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Transcription factor EB controls metabolic flexibility during exercise
Full text linkSkeletal muscle is the most abundant tissue in the whole organism representing more than 40% of the total body mass. This organ is responsible for the 30% of metabolic rate in basal condition, suggesting its great relevance not only for locomotor activity, but also for the control of whole body metabolism. Indeed skeletal muscle is a highly dynamic tissue that modulates its metabolism and mass as a consequence of different physiopathological conditions. One stimulus that triggers major adaptations is exercise, which is also well known to activate autophagy (Grumati, Coletto, Schiavinato, et al., 2011). Physical exercise elicits several beneficial effects acting on mitochondrial content/function, fatty acid oxidation and glucose uptake; however it is considered a disruptive trigger for myofiber homeostasis that needs to be counterbalanced through the activation of transcriptionally regulated pathways ready to contrast mechanical and metabolic stresses produced during contraction. The role of FoxOs transcription factors and TFEB in regulating protein breakdown and autophagy is known (Milan et al., 2015; Settembre et al., 2011). However the role of TFEB in skeletal muscle and its possible effects in controlling exercise-dependent adaptations in this tissue were not proved.
TFEB has been proposed as the key factor that coordinates autophagy to lysosomal biogenesis in cell culture, with different evidences showing the regulation of its activity. In particular it is known that an mTORC1 phosphorylation is able to prevent TFEB function by retaining it in the cytoplasm. However, there were no evidences concerning the possible phosphatases involved in TFEB activation.
Using a cellular high content screening able to monitor TFEB nuclear translocation during starvation, we identified PPP3CB, the catalytic subunit of calcineurin, as one of the highest hit for TFEB nuclear relocalization. We demonstrated that calcineurin activity is necessary and sufficient to push TFEB in the nucleus, where it can complete its function. Nevertheless, calcineurin is known to be active in skeletal muscle during contraction as a consequence of calcium oscillations. For this reason we wondered whether calcineurin activity could affect TFEB translocation also in adult skeletal muscle during exercise.
Using muscles transfected with a TFEB-GFP reporter, we demonstrated that calcineurin activity is necessary and sufficient to promote TFEB nuclear translocation even in adult skeletal muscle during coxntraction.
However, the physiological meaning of this nuclear translocation in skeletal muscle remained to be addressed.
To answer this question we used gain and loss of function approaches, by mean of viral infection of TFEB overesxpressing vectors, muscle specific TFEB knockout animals and tamoxifen inducible muscle specific TFEB transgenic animals.
From microarray analysis of muscles overexpressing and lacking TFEB, we realized that the major pathways affected by genetic manipulation are related to mitochondrial biogenesis and function, lipid utilization and glucose homeostasis. Thus we started to dissect the function of TFEB in skeletal muscle proving that its activation is required for mitochondrial biogenesis that is indeed increased in transgenic muscle. We also found an augmented mitochondrial number and size in transgenic muscle, with only a small percentage of dysfunctional mitochondria in KO animals.
These changes were paralleled by a TFEB signature in gene expression of genes involved in mitochondrial biogenesis and functionality. Moreover, these morphometric and gene expression evidences correlate with increased mitochondrial respiration and higher activity of respiratory chain complexes. For this reason transgenic muscles produce more ATP than normal mice, while KO muscles have a lower ATP synthesis because mitochondria present a leak in mitochondrial membrane that dissipate membrane potential.
Nevertheless, in order to understand if TFEB is able to promote this mitochondrial program independently from PGC1α, we checked the expression of NRF1/2, TFAM and other genes involved in mitochondrial biogenesis in a model of PGC1α ablation during TFEB overexpression. These data, and complexes activity measurements, demonstrate that TFEB is able per se to activate the transcriptional program directly binding to NRF1 and NRF2 genes promoters without the need of the transcriptional co-activator.
At this point, we challenged mice with exercise finding that transgenic mice are more resistant to exhaustive contraction than control; conversely muscle specific TFEB-KO animals display pronounced exercise intolerance due to their lack in ATP production.
In order to better explain this latter finding, thanks to metabolic measurements we realized that KO muscles rely more on glucose oxidation both in basal condition and during the first phases of exercise thus explaining the observed exercise intolerance triggered by glycogen storage depletion. Furthermore lactate quantification in serum before and after exercise suggests that KO animal depend more on anaerobic glycolysis with respect to control and transgenic counterpart. To deeply investigate the role of glucose oxidation that seems the cause of exercise intolerance, we monitored glycogen levels in muscle of KO animals in resting condition, revealing a reduction of glycogen storage. For this reason after the early stages of exercise TFEB-KO animals need to rapidly shift their metabolism to fatty acid oxidation that however cannot support energy demand because of the presence of dysfunctional mitochondria.
Altogether these findings indicate that TFEB is impinging more on metabolism rather than autophagy, that indeed is not affected by TFEB genetic modulation; more in detail TFEB seems to significantly modulate muscular glucose homeostasis that is altered in KO animals. Reduced glucose uptake and glycogen synthesis during EU clamps explains why glycogen storages are depleted in KO animals, while the transgenic counterpart present more glycogen accumulation. This phenotypic effect is paralleled by a change in glucose related genes expression, with higher levels of glucose transporters and glycogen synthesis regulator in transgenic muscles, even in the absence of PGC1α. Nevertheless TFEB overexpression is also able to drive factors such as nNOS and AMPK activity, thus modulating not only the expression but also the signalling pathways related to glucose homeostasis.
In conclusion all these findings strongly support a new vision of TFEB as master regulator of metabolic flexibility during physical exercise in a PGC1α-independent fashion
Le frodi nel comparto ittico
No full textIl comparto ittico. Fattori predisponenti le frodi ittiche. Tipologie di frodi ittiche. La pesca illegale, non registrata e non regolamentata. Frodi ittiche commerciali. Alterazione delle etichette. Trattamenti fraudolenti per modificare lo stato di freschezza e l'aspetto dei prodotti della pesca. Frodi sanitarie. Tecniche di laboratorio. Tecniche di laboratorio per l'identificazione di specie. Tecniche di laboratorio per la discriminazione del prodotto fresco -congelato. Esame delle modificazioni micro-anatomiche: esame istologico ed ematologico. Tecniche analitiche alternative
Diagnostica Analitica degli Alimenti in Igiene e tecnologie degli alimenti di origine animale
No full textRecent strandings of the giant jellyfish Rhizostoma luteum Quoy and Gaimard, 1827 (Cnidaria: Scyphozoa: Rhizostomeae) on the Atlantic and Mediterranean coasts
No full textWe present reports of sightings of living and stranded specimens of Rhizostoma luteum on the Atlantic coast of Morocco and along the south shore of the Iberian Peninsula in June–July 2012 and in January–February 2013. During summer 2012 and following the dominant currents, the jellyfish first appeared in the Gulf of Cadiz west of the Strait of Gibraltar. Subsequently, seven additional sightings were reported east of the Strait, in the Alboran Sea. In winter 2013, another event of stranded individuals of this species occurred in the Gulf of Cadiz. A phylogenetic analysis performed on the mitochondrial cytochrome c oxidase I (COI) gene sequence in specimens from both stranding events confirmed the morphological classification, ratifying that R. luteum differs from Rhizostoma octopus and Rhizostoma pulmo. This study records the presence of this species for the first time in 60 years.This work was financially supported by Spanish MIC Plan Nacional Project JELLY-PHYS (CTM2011-22586), Ministerio de Agricultura, Alimentación y Medio Ambiente (049/2010), Consejería de Ciencia, Innovación y Empresa Junta de Andalucía (Observatorio del Estrecho), E.U. MarinERA Project MedEX (CTM2008-04036-E/MAR), PERSEUS (FP7-287600). A research grant to L. P. from Ramón y Cajal Programme of the Spanish MIC and a JAE-Doc contract (#X0SC000087) to D. M. from the Spanish Council for Scientific Research (CSIC) are also acknowledged.Peer Reviewe
Two alternative multiplex PCRs for identification of the seven species of anglerfish (Lophius spp) using an end point or a melting curve analysis real-time protocol
No full textAnglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species
recettore mineralcorticoide ed organo adiposo: implicazioni cliniche e terapeutiche
No full textil recettore mineralcorticoide (MR) è stato recentemente identificato in diversi tessuti extra-renali, tra cui il tessuto adiposo, ove svolge funzioni importanti nel controllo della funzione adipocitaria. l'espressione di MR a livello del tessuto adiposo è più elevata nell'obesità; ciò suggerisce che la sua eccessiva attivazione determina un'alterazione della funzione dell'adipocita. quest'articolo discute le possibili applicazioni degli antagonisti di MR nell'obesità e nelle sue complicazioni metaboliche
Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin? A systematic review
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