently untreatable disorder.
Asunto(s)
Sistema Nervioso Central/anomalías , Lipogranulomatosis de Farber/complicaciones , Lipogranulomatosis de Farber/patología , Malformaciones del Sistema Nervioso/etiología , Malformaciones del Sistema Nervioso/patología , Ceramidasa Ácida/metabolismo , Animales , Conducta Animal , Sistema Nervioso Central/patología , Cerebelo/patología , Cerebelo/ultraestructura , Cerebro/patología , Cerebro/ultraestructura , Homocigoto , Hidrocefalia/patología , Ratones , Ratones Transgénicos , Actividad Motora , Neuronas/patología , Neuronas/ultraestructura , Fenotipo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingolípidos/metabolismo , Factores de TiempoRESUMEN
Fumonisin B1 (FB1) is a well-known inhibitor of de novo sphingolipid biosynthesis, due to its ability to inhibit ceramide synthases (CerS) activity. In mammals, this toxin triggers broad clinical symptoms with multi-organ dysfunction such as hepatotoxicity or pulmonary edema. The molecular mechanism of CerS inhibition by FB1 remains unknown. Due to the existence of six mammalian CerS isoforms with a tissue-specific expression pattern, we postulated that the organ-specific adverse effects of FB1 might be due to different CerS isoforms. The sphingolipid contents of lung and liver were compared in normal and FB1-exposed piglets (gavage with 1.5 mg FB1/kg body weight daily for 9 days). The effect of the toxin on each CerS was deduced from the analysis of its effects on individual ceramide (Cer) and sphingomyelin (SM) species. As expected, the total Cer content decreased by half in the lungs of FB1-exposed piglets, while in contrast, total Cer increased 3.5-fold in the livers of FB1-exposed animals. Our data also indicated that FB1 is more prone to bind to CerS4 and CerS2 to deplete lung and to enrich liver in d18:1/C20:0 and d18:1/C22:0 ceramides. It also interact with CerS1 to enrich liver in d18:1/C18:0 ceramides. Cer levels were counterbalanced by those of SM. In conclusion, these results demonstrate that the specificity of the effects of FB1 on tissues and organs is due to the effects of the toxin on CerS4, CerS2, and CerS1.
Asunto(s)
Fumonisinas/toxicidad , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Esfingosina N-Aciltransferasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/toxicidad , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Esfingolípidos/biosíntesis , Esfingosina N-Aciltransferasa/metabolismo , Porcinos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismoRESUMEN
A new sphingolipid hybrid molecule was designed to assemble, within a tail-to-tail double-chain structure, the ceramide hydrophilic moiety and the tetrahydrofuran pharmacophore of jaspine B, a natural product known to interfere with sphingolipid metabolism. This compound was prepared through acylation of sphingosine with a jaspine B derivative bearing a COOH group in the terminal position of the aliphatic backbone. This new hybrid molecule was evaluated for its capacities to affect melanoma cell viability and sphingolipid metabolism. While retaining the cytotoxicity of ceramide itself, this compound was shown to lower the sphingomyelin cellular levels and significantly enhance the production of sphingosine-1-phosphate, thus representing a novel sphingolipid metabolism modulator.
Asunto(s)
Productos Biológicos/farmacología , Ceramidas/farmacología , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Productos Biológicos/química , Productos Biológicos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ceramidas/química , Ceramidas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Conformación Molecular , Esfingolípidos/química , Esfingosina/química , Esfingosina/metabolismo , Esfingosina/farmacología , Relación Estructura-ActividadRESUMEN
INTRODUCTION: Production of isoprostanes (IsoPs) is enhanced after acute, intense, and prolonged exercise, in untrained subjects. This effect is greater in older subjects. The present study aims to delineate the profile of acute-exercise-induced IsoPs levels in young and older endurance-trained subjects. METHODS: All included subjects were male, young (n = 6; 29 yrs ± 5.7) or older (n = 6; 63.7 yrs ± 2.3), and competitors. The kinetics of F2-IsoPs in blood-sera was assessed at rest, for the maximal aerobic exercise power (MAP) corresponding to the cardio-respiratory fitness index and after a 30-min recovery period. RESULTS: No significant time effect on F2-IsoPs kinetics was identified in young subjects. However, in older athletes, F2-IsoPs blood-concentrations at the MAP were higher than at rest, whereas these blood-concentrations did not differ between rest and after the 30-min recovery period. CONCLUSION: Because plasma glutathione (GSH) promotes the formation of some F2-IsoPs, we suggest that the surprising decrease in F2-IsoPs levels in older subjects would be caused by decreased GSH under major ROS production in older subjects. We argue that the assessment F2-IsoPs in plasma as biomarkers of the aging process should be challenged by exercise to improve the assessment of the functional response against reactive oxygen species in older subjects.
RESUMEN
Sphingolipids are sphingoid base-containing lipids, among which some metabolites behave as bioactive molecules in various biological processes, including cell death. Whereas ceramide is now viewed as an anti-oncometabolite, leading to cancer cell death, CD95L-induced apoptosis is associated with sphingolipid changes, which likely contribute to caspase-dependent signaling pathway activation. Here, we describe Liquid Chromatography-high resolution mass spectrometry method (LC-HRMS) to analyze sphingolipid metabolism changes triggered by CD95L.
Asunto(s)
Cromatografía Liquida , Proteína Ligando Fas/metabolismo , Metabolismo de los Lípidos , Espectrometría de Masas , Esfingolípidos/metabolismo , Apoptosis , Ceramidas/metabolismo , Unión ProteicaRESUMEN
Diabetic nephropathy (DN) is a major cause of chronic kidney disease that frequently leads to end stage renal failure. Lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are lysophospholipid mediators shown to accumulate in kidney and to promote renal inflammation and tubulo-interstitial fibrosis in diabetic rodent models. Here we assessed whether LPA and LPC were associated to the development of nephropathy in diabetic human patients. Several molecular species of LPA and LPC were quantified by LC/MS-MS in urine and plasma from type 2 diabetic patients with (cases; n=41) or without (controls, n=41) nephropathy symptoms (micro/macro-albuminuria and eGFR<60ml/min/1.73m2). Cases and controls were matched for sex, age and diabetes duration. Six species were detected in urine for both LPA and LPC, LPA16:0, LPA20:4, LPC16:0, LPC18:0, LPC18:1, and LPC18:2 that were significantly more concentrated in cases than in controls. Total LPC and LPA (sum of detected species) were significantly and exclusively associated with albuminuria (P<0.0001 and P=0.0009 respectively) and were significantly higher in the 3rd when compared to the 1st albuminuria tertile in cases. Plasma lysophospholipids showed a different species profile urine and their concentrations were not different between cases and controls. In conclusion, urine concentration of lysophospholipids increases in diabetic patients with DN as the likely result of their co-excretion with albumin combined with possible local production by kidney. Because LPA and LPC are known to promote renal inflammation and tubulo-interstitial fibrosis, their increased production in DN could participate to the development of kidney damage associated with diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/fisiopatología , Riñón/fisiopatología , Lisofosfatidilcolinas/orina , Lisofosfolípidos/orina , Insuficiencia Renal/complicaciones , Regulación hacia Arriba , Anciano , Albuminuria/etiología , Biomarcadores/sangre , Biomarcadores/orina , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/orina , Femenino , Tasa de Filtración Glomerular , Humanos , Lisofosfatidilcolinas/sangre , Lisofosfolípidos/sangre , Masculino , Persona de Mediana Edad , Proyectos Piloto , Eliminación Renal , Insuficiencia Renal/fisiopatología , Índice de Severidad de la EnfermedadRESUMEN
Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain.
Asunto(s)
Química Encefálica , Ácidos Grasos Omega-3/análisis , Ácidos Grasos Omega-6/análisis , Isoprostanos/análisis , Hígado/química , Animales , Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-6/sangre , Humanos , Isoprostanos/sangre , Límite de Detección , Ratones , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment.
Asunto(s)
Albuminuria/orina , Riñón/metabolismo , Lisofosfolípidos/orina , Nefritis Intersticial/orina , Proteínas del Tejido Nervioso/metabolismo , Fosfatidato Fosfatasa/metabolismo , Insuficiencia Renal Crónica/orina , Albuminuria/genética , Albuminuria/patología , Albuminuria/fisiopatología , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Fibrosis , Expresión Génica , Riñón/patología , Riñón/fisiopatología , Lisofosfolípidos/sangre , Ratones , Nefrectomía , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Nefritis Intersticial/fisiopatología , Proteínas del Tejido Nervioso/genética , Fosfatidato Fosfatasa/genética , Fosforilación , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
CONTEXT: Skeletal muscle insulin resistance is proposed to result from impaired skeletal muscle lipid oxidative capacity. However, there is no evidence indicating that muscle lipid oxidative capacity is impaired in healthy otherwise insulin-resistant individuals. OBJECTIVE: The objective of the study was to assess muscle lipid oxidative capacity in young, nonobese, glucose-tolerant, insulin-resistant vs insulin-sensitive individuals. DESIGN AND VOLUNTEERS: In 13 insulin-sensitive [by Matsuda index (MI) (22.6 ± 0.6 [SE] kg/m(2)); 23 ± 1 years; MI 5.9 ± 0.1] and 13 insulin-resistant (23.2 ± 0.6 kg/m(2); 23 ± 3 years; MI 2.2 ± 0.1) volunteers, skeletal muscle biopsy, blood extraction before and after an oral glucose load, and dual-energy x-ray absorptiometry were performed. MAIN OUTCOME MEASURES: Skeletal muscle mitochondrial to nuclear DNA ratio, oxidative phosphorylation protein content, and citrate synthase and ß-hydroxyacyl-CoA dehydrogenase activities were assessed. Muscle lipids and palmitate oxidation ((14)CO2 and (14)C-acid soluble metabolites production) at 4 [1-(14)C]palmitate concentrations (45-520 µM) were also measured. RESULTS: None of the muscle mitochondrial measures showed differences between groups, except for a higher complex V protein content in insulin-resistant vs insulin-sensitive volunteers (3.5 ± 0.4 vs 2.2 ± 0.4; P = .05). Muscle ceramide content was significantly increased in insulin-resistant vs insulin-sensitive individuals (P = .04). Total palmitate oxidation showed a similar concentration-dependent response in both groups (P = .69). However, lipid oxidative efficiency (CO2 to (14)C-acid soluble metabolites ratio) was enhanced in insulin-resistant vs insulin-sensitive individuals, particularly at the highest palmitate concentration (0.24 ± 0.04 vs 0.12 ± 0.02; P = .02). CONCLUSIONS: We found no evidence of impaired muscle mitochondrial oxidative capacity in young, nonobese, glucose-tolerant, otherwise insulin-resistant vs insulin-sensitive individuals. Enhanced muscle lipid oxidative efficiency in insulin resistance could be a potential mechanism to prevent further lipotoxicity.
Asunto(s)
Peso Corporal Ideal , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Músculo Esquelético/metabolismo , Adolescente , Adulto , Eficiencia , Femenino , Humanos , Peso Corporal Ideal/fisiología , Masculino , Mitocondrias Musculares/metabolismo , Músculo Esquelético/patología , Oxidación-Reducción , Fosforilación Oxidativa , Regulación hacia Arriba , Adulto JovenRESUMEN
Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment (AU)
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