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• W2565398910 abstract "•TFEB regulates mitochondrial biogenesis and function in muscle•Glucose homeostasis in skeletal muscle requires TFEB•The effects of TFEB on muscle metabolism are independent from PGC1α•TFEB coordinates metabolic flexibility during physical exercise The transcription factor EB (TFEB) is an essential component of lysosomal biogenesis and autophagy for the adaptive response to food deprivation. To address the physiological function of TFEB in skeletal muscle, we have used muscle-specific gain- and loss-of-function approaches. Here, we show that TFEB controls metabolic flexibility in muscle during exercise and that this action is independent of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Indeed, TFEB translocates into the myonuclei during physical activity and regulates glucose uptake and glycogen content by controlling expression of glucose transporters, glycolytic enzymes, and pathways related to glucose homeostasis. In addition, TFEB induces the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. This coordinated action optimizes mitochondrial substrate utilization, thus enhancing ATP production and exercise capacity. These findings identify TFEB as a critical mediator of the beneficial effects of exercise on metabolism. The transcription factor EB (TFEB) is an essential component of lysosomal biogenesis and autophagy for the adaptive response to food deprivation. To address the physiological function of TFEB in skeletal muscle, we have used muscle-specific gain- and loss-of-function approaches. Here, we show that TFEB controls metabolic flexibility in muscle during exercise and that this action is independent of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Indeed, TFEB translocates into the myonuclei during physical activity and regulates glucose uptake and glycogen content by controlling expression of glucose transporters, glycolytic enzymes, and pathways related to glucose homeostasis. In addition, TFEB induces the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. This coordinated action optimizes mitochondrial substrate utilization, thus enhancing ATP production and exercise capacity. These findings identify TFEB as a critical mediator of the beneficial effects of exercise on metabolism. Exercise elicits several beneficial effects by acting on mitochondrial content/function, fatty acid oxidation, and glucose homeostasis (Hawley, 2002Hawley J.A. Adaptations of skeletal muscle to prolonged, intense endurance training.Clin. Exp. Pharmacol. Physiol. 2002; 29: 218-222Crossref PubMed Scopus (204) Google Scholar, Holloszy and Coyle, 1984Holloszy J.O. Coyle E.F. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences.J. Appl. Physiol. 1984; 56: 831-838Crossref PubMed Scopus (1423) Google Scholar, Holloszy et al., 1998Holloszy J.O. Kohrt W.M. Hansen P.A. The regulation of carbohydrate and fat metabolism during and after exercise.Front. Biosci. 1998; 3: D1011-D1027Crossref PubMed Google Scholar). Indeed, muscle activity is important to counteract disease progression in diabetes, obesity, and metabolic syndrome. The signaling pathways that control the contraction-mediated beneficial effects on mitochondria and glucose/lipid homeostasis are distinct from insulin signaling and mainly rely on AMPK and PGC1α. We have recently found that exercise leads to nuclear translocation of the helix-loop-helix leucine zipper transcription factor EB (TFEB) (Medina et al., 2015Medina D.L. Di Paola S. Peluso I. Armani A. De Stefani D. Venditti R. Montefusco S. Scotto-Rosato A. Prezioso C. Forrester A. et al.Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB.Nat. Cell Biol. 2015; 17: 288-299Crossref PubMed Scopus (776) Google Scholar), an important regulator of lysosomal biogenesis and autophagy (Sardiello et al., 2009Sardiello M. Palmieri M. di Ronza A. Medina D.L. Valenza M. Gennarino V.A. Di Malta C. Donaudy F. Embrione V. Polishchuk R.S. et al.A gene network regulating lysosomal biogenesis and function.Science. 2009; 325: 473-477Crossref PubMed Scopus (1568) Google Scholar, Settembre et al., 2011Settembre C. Di Malta C. Polito V.A. Garcia Arencibia M. Vetrini F. Erdin S. Erdin S.U. Huynh T. Medina D. Colella P. et al.TFEB links autophagy to lysosomal biogenesis.Science. 2011; 332: 1429-1433Crossref PubMed Scopus (2009) Google Scholar). Upregulation of TFEB has been found in several tissues after food deprivation, including liver and skeletal muscle. We have previously shown that in liver, TFEB regulates genes involved in lipid catabolism, fatty acid oxidation, and ketogenesis (Settembre et al., 2013Settembre C. De Cegli R. Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh T. Carissimo A. Palmer D. Klisch T.J. et al.TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.Nat. Cell Biol. 2013; 15: 647-658Crossref PubMed Scopus (630) Google Scholar). Some of these effects are elicited by TFEB-mediated induction of PGC1α (Settembre et al., 2013Settembre C. De Cegli R. Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh T. Carissimo A. Palmer D. Klisch T.J. et al.TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.Nat. Cell Biol. 2013; 15: 647-658Crossref PubMed Scopus (630) Google Scholar), a transcriptional coactivator, which interacts with and enhances the activity of transcription factors involved in mitochondrial biogenesis, glucose homeostasis, and lipid oxidation (Kelly and Scarpulla, 2004Kelly D.P. Scarpulla R.C. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function.Genes Dev. 2004; 18: 357-368Crossref PubMed Scopus (977) Google Scholar, Puigserver et al., 1998Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3070) Google Scholar). In the presence of nutrients, TFEB is sequestered in the cytoplasm by mTORC1-mediated phosphorylation, whereas in nutrient-depleted conditions, mTORC1 is inactive and dephosphorylated TFEB translocates to the nucleus, where it induces the transcription of target genes (Martina et al., 2012Martina J.A. Chen Y. Gucek M. Puertollano R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB.Autophagy. 2012; 8: 903-914Crossref PubMed Scopus (785) Google Scholar, Roczniak-Ferguson et al., 2012Roczniak-Ferguson A. Petit C.S. Froehlich F. Qian S. Ky J. Angarola B. Walther T.C. Ferguson S.M. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis.Sci. Signal. 2012; 5: ra42Crossref PubMed Scopus (839) Google Scholar, Settembre et al., 2012Settembre C. Zoncu R. Medina D.L. Vetrini F. Erdin S. Erdin S. Huynh T. Ferron M. Karsenty G. Vellard M.C. et al.A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB.EMBO J. 2012; 31: 1095-1108Crossref PubMed Scopus (1250) Google Scholar). The dephosphorylation of TFEB is mediated by the calcium-dependent phosphatase, calcineurin, which is necessary for TFEB activation (Medina et al., 2015Medina D.L. Di Paola S. Peluso I. Armani A. De Stefani D. Venditti R. Montefusco S. Scotto-Rosato A. Prezioso C. Forrester A. et al.Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB.Nat. Cell Biol. 2015; 17: 288-299Crossref PubMed Scopus (776) Google Scholar). Importantly, exercise-dependent calcium influx activates calcineurin, which dephosphorylates TFEB, leading to nuclear localization. The calcineurin-mediated induction of TFEB is independent from mTORC1 activity, indicating that calcium-dependent signaling is a rate-limiting step of TFEB activation (Medina et al., 2015Medina D.L. Di Paola S. Peluso I. Armani A. De Stefani D. Venditti R. Montefusco S. Scotto-Rosato A. Prezioso C. Forrester A. et al.Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB.Nat. Cell Biol. 2015; 17: 288-299Crossref PubMed Scopus (776) Google Scholar). Previous studies implicated calcineurin in a variety of physiological processes, in particular in skeletal muscle adaptation to exercise (Gehlert et al., 2015Gehlert S. Bloch W. Suhr F. Ca2+-dependent regulations and signaling in skeletal muscle: from electro-mechanical coupling to adaptation.Int. J. Mol. Sci. 2015; 16: 1066-1095Crossref PubMed Scopus (108) Google Scholar). Muscle-specific transgenic mice that overexpress an activated form of calcineurin show increased glucose uptake, glycogen accumulation, and lipid oxidation (Long et al., 2007Long Y.C. Glund S. Garcia-Roves P.M. Zierath J.R. Calcineurin regulates skeletal muscle metabolism via coordinated changes in gene expression.J. Biol. Chem. 2007; 282: 1607-1614Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Interestingly, calcineurin promotes the nuclear translocation of another family of transcription factors, NFAT, which, depending on the type of physical activity, modulate the expression of the different myosin isoforms (Calabria et al., 2009Calabria E. Ciciliot S. Moretti I. Garcia M. Picard A. Dyar K.A. Pallafacchina G. Tothova J. Schiaffino S. Murgia M. NFAT isoforms control activity-dependent muscle fiber type specification.Proc. Natl. Acad. Sci. USA. 2009; 106: 13335-13340Crossref PubMed Scopus (113) Google Scholar, Long et al., 2007Long Y.C. Glund S. Garcia-Roves P.M. Zierath J.R. Calcineurin regulates skeletal muscle metabolism via coordinated changes in gene expression.J. Biol. Chem. 2007; 282: 1607-1614Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, McCullagh et al., 2004McCullagh K.J. Calabria E. Pallafacchina G. Ciciliot S. Serrano A.L. Argentini C. Kalhovde J.M. Lømo T. Schiaffino S. NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching.Proc. Natl. Acad. Sci. USA. 2004; 101: 10590-10595Crossref PubMed Scopus (159) Google Scholar). Here we show that the calcineurin-TFEB axis plays a major role in the metabolic adaptations that occur during physical exercise. By using gain- and loss-of-function approaches, we show that TFEB regulates mitochondrial biogenesis and glucose uptake independently of PGC1α. Indeed, TFEB controls genes involved in glucose metabolism such as GLUT1 and GLUT4, hexokinase I and II, TBC1 domain family member 1 (TBC1D1), and glycogen synthase (GYS), leading to glycogen accumulation to sustain energy production during exercise. We previously demonstrated that TFEB promotes lipid catabolism in the liver and protects against diet-induced weight gain and insulin resistance (Settembre et al., 2013Settembre C. De Cegli R. Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh T. Carissimo A. Palmer D. Klisch T.J. et al.TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.Nat. Cell Biol. 2013; 15: 647-658Crossref PubMed Scopus (630) Google Scholar). Here we studied the physiological relevance of TFEB in skeletal muscle, an important insulin- and autophagy-dependent tissue (Grumati and Bonaldo, 2012Grumati P. Bonaldo P. Autophagy in skeletal muscle homeostasis and in muscular dystrophies.Cells. 2012; 1: 325-345Crossref PubMed Scopus (59) Google Scholar, Mammucari et al., 2007Mammucari C. Milan G. Romanello V. Masiero E. Rudolf R. Del Piccolo P. Burden S.J. Di Lisi R. Sandri C. Zhao J. et al.FoxO3 controls autophagy in skeletal muscle in vivo.Cell Metab. 2007; 6: 458-471Abstract Full Text Full Text PDF PubMed Scopus (1440) Google Scholar, Masiero et al., 2009Masiero E. Agatea L. Mammucari C. Blaauw B. Loro E. Komatsu M. Metzger D. Reggiani C. Schiaffino S. Sandri M. Autophagy is required to maintain muscle mass.Cell Metab. 2009; 10: 507-515Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar). Transcriptome analysis was performed by whole-genome gene expression profiling experiments (SuperSeries-GSE62980) in skeletal muscle from both TFEB-overexpressing and TFEB knockout (KO) mice. Overexpression of Tcfeb, the murine homolog of human TFEB, in muscle was achieved by means of intramuscular viral-mediated gene transfer using the adeno-associated virus (AAV) system. Adult mice were injected intramuscularly with either AAV2.1-CMV-TFEB or AAV2.1-CMV-GFP control vector and animals were sacrificed after 21 days, a time that allows efficient TFEB expression (Figure S1A, available online). Muscle-specific conditional TFEB KO mice were generated by crossing Tcfeb floxed (Settembre et al., 2013Settembre C. De Cegli R. Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh T. Carissimo A. Palmer D. Klisch T.J. et al.TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.Nat. Cell Biol. 2013; 15: 647-658Crossref PubMed Scopus (630) Google Scholar) with MLC1f-Cre transgenic mice (Bothe et al., 2000Bothe G.W. Haspel J.A. Smith C.L. Wiener H.H. Burden S.J. Selective expression of Cre recombinase in skeletal muscle fibers.Genesis. 2000; 26: 165-166Crossref PubMed Scopus (114) Google Scholar). Efficiency and specificity of the gene deletion were confirmed by quantitative real-time PCR analysis on multiple tissues (Figure S1B). Overexpression of TFEB in muscle resulted in the upregulation of 1,514 genes and the downregulation of 1,109 genes (GSE62975), while genetic ablation of TFEB increased 496 genes and suppressed 458 genes (GSE62976). The up- or downregulated genes are highlighted in red and green, respectively, in Tables S1 and S2. To identify the main cellular compartments (CCs) and the principal biological process (BPs) for which the TFEB-dependent genes were enriched, we performed a gene ontology enrichment analysis (GOEA). The GOEA was performed on the lists of genes whose expression was either increased or decreased in transfected muscle or in the TFEB KO mice. Interestingly, several gene categories related to cellular metabolism, including lipid and glucose homeostasis, were found upregulated in TFEB-overexpressing muscle and downregulated in TFEB KO (Figure 1A; Table S3). Strikingly, genes involved in mitochondrial biogenesis were oppositely regulated by gain- and loss-of-function approaches. Indeed, 38 genes involved in mitochondrial function were induced in AAV2.1-TFEB-infected muscles (Table S4), while 73 genes were inhibited in TFEB KO muscles (Table S5). To better identify the network of genes regulated by TFEB in muscle, we performed sequence analysis to identify putative TFEB target sites, previously referred to as CLEAR sites (coordinated lysosomal expression and regulation) (Palmieri et al., 2011Palmieri M. Impey S. Kang H. di Ronza A. Pelz C. Sardiello M. Ballabio A. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways.Hum. Mol. Genet. 2011; 20: 3852-3866Crossref PubMed Scopus (582) Google Scholar), in the promoter regions of the downregulated genes in TFEB KO mice. Interestingly, we found that 79% of these genes contain a CLEAR sequence and are, therefore, potential direct targets of TFEB (Table S6). To examine potential effects of TFEB in mitochondrial function, we analyzed mitochondrial morphology in muscles overexpressing or lacking TFEB. Electron microscopy (EM) analyses showed a striking increase of mitochondrial density and volume in TFEB-overexpressing muscles (Figures 1B, 1C, 1E, and 1F). Interestingly, mitochondrial density and size were normal in the TFEB KO muscles (Figures 1E and 1F). Consistent with the EM data, increase of mitochondrial DNA (mtDNA) was found in TFEB transgenic muscles, while no differences were observed in TFEB KO muscles (Figure 1G). However, while the cristae shape, matrix density, and outer membrane morphology were normal in TFEB-overexpressing muscles (Figure 1C), abnormalities were found in approximately 10% of the mitochondria from TFEB KO muscles (Figures 1D and 1H). An increase in the number of mitochondria was also observed in C2C12 muscle cells transfected with TFEB-GFP, as detected by immunofluorescence confocal and confirmed by EM analyses (Figures S2A and S2B). Quantitative real-time PCR also revealed an increase of mtDNA content in TFEB-overexpressing cells (Figure S2C). Importantly, quantitative real-time PCR analysis revealed that TFEB overexpression in muscle and in C2C12 cells induces the expression of many genes involved in mitochondrial biogenesis and function, including the master gene of mitochondrial biogenesis, PGC1α, a known direct target of TFEB (Settembre et al., 2013Settembre C. De Cegli R. Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh T. Carissimo A. Palmer D. Klisch T.J. et al.TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.Nat. Cell Biol. 2013; 15: 647-658Crossref PubMed Scopus (630) Google Scholar) (Figures 2A and S2D). Moreover, another PGC-1 family member, PGC1β, was also upregulated by TFEB overexpression. Consistently, we found a significant induction of peroxisome proliferator-activated receptor α (PPARα), PPARβ/δ, and PPARγ in TFEB-overexpressing muscles. However, TFEB deletion did not affect the expression of PGC1α/β and PPAR genes, with the exception of PPARα, which was downregulated. In order to elucidate the possible mechanisms underlying the induction of mitochondrial biogenesis observed in TFEB-transfected muscles, we examined the expression of nuclear respiratory factors 1 and 2 (NRF1and NRF2). The mRNA levels of NRF2 were increased, as well as NRF downstream genes, including mitochondrial transcription factor A (TFAM). Chromatin immunoprecipitation (ChIP) experiments confirmed the direct recruitment of TFEB on NRF1 and NRF2 promoters (Figure 2B), but not on TFAM promoter (data not shown). Finally, overexpression of TFEB in skeletal muscle increased the expression of mitochondrial enzymes. Subunits of the four respiratory chain complexes and the ATP synthase, as well as genes encoding electron transport and tricarboxylic acid cycle proteins, were induced by TFEB overexpression and were reduced by TFEB deletion (Figure 2A). Importantly, immunoblotting analyses confirmed the increase of complex I (NDUFA9), complex II (SDHA), and complex IV (COX5a) proteins in TFEB-transfected muscles (Figures 2C and 2D). TFEB deletion did not alter NDUFA9 and COX5a expression but significantly reduced the level of SDHA protein (Figures 2C and 2D). To better characterize the involvement of TFEB in mitochondrial respiration, we analyzed the specific activities of enzymes involved in oxidative phosphorylation. Biochemical analysis of muscle samples infected with AAV2.1-TFEB compared to wild-type (WT) muscles showed increase of citrate synthase, mitochondrial respiratory chain complex I (CI), CII, CIII, and CIV activities (Figure 2E). Consistent with the western blot analyses, TFEB deletion led to decrease of CII activity, the complex that contains the SDHA flavoprotein, while the other respiratory complexes had normal activities (Figure 2E). The changes in respiratory chain activities were corroborated by histochemical analyses for COX and SDH activity in AAV2.1-TFEB-transfected and TFEB KO muscles. Indeed, only SDH activity was greatly reduced in the absence of TFEB, while both COX and SDH were increased in TFEB-overexpressing muscles (Figure 2F). To further investigate the role of TFEB in mitochondrial function, we generated an inducible muscle-specific transgenic mouse line. Acute activation of TFEB by tamoxifen treatment in adult mice recapitulated the phenotype of AAV2.1-TFEB overexpression on mitochondria biogenesis (Figure S3). Consistent with the increase of SDH and respiratory chain complex activity, mitochondrial respiration was significantly enhanced by TFEB expression in adult transgenic muscles (Figures S3A and S3B). We next assessed whether these TFEB-mediated changes of mitochondrial morphology and function had any impact on energy production. ATP levels were higher in TFEB-transfected muscles and lower in TFEB-deficient muscles compared to controls (Figure 2G). To understand the mechanisms underlying the significant decrease of ATP in TFEB KO muscles, we checked mitochondrial function in these animals. Fluorescent dyes, like tetramethylrhodamine methyl ester (TMRM), monitor the mitochondrial membrane potential (Δψm), the critical parameter that drives ATP production. Therefore, we checked the status of Δψm in isolated adult fibers of WT and TFEB KO muscles. As expected, in control mice oligomycin-dependent inhibition of ATP synthase did not alter Δψm (Figure 2H), and mitochondrial depolarization was achieved after membrane permeabilization by the protonophore carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP). Conversely, mitochondria of TFEB null fibers underwent a significant depolarization after oligomycin treatment (Figure 2G), suggesting that these fibers were at least in part relying on reverse activity of ATP synthase to preserve their membrane potential as a consequence of proton membrane leak. In addition, oxidative stress, revealed by protein carbonylation, was significantly higher in TFEB KO mice than controls (Figures 2I and S3C). Both PGC1α and PGC1β are master regulators of mitochondrial biogenesis and oxidative metabolism. However, recent findings suggest the presence of an independent pathway that regulates mitochondrial biogenesis during exercise (Rowe et al., 2012Rowe G.C. El-Khoury R. Patten I.S. Rustin P. Arany Z. PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle.PLoS ONE. 2012; 7: e41817Crossref PubMed Scopus (95) Google Scholar). Therefore, we examined whether TFEB is the missing sensor of physical activity that coordinates the metabolic responses independently of PGC1α. First, we checked expression and localization of endogenous TFEB in PGC1α KO mice before and after exercise. TFEB was expressed at lower levels and more cytosolic in PGC1α KO mice when compared to controls (Figures 3A and 3B ). Importantly, exercise restored a normal TFEB expression, induced TFEB nuclear translocation, and triggered upregulation of genes related to mitochondrial biogenesis (Figures 3C and S4A). EM analysis of overexpressed TFEB in PGC1α KO mice revealed increased mitochondrial volume and density (Figure S4B). TFEB overexpression in PGC1α KO muscle also resulted in increased COX and SDH activity along with increased activities of CI, II, III, and IV (Figures S4C and S4D). The levels of PGC1α targets such as TFAM, NRF1, and NRF2 were also significantly upregulated by TFEB overexpression, even in the absence of PGC1α (Figure S5A). Importantly, systemic delivery of TFEB in muscle-specific PGC1α KO mice improved their exercise tolerance (Figure 3D). Indeed, TFEB expression was able to restore normal fatigue index when expressed in PGC1α KO muscle (Figure 3E). Altogether, these findings suggest that the induction of mitochondrial biogenesis in TFEB-overexpressing muscles does not depend on the presence of PGC1α. To determine whether PGC1β may compensate for the lack of PGC1α, we measured the effect of TFEB overexpression on mitochondrial biogenesis in cells that were silenced for PGC1α and PGC1β. Importantly, inhibition of both PGC1 factors did not prevent or reduce TFEB-mediated induction of genes related to mitochondrial biogenesis and mitochondrial respiratory chain activity (Figure S5B). Physical activity has a major impact on glucose homeostasis and mitochondrial biogenesis and function. Therefore, we checked whether acute exhausting versus mild and chronic exercise regiments are equally able to activate TFEB. As a readout of TFEB activation, we monitored its nuclear localization. While acute exhausting contraction led to nuclear translocation of TFEB (Figure 4A), mild exercise did not (Figure 4B). However, 7 weeks of training with progressive increase of intensity without reaching exhaustion induced a massive TFEB nuclear translocation with concomitant cytosolic depletion (Figure 4B). Therefore, intensity and duration of training are critical factors that affect TFEB nuclear translocation and transcriptional regulation of genes related to mitochondrial biogenesis and function (Figure 4C). To determine the physiological consequences of TFEB translocation during physical activity, we examined exercise performance in both TFEB KO and inducible muscle-specific TFEB transgenic mice. High-intensity exercise revealed significant training intolerance of TFEB KO mice compared to controls (Figure 5A). Conversely, acute muscle-specific activation of TFEB enhanced physical performance (Figure 5A). To better understand the exercise intolerance of the TFEB KO mice, we examined energy expenditure during treadmill running. While WT mice maintained constant levels of energy expenditure during physical activity, the TFEB KO mice displayed a drop after 15 min of physical exercise (Figure 5B). Metabolic analyses revealed that in basal condition, TFEB KO mice have a higher respiratory exchange rate (RER) than controls (Figure 5C). These data suggest that TFEB KO mice depend on glucose oxidation more than controls. In addition, while WT mice maintained a relatively constant RER during running period, TFEB KO mice showed a drop in RER after 20 min (Figure 5C). This decrease indicates a shift in substrate usage from glucose to fat metabolism. Finally, we measured glucose and fatty acid levels in muscle and blood from TFEB KO and TFEB transgenic mice before and after exercise. TFEB KO mice showed lower blood glucose levels compared to WT mice in basal condition (Figure 5D). Exercise caused a 50% reduction of blood glucose in TFEB KO, TFEB transgenic, and control mice (Figure 5D). Insulin levels mirrored the changes of blood glucose, as they were reduced in basal condition in TFEB KO mice and dropped after exercise in the different genotypes (Figure 5E). TFEB KO mice are hypoglycemic, contain dysfunctional mitochondria, and produce less ATP. Thus, we reasoned that they use anaerobic glycolysis to produce energy. Consistent with this hypothesis, we found higher levels of lactate in the blood of TFEB KO mice before and after exercise compared to controls (Figure 5F). Conversely, lactate of transgenic mice was already lower than controls in resting condition and did not increase after exercise (Figure 5F). Therefore, TFEB transgenic mice better utilize glucose for energy production. To further confirm this finding, we measured glycogen levels in muscle. Glycogen levels were remarkably lower in TFEB KO mice and higher in TFEB transgenic in basal condition compared to WT. Enzymatic quantification showed that glycogen content was 3-fold less after TFEB ablation (Figure 5G), while it was 10-fold higher after TFEB overexpression compared to controls (Figure 5G). This was confirmed by periodic acid-Schiff (PAS) staining (Figure 5H). Exercise led to glycogen consumption in both TFEB KO muscles and controls (Figure 5G). The lower glycogen content detected in sedentary TFEB KO muscles explains the decrease of RER observed after a 15 min exercise (Figure 5C). Since glycogen is rapidly depleted in TFEB KO mice, the additional need for energy during exhausting exercise requires a switch from glycolysis to fatty acid oxidation. Consistently, while blood free fatty acid concentrations were reduced after exercise in both TFEB KO mice and controls (Figure 5I) and blood levels of non-esterified fatty acids (NEFAs) did not differ between genotypes (Figure 5I), their muscle content dramatically decreased after exercise only in TFEB KO mice (Figure 5J). Importantly, ketones did not differ between TFEB KO and controls (Figure 5K). These findings confirm a change in metabolic flexibility in the absence of TFEB that forced muscle cells to use lipids for ATP production. The exhaustion of the lipid fuel in KO mice results in inability to maintain the same exercise intensity of controls. We and others have found that autophagy is important for mitochondrial quality control and is activated by exercise to clear dysfunctional mitochondria (Lo Verso et al., 2014Lo Verso F. Carnio S. Vainshtein A. Sandri M. Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity.Autophagy. 2014; 10: 1883-1894Crossref PubMed Scopus (100) Google Scholar). Thus, we checked whether TFEB controls autophagy in adult skeletal muscles. Surprisingly, TFEB activation was not sufficient to enhance autophagy flux and TFEB deletion did not impair autophagy flux in the presence or absence of nutrients (Figures S6A and S6B). Moreover, activation of TFEB did not induce protein breakdown and muscle loss. In fact, most of the atrophy-related genes belonging to the ubiquitin proteasome and autophagy-lysosome systems were not induced by TFEB expression (Figure S6C). Similarly, mitophagy genes were not upregulated. Since protein degradation is not affected by TFEB activation, we checked whether genes related to protein synthesis were modulated by TFEB. However, when we checked a cross-sectional area, we found a shift toward smaller size in transgenic mice, suggesting that protein synthesis was not induced. This decrease in fiber size is due to a metabolic shift because oxidative fibers are smaller than glycolytic skeletal muscle fibers (Figure S7A). Furthermore, we did not find any significant difference in myosin distribution between transgenic and control muscles (Figure S7B). Therefore, TFEB controls myofiber metabolism, but not myosin content/type, independently of autophagy or proteostasis. Because muscles from TFEB KO mice contain lower glycogen levels than controls, we reasoned that they may have abnormal regulation" @default.
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• W2565398910 title "Transcription Factor EB Controls Metabolic Flexibility during Exercise" @default.
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