Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts

BACKGROUND: Most cancer cells are characterized by mobile lipids visible on proton NMR ((1)H-NMR), these being comprised mainly of methyl and methylene signals from lipid acyl chains. Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids (methylene and methyl, respectively), which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals. RESULTS: The proportion of esterified cholesterol was found to be higher in K562-sensitive cells than in resistant cells, while the total cholesterol content was identical in both cell lines. Cholesterol extraction in the K562 wild type (K562wt) cell line and its MDR counterpart (K562adr), using methyl-β-cyclodextrin, was accompanied by a rise of mobile lipids in K562wt cells only. The absence of caveolae was checked by searching for the caveolin-1 protein in K562wt and K562adr cells. However, cholesterol was enriched in another membrane microdomain designated as "detergent-insoluble glycosphingomyelin complexes" or rafts. These microdomains were studied after extraction with triton X-100, a mild non-ionic detergent, revealing mobile lipid signals preserved only in the K562wt spectra. Moreover, following perturbation/disruption of these microdomains using sphingomyelinase, mobile lipids increased only in K562wt cells. CONCLUSION: These results suggest that cholesterol and sphingomyelin are involved in mobile lipid generation via microdomains of detergent-insoluble glycosphingomyelin complexes such as rafts. Increasing our knowledge of membrane microdomains in sensitive and resistant cell lines may open up new possibilities in resistance reversion

Protein catabolism and high lipid metabolism associated with long-distance exercise are revealed by plasma NMR metabolomics in endurance horses.

International audienceDuring long distance endurance races, horses undergo high physiological and metabolic stresses. The adaptation processes involve the modulation of the energetic pathways in order to meet the energy demand. The aims were to evaluate the effects of long endurance exercise on the plasma metabolomic profiles and to investigate the relationships with the individual horse performances. The metabolomic profiles of the horses were analyzed using the non-dedicated methodology, NMR spectroscopy and statistical multivariate analysis. The advantage of this method is to investigate several metabolomic pathways at the same time in a single sample. The plasmas were obtained before exercise (BE) and post exercise (PE) from 69 horses competing in three endurance races at national level (130-160 km). Biochemical assays were also performed on the samples taken at PE. The proton NMR spectra were compared using the supervised orthogonal projection on latent structure method according to several factors. Among these factors, the race location was not significant whereas the effect of the race exercise (sample BE vs PE of same horse) was highly discriminating. This result was confirmed by the projection of unpaired samples (only BE or PE sample of different horses). The metabolomic profiles proved that protein, energetic and lipid metabolisms as well as glycoproteins content are highly affected by the long endurance exercise. The BE samples from finisher horses could be discriminated according to the racing speed based on their metabolomic lipid content. The PE samples could be discriminated according to the horse ranking position at the end of the race with lactate as unique correlated metabolite. As a conclusion, the metabolomic profiles of plasmas taken before and after the race provided a better understanding of the high energy demand and protein catabolism pathway that could expose the horses to metabolic disorders

Understanding the response to endurance exercise using a systems biology approach: combining blood metabolomics, transcriptomics and miRNomics in horses

BACKGROUND: Endurance exercise in horses requires adaptive processes involving physiological, biochemical, and cognitive-behavioral responses in an attempt to regain homeostasis. We hypothesized that the identification of the relationships between blood metabolome, transcriptome, and miRNome during endurance exercise in horses could provide significant insights into the molecular response to endurance exercise. For this reason, the serum metabolome and whole-blood transcriptome and miRNome data were obtained from ten horses before and after a 160 km endurance competition.[br/] RESULTS: We obtained a global regulatory network based on 11 unique metabolites, 263 metabolic genes and 5 miRNAs whose expression was significantly altered at T1 (post- endurance competition) relative to T0 (baseline, pre-endurance competition). This network provided new insights into the cross talk between the distinct molecular pathways (e.g. energy and oxygen sensing, oxidative stress, and inflammation) that were not detectable when analyzing single metabolites or transcripts alone. Single metabolites and transcripts were carrying out multiple roles and thus sharing several biochemical pathways. Using a regulatory impact factor metric analysis, this regulatory network was further confirmed at the transcription factor and miRNA levels. In an extended cohort of 31 independent animals, multiple factor analysis confirmed the strong associations between lactate, methylene derivatives, miR-21-5p, miR-16-5p, let-7 family and genes that coded proteins involved in metabolic reactions primarily related to energy, ubiquitin proteasome and lipopolysaccharide immune responses after the endurance competition. Multiple factor analysis also identified potential biomarkers at T0 for an increased likelihood for failure to finish an endurance competition.[br/] CONCLUSIONS: To the best of our knowledge, the present study is the first to provide a comprehensive and integrated overview of the metabolome, transcriptome, and miRNome co-regulatory networks that may have a key role in regulating the metabolic and immune response to endurance exercise in horses

Publisher Correction: Unraveling the effects of the gut microbiota composition and function on horse endurance physiology

Correction de l'article DOI: 10.1038/s41598-019-46118-

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis

Background: Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints. Results: Two lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha). Conclusion: This study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans

Rôle du Cholestérol, du 24S-hydroxycholestérol et des microdomaines sur la fonction de la P-glycoprotéine (possible implication dans la maladie d'Alzheimer)

La maladie d Alzheimer (MA) est la plus fréquente des démences séniles. Des études montrent que certains mécanismes physiopathologiques de cette maladie font intervenir la cascade amyloïde à laquelle participent les peptides Ab. Ces peptides sont des substrats des transporteurs membranaires de la barrière hémato-encéphalique (BHE) tels que la P-gp. Par ailleurs, les liens relevés entre le cholestérol, ses métabolites et la MA sont encore mal connus. Des explorations par résonance magnétique nucléaire (RMN) pourraient mettre en évidence des modifications du métabolisme du cholestérol ou d autres voies dans la MA. Le premier but de ce travail est d explorer l effet du cholestérol et de son métabolite cérébral, le 24S-hydroxy-cholestérol (24S-OH) sur l activité de transport de la P-gp. En outre, les perturbations métaboliques provoquées par le 24S-OH ont été étudiées par la spectrométrie RMN, grâce à l analyse de fragments de cortex cérébral de patients. Nous avons pu comparer par RMN les contenus en lipides et en métabolites hydrosolubles dans ces échantillons provenant de patients atteints de la MA et ceux atteints de sclérose latérale amyotrophique (SLA). Notre étude suggère que la P-gp et la cavéoline-1 modulent le taux du cholestérol cellulaire et la distribution du cholestérol dans la membrane plasmique de manière énergie dépendante uniquement lorsqu elles coexistent. Par ailleurs, le 24S-OH module la fonction de la P-gp. Nos résultats suggèrent pour la première fois une inhibition de la P-gp par un métabolite du cholestérol. Enfin, la métabolomique par RMN a permis de montrer que certaines voies métaboliques sont différents dans le cortex frontal de patients souffrant de la MA et le cortex frontal de patients atteints de SLA.Alzheimer s disease (AD) is the most common dementia in elderly patients. Many studies have shown the importance of the amyloïd cascade in the pathology mechanisms of AD including the Ab peptides. These peptides are substrates of many membrane transporters such as P-glycoprotein over-expressed in the blood-brain barrier (BBB). However, the existing relations between the cholesterol, its metabolites and AD are still poorly understood. Investigations by nuclear magnetic resonance (NMR) may evidence cholesterol metabolism impairment in AD as well as involvement of other metabolic pathways. The first goal of this work was to explore the effects of cholesterol and its brain metabolite, the 24S-hydroxycholesterol (24S-OH), on the transport activity of P-gp. Finally, through the NMR analysis of cerebral cortex fragments of patients, we studied the changes of lipid content and water-soluble metabolites of samples from patients with AD and those from patients with Amyotrophic Lateral Sclerosis (ALS). Our study suggests that P-gp and caveolin-1 modulate the rate of cellular cholesterol and its distribution within the plasma membrane, with an ATP-dependent mechanism only when this two proteins coexist. Moreover, 24S-OH modulates the P-gp function. For the first time an inhibition of P-gp by a metabolite of cholesterol was suggested. At least, NMR metabolomics has shown that some metabolic pathways are altered differently in the frontal cortex of patients with AD and the frontal cortex of patients with ALS.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

La métabolomique par RMN : principes et exploration des effets des courses hippiques d’endurance sur le métabolome plasmatique.

National audienc

La spectroscopie par résonance magnétique nucléaire du proton appliquée aux liquides biologiques et la métabonomique

[Résumé en français] La métabonomique utilise principalement la SRM comme outil de détection des métabolites associé à des modèles d analyse statistique particuliers comme l analyse en composante principale et d autres méthodes d analyse multiparamétrique. Cette approche a pour but de créer des profils métaboliques en fonction de caractéristiques physiologiques, d un état pathologique, de la prise d un médicament ou d un toxique. Les études que nous présentons dans cette thèse sont issues directement d applications cliniques de la SRM proton et devraient conduire le clinicien à s intéresser à cette technologie. Nous avons pu montrer, à partir d échantillons d urine, de sérum, de liquide synovial ou de liquide d ascite, qu il était possible de comprendre ou de découvrir des métabolismes tissulaires ou des effets toxiques des médicaments. Ainsi, nous avons exploré la fonction rénale de patients de réanimation traités par des antibiotiques potentiellement néphrotoxiques - l effet des antibiotiques sur le liquide d ascite de patients cirrhotiques - l influence de l alimentation sur les métabolites plasmatiques. Nous avons aussi observé les différences de composition biochimique entre le liquide articulaire et le sérum de patients atteints de polyarthrite rhumatoïde et aussi les effets du paracétamol sur les métabolites urinaires chez des patients subissant une hépatectomie chirurgicale. Des modèles animaux ont été utilisés pour comprendre les effets de l oxygène sur les métabolites du cerveau ou les effets des solutés de remplissage sur le rein.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Transport et accumulation de médicaments à base de métaux ou métalloïdes dans des cellules cancéreuses et dans les parasites de type Leishmania

[Résumé en français] parmi les chimiothérapies anticancéreuses utilisées, les complexes métalliques dont le cisplatine ou cis diamine-dichloro platine (CDDP) se sont avérés très efficaces. Des médicaments à base de métaux et métalloïdes sont aussi utilisés pour les traitements antiparasitaires dont les traitements anti Leishmania. Cependant, malgré la variété des médicaments utilisés dans le traitement de ces affections, les cellules deviennent résistantes à ceux-ci. Afin d'apporter des informations sur les mécanismes d'action des médicaments à base de métaux et métalloïdes, le but de notre travail à été : 1)- de déterminer quels sont, dans les cellules, les sites d'accumulation des composés à base de Pt2+ ; 2)- de contribuer à la compréhension des mécanismes d'entrée du platine dans les cellules ; 3)- de comprendre l'efficacité des différents composés à base d'antimoine dans l'activité leishmanicide in vivo ou in vitro et sur l'activité cytotoxique dans les cellules surexprimant ou non la MRP1.Les résultats obtenus ont montré que le platine s'accumule dans les mitochondries et que cette accumulation dépend aussi bien de l'énergie que du potentiel de la membrane mitochondriale. Nous avons aussi montré que le platine pourrait interagir avec l'un, au moins, des transporteurs du glucose, notamment le transporteur Na+/glucose. Concernant les complexes d'antimoine, nos résultats ont montré que l'antimoine trivalent (SbIII) contenu dans les complexes d'antimoine pentavalent (SbV) est responsable de leur activité cytotoxique ou leishmanicide. D'autre part, l'efficacité des complexes antimoniés pentavalents est réduite par leur complexation avec la N-méthyl D-glucamine.PARIS13-BU Sciences (930792102) / SudocSudocFranceF