RESUMO
BACKGROUND: Sestrins (SESN1-3) act as proximal sensors in leucine-induced activation of the protein kinase mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), a key regulator of cell growth and metabolism. OBJECTIVE: In the present study, the hypothesis that SESNs also mediate glucose-induced activation of mTORC1 was tested. METHODS: Rats underwent overnight fasting, and in the morning, either saline or a glucose solution (4 gâ kg-1 BW/10 mLâ kg-1) was administered by oral gavage; mTORC1 activation in the tibialis anterior muscle was assessed. To further assess the mechanism through which glucose promotes mTORC1 activation, wild-type (WT) HEK293T and HEK293T cells lacking either all 3 SESNs (SESNTKO) or hexokinase 2 (HK2KO) were deprived of glucose, followed by glucose addback, and mTORC1 activation was assessed. In addition, glucose-induced changes in the association of the SESNs with components of the GAP activity toward the Rags (GATOR2) complex and with hexokinase 2 (HK2) were assessed by co-immunoprecipitation. One- and two-way ANOVA with Tukey post hoc comparisons were used. RESULTS: Glucose administration to fasted rats promoted mTORC1 activation. Similarly, glucose readdition (GluAB) to the medium of glucose-deprived WT cells also promoted mTORC1 activation. By contrast, SESNTKO cells demonstrated attenuated mTORC1 activation following GluAB compared with WT cells. Interestingly, HK2 associated with all 3 SESNs in a glucose-dependent manner, i.e., HK2 abundance in SESN immunoprecipitates was high in cells deprived of glucose and decreased in response to GluAB. Moreover, similar to SESNTKO cells, the sensitivity of mTORC1 to GluAB was attenuated in HK2KO cells compared with WT cells. CONCLUSIONS: The results of this study demonstrate that the SESNs and HK2 play important roles in glucose-induced mTORC1 activation in HEK293T cells. However, unlike leucine-induced mTORC1 activation, the effect was independent of the changes in SESN-GATOR2 interaction, and instead, it was associated with alterations in the association of SESNs with HK2.
Assuntos
Transdução de Sinais , Serina-Treonina Quinases TOR , Ratos , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células HEK293 , Serina-Treonina Quinases TOR/metabolismo , Leucina/farmacologia , Sestrinas/metabolismo , Hexoquinase/metabolismo , Hexoquinase/farmacologia , Glucose/farmacologiaRESUMO
Leucine and insulin-like growth factor-1 (IGF-1) are important regulators of protein synthesis in skeletal muscle. The mechanistic target of rapamycin complex 1 (mTORC1) is of particular importance in their mechanism of action. In the present study, pathways through which leucine and IGF-1 converge to mediate activation of mTORC1 were examined in L6 myoblasts that were deprived of leucine and serum followed by readdition of either leucine or IGF-1. Compared with leucine- and serum-deprived myoblasts, IGF-1, but not leucine, promoted phosphorylation of protein kinase B (AKT), tuberous sclerosis complex 2 (TSC2), and the autophosphorylation site on mTOR (S2481) and also stimulated mTOR kinase activity in mTOR immunoprecipitated samples. Both leucine and IGF-1 promoted phosphorylation of mTOR on S2448. The association of mTOR with the regulatory-associated protein of mTOR (Raptor) was altered by IGF-1 treatment and trended (P = 0.065) to be altered by leucine treatment. Alterations in the association of mTOR with Raptor were proportional to changes in phosphorylation of the mTOR substrates, eIF4E-binding protein 1 (4E-BP1), and ribosomal protein S6 Kinase-ß1 (p70S6K1). Surprisingly, leucine, but not IGF-1, stimulated protein synthesis suggesting a unique role for nutrients in regulating protein synthesis. Overall, the results are consistent with a model whereby IGF-1 stimulates phosphorylation of 4E-BP1 and p70S6K1 in L6 myoblasts through an AKT-TSC2-mTORC1 signaling pathway that also involves changes in the interaction between mTOR and Raptor. In contrast, leucine signaling to mTOR results in alterations in certain mTOR phosphorylation sites and the interaction between mTOR and Raptor and stimulates protein synthesis.
Assuntos
Fator de Crescimento Insulin-Like I , Proteínas Proto-Oncogênicas c-akt , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Leucina/metabolismo , Leucina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mioblastos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O-GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, â¼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.
Assuntos
Proteínas de Transporte/metabolismo , Retinopatia Diabética/metabolismo , Proteínas do Olho/metabolismo , Mitocôndrias/metabolismo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Retina/metabolismo , Acilação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Fatores de Iniciação em Eucariotos , Proteínas do Olho/genética , Feminino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Consumo de Oxigênio/efeitos dos fármacos , Fosfoproteínas/genética , Piranos/farmacologia , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Retina/patologia , Tiazóis/farmacologiaRESUMO
Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.
RESUMO
BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Leucina/administração & dosagem , Leucina/deficiência , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Peroxidases/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidases/genética , Fatores de Transcrição/genéticaRESUMO
Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células HEK293 , Humanos , Técnicas In Vitro , RatosRESUMO
Background: Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation.Objective: We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing.Methods: Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T (Tnnt3). Tnnt3 splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition.Results: Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. Tnnt3 transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in Tnnt3 alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study.Conclusions: Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in Tnnt3 alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in Tnnt3 alternative splicing.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Dieta Hiperlipídica , Gorduras na Dieta/farmacologia , Ácidos Graxos/farmacologia , Proteínas Musculares/genética , Músculo Esquelético/efeitos dos fármacos , Precursores de RNA/metabolismo , Tecido Adiposo/metabolismo , Animais , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/farmacologia , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/metabolismo , Ratos Sprague-Dawley , Transcriptoma/efeitos dos fármacos , Troponina T/genética , Troponina T/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) is considered a major role player in the pathogenesis of diabetic retinopathy, yet the mechanisms regulating its expression are not fully understood. Our laboratory previously demonstrated that diabetes-induced VEGF expression in the retina was dependent on the repressor of mRNA translation 4E-BP1. Interaction of 4E-BP1 with the cap-binding protein eIF4E regulates protein expression by controlling the selection of mRNAs for translation. The process is regulated by the master kinase mTOR in complex 1 (mTORC1), which phosphorylates 4E-BP1, thus promoting its disassociation from eIF4E. In the present study, we investigated the role of the Akt/mTORC1 repressor REDD1 (regulated in development and DNA damage) in diabetes-induced VEGF expression. REDD1 expression was induced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in Müller cells concomitant with increased VEGF expression. In Müller cells, hyperglycemic conditions attenuated global rates of protein synthesis and cap-dependent mRNA translation concomitant with up-regulated cap-independent VEGF mRNA translation, as assessed by a bicistronic luciferase reporter assay. Hyperglycemic conditions also attenuated mTORC1 signaling and enhanced 4E-BP1 binding to eIF4E. Furthermore, ectopic expression of REDD1 in Müller cells was sufficient to promote both increased 4E-BP1 binding to eIF4E and VEGF expression. Whereas the retina of wild-type mice exhibited increased expression of VEGF and tumor necrosis factor alpha (TNF-α) 4 weeks after streptozotocin administration, the retina of REDD1 knock-out mice failed to do so. Overall, the results demonstrate that REDD1 contributes to the pathogenesis of diabetes in the retina by mediating the pathogenic effects of hyperglycemia.
Assuntos
Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Hiperglicemia/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The overload-induced increase in muscle mass is accompanied by protein accretion; however, the initiating events are poorly understood. Regulated in Development and DNA Damage 1 (REDD1), a repressor of the mechanistic target of rapamycin in complex 1 (mTORC1), blunts the elevation in protein synthesis induced by acute muscle contractions. Therefore, this study was designed to determine whether REDD1 alters the rate of the overload-induced increase in muscle mass. Wild-type (WT) and REDD1-null mice underwent unilateral functional overload (OV) of the plantaris, while the contralateral sham leg served as a control. After 3 and 5 days of OV, puromycin incorporation was used as a measurement of protein synthesis. The percent increase in plantaris wet weight and protein content was greater in REDD1-null mice after 3, 5, and 10 days OV. The overload-stimulated rate of protein synthesis in the plantaris was similar between genotypes after 3 days OV, but translational capacity was lower in REDD1-null mice, indicating elevated translational efficiency. This was likely due to elevated absolute mTORC1 signaling [phosphorylation of p70S6K1 (Thr-389) and 4E-BP1 (Ser-65)]. By 5 days of OV, the rate of protein synthesis in REDD1-null mice was lower than WT mice with no difference in absolute mTORC1 signaling. Additionally, markers of autophagy (LC3II/I ratio and p62 protein) were decreased to a greater absolute extent after 3 days OV in REDD1-null mice. These data suggest that loss of REDD1 augments the rate of the OV-induced increase in muscle mass by altering multiple protein balance pathways.
Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão/fisiologia , Fatores de Transcrição/genéticaRESUMO
UNLABELLED: Defective autophagy is implicated in the pathogenesis of nonalcoholic fatty liver diseases (NAFLD) through poorly defined mechanisms. Cardiolipin is a mitochondrial phospholipid required for bioenergetics and mitophagy from yeast to mammals. Here we investigated a role for ALCAT1 in the development of NAFLD. ALCAT1 is a lysocardiolipin acyltransferase that catalyzes pathological cardiolipin remodeling in several aging-related diseases. We show that the onset of diet-induced NAFLD caused autophagic arrest in hepatocytes, leading to oxidative stress, mitochondrial dysfunction, and insulin resistance. In contrast, targeted deletion of ALCAT1 in mice prevented the onset of NAFLD. ALCAT1 deficiency also restored mitophagy, mitochondrial architecture, mitochondrial DNA (mtDNA) fidelity, and oxidative phosphorylation. In support of a causative role of the enzyme in a mitochondrial etiology of the disease, hepatic ALCAT1 expression was significantly up-regulated in mouse models of NAFLD. CONCLUSION: Forced expression of ALCAT1 in primary hepatocytes led to multiple defects that are highly reminiscent of NAFLD, including steatosis, defective autophagy, and mitochondrial dysfunction, linking pathological cardiolipin remodeling by ALCAT1 to the pathogenesis of NAFLD.
Assuntos
Aciltransferases/metabolismo , Mitofagia , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Autofagia , Fibrose , Hepatócitos/fisiologia , Lipogênese , Fígado/patologia , Masculino , Camundongos Knockout , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse OxidativoRESUMO
The mammalian target of rapamycin (mTOR) plays an important role in controlling islet ß-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic ß-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet ß-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines ß-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet ß-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.
Assuntos
Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Tamanho Celular , Células Secretoras de Insulina/metabolismo , Luciferases , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Estatísticas não ParamétricasRESUMO
In a previous study (Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, and Jefferson LS. Am J Physiol Endocrinol Metab 304: E229-236, 2013.), we observed a rapid (i.e., 1-3 days) immobilization-induced repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling in hindlimb skeletal muscle of young (2-mo-old) rats that was associated with elevated expression of regulated in development and DNA-damage response (REDD) 1 and REDD2. The present study extends that observation to include an assessment of those parameters in soleus muscle of the immobilized hindlimb of various-aged rats as well as in response to remobilization. Male Sprague-Dawley rats aged 2, 9, and 18 mo were subjected to unilateral hindlimb immobilization for 7 days, whereas one group of the 9-mo-old animals underwent 7 days of remobilization. Soleus muscle mass-to-body mass ratio declined with age, with the loss of muscle mass following hindlimb immobilization being inversely proportional to age. Compared with 2-mo-old rats, the older rats exhibited reduced mTORC1 signaling in the nonimmobilized limb in association with elevated REDD2, but not REDD1, mRNA expression. In the 2-mo-old rats, 7 days of hindlimb immobilization attenuated mTORC1 signaling and induced REDD2, but not REDD1, mRNA expression. In contrast, hindlimb immobilization did not further attenuate the age-related reduction in mTORC1 signaling nor further enhance the age-related induction of REDD2 mRNA expression in 9- and 18-mo-old rats. Across ages, REDD1 mRNA was not impacted by immobilization. Finally, remobilization elevated mTORC1 signaling and lowered REDD2 mRNA expression, with no impact on REDD1 gene expression. In conclusion, changes in mTORC1 signaling associated with aging, immobilization, and remobilization were inversely proportional to alterations in REDD2 mRNA expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Imobilização/fisiologia , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Nucleares/genética , Fosforilação , RNA Mensageiro/química , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Análise de Regressão , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: In skeletal muscle, the nutrient-induced stimulation of protein synthesis requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Expression of the repressor of mTORC1 signaling, regulated in development and DNA damage 1 (REDD1), is elevated in muscle during various atrophic conditions and diminished under hypertrophic conditions. The question arises as to what extent REDD1 limits the nutrient-induced stimulation of protein synthesis. OBJECTIVE: The objective was to examine the role of REDD1 in limiting the response of muscle protein synthesis and mTORC1 signaling to a nutrient stimulus. METHODS: Wild type REDD1 gene (REDD1(+/+)) and disruption in the REDD1 gene (REDD1(-/-)) mice were feed deprived for 16 h and randomized to remain feed deprived or refed for 15 or 60 min. The tibialis anterior was then removed for analysis of protein synthesis and mTORC1 signaling. RESULTS: In feed-deprived mice, protein synthesis and mTORC1 signaling were significantly lower in REDD1(+/+) than in REDD1(-/-) mice. Thirty minutes after the start of refeeding, protein synthesis in REDD1(+/+) mice was stimulated by 28%, reaching a value similar to that observed in feed-deprived REDD1(-/-) mice, and was accompanied by increased phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) by 81%, 167%, and 207%, respectively. In refed REDD1(-/-) mice, phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) were significantly augmented above the values observed in refed REDD1(+/+) mice by 258%, 405%, and 401%, respectively, although protein synthesis was not coordinately increased. Seventy-five minutes after refeeding, REDD1 expression in REDD1(+/+) mice was reduced (â¼15% of feed-deprived REDD1(+/+) values), and protein synthesis and mTORC1 signaling were not different between refed REDD1(+/+) mice and REDD1(-/-) mice. CONCLUSIONS: The results show that REDD1 expression limits protein synthesis in mouse skeletal muscle by inhibiting mTORC1 signaling during periods of feed deprivation and that a reduction in its expression is necessary for maximal stimulation of protein synthesis in response to refeeding.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Micronutrientes/administração & dosagem , Complexos Multiproteicos/genética , Proteínas Musculares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The chronic activation of the mechanistic (mammalian) target of rapamycin in complex 1 (mTORC1) in response to excess nutrients contributes to obesity-associated pathologies. OBJECTIVE: To understand the initial events that ultimately lead to obesity-associated pathologies, the present study assessed mTORC1 responses in the liver after a relatively short exposure to a high-fat diet (HFD). METHODS: Male, obesity-prone rats were meal-trained to consume either a control (CON; 10% of energy from fat) diet or an HFD (60% of energy from fat) for 2 wk. Livers were collected and analyzed for mTORC1 signaling [assessed by changes in phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1)] and potential regulatory mechanisms, including changes in the association of Ras-related GTP binding (Rag) A and RagC with mechanistic target of rapamycin (mTOR) and expression of Sestrin1, Sestrin2, and Sestrin3. RESULTS: Feeding-induced activation of mTORC1 was blunted in the livers of rats fed the HFD compared with those fed the CON diet (p70S6K1 phosphorylation, 19% of CON; 4E-BP1 phosphorylation, 61% of CON). The attenuated response was not due to a change in a kinase also referred to as protein kinase B (Akt) signaling but rather to resistance to amino acid-induced activation of mTORC1, as evidenced by a reduction in the interaction of RagA (69% of CON) and RagC (66% of CON) with mTOR and enhanced expression of the mTORC1 repressors Sestrin2 (132% of CON) and Sestrin3 (143% of CON). The consumption of an HFD led to impaired amino acid-induced activation of mTORC1 as assessed in livers perfused in situ with medium containing various concentrations of amino acids. CONCLUSIONS: These results in rats support a model in which the initial response of the liver to an HFD is an attenuation of, rather than the expected activation of, mTORC1. The initial response likely represents a counterregulatory mechanism to handle the onset of excess nutrients and is caused by enhanced expression of Sestrin2 and Sestrin3, which, in turn, leads to impaired Rag signaling, resulting in resistance to amino acid-induced activation of mTORC1.
Assuntos
Aminoácidos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Glicemia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Imunoprecipitação , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Obesidade/tratamento farmacológico , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genéticaRESUMO
In this study, the interaction of mTORC1 with its downstream targets p70S6K1 and 4E-BP1 was evaluated in both mouse liver and mouse embryonic fibroblasts following combined disruption of the genes encoding 4E-BP1 and 4E-BP2. Phosphorylation of p70S6K1 was dramatically elevated in the livers of mice lacking 4E-BP1 and 4E-BP2 following feeding-induced activation of mTORC1. Immunoprecipitation of mTORC1 suggested that elevated phosphorylation was the result of enhanced interaction of p70S6K1 with raptor. These findings were extended to a cell culture system wherein loss of 4E-BP1 and 4E-BP2 resulted in elevated interaction of p70S6K1 with IGF1-induced activation of mTORC1 in conjunction with an enhanced rate of p70S6K1 phosphorylation at Thr-389. Furthermore, cotransfecting HA-p70S6K1 with 4E-BP1, but not 4E-BP1(F114A), reduced recovery of mTORC1 in HA-p70S6K1 immunoprecipitates. Together, these findings support the conclusion that, in the absence of 4E-BP proteins, mTORC1-mediated phosphorylation of p70S6K1 is elevated by a reduction in competition between the two substrates for interaction with raptor.
Assuntos
Proteínas de Transporte/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Fibroblastos/metabolismo , Imunoprecipitação , Fígado/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Mutação , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Proteína Regulatória Associada a mTOR , Transdução de Sinais , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
The present project was designed to investigate phosphorylation of p70S6K1 in an animal model of skeletal muscle overload. Within 24 h of male Sprague-Dawley rats undergoing unilateral tenotomy to induce functional overloading of the plantaris muscle, phosphorylation of the Thr³89 and Thr4²¹/Ser4²4 sites on p70S6K1 was significantly elevated. Since the Thr4²¹/Ser4²4 sites are purportedly mammalian target of rapamycin complex 1 (mTORC1) independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF-I treatment of serum-deprived HEK-293E cells as an in vitro model system, because IGF-I promotes phosphorylation of p70S6K1 on both the Thr³89 and Thr4²¹/Ser4²4 sites in skeletal muscle and in cells in culture. We found that, whereas the mTOR inhibitor TORIN2 prevented the IGF-I-induced phosphorylation of the Thr4²¹/Ser4²4 sites, it surprisingly enhanced phosphorylation of these sites during serum deprivation. JNK inhibition with SP600125 attenuated phosphorylation of the Thr4²¹/Ser4²4 sites, and in combination with TORIN2 both the effect of IGF-I and the enhanced Thr4²¹/Ser4²4 phosphorylation during serum deprivation were ablated. In contrast, both JNK activation with anisomycin and knockdown of the mTORC2 subunit rictor specifically stimulated phosphorylation of the Thr4²¹/Ser4²4 sites, suggesting that mTORC2 represses JNK-mediated phosphorylation of these sites. The role of JNK in mediating p70S6K1 phosphorylation was confirmed in the animal model noted above, where rats treated with SP600125 exhibited attenuated Thr4²¹/Ser4²4 phosphorylation. Overall, the results provide evidence that the mTORC1 and JNK signaling pathways coordinate the site-specific phosphorylation of p70S6K1. They also identify a novel role for mTORC1 and mTORC2 in the inhibition of JNK.
Assuntos
Transtornos Traumáticos Cumulativos/metabolismo , Modelos Animais de Doenças , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Transtornos Traumáticos Cumulativos/fisiopatologia , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Fosforilação/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína Companheira de mTOR Insensível à Rapamicina , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/química , Serina/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Treonina/metabolismoRESUMO
Regulated in DNA damage and development 1 (REDD1) is a repressor of mTOR complex 1 (mTORC1) signaling. In humans, REDD1 mRNA expression in skeletal muscle is repressed following resistance exercise in association with activation of mTORC1. However, whether REDD1 protein expression is also reduced after exercise and if so to what extent the loss contributes to exercise-induced activation of mTORC1 is unknown. Thus, the purpose of the present study was to examine the role of REDD1 in governing the response of mTORC1 and protein synthesis to a single bout of muscle contractions. Eccentric contractions of the tibialis anterior were elicited via electrical stimulation of the sciatic nerve in male mice in either the fasted or fed state or in fasted wild-type or REDD1-null mice. Four hours postcontractions, mTORC1 signaling and protein synthesis were elevated in fasted mice in association with repressed REDD1 expression relative to nonstimulated controls. Feeding coupled with contractions further elevated mTORC1 signaling, whereas REDD1 protein expression was repressed compared with either feeding or contractions alone. Basal mTORC1 signaling and protein synthesis were elevated in REDD1-null compared with wild-type mice. The magnitude of the increase in mTORC1 signaling was similar in both wild-type and REDD1-null mice, but, unlike wild-type mice, muscle contractions did not stimulate protein synthesis in mice deficient for REDD1, presumably because basal rates were already elevated. Overall, the data demonstrate that REDD1 expression contributes to the modulation of mTORC1 signaling following feeding- and contraction-induced activation of the pathway.
Assuntos
Regulação para Baixo , Complexos Multiproteicos/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Animais , Estimulação Elétrica , Ativação Enzimática , Regulação da Expressão Gênica , Membro Posterior , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/biossíntese , Músculo Esquelético/enzimologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fatores de Transcrição/genéticaRESUMO
The protein kinase Mechanistic Target of Rapamycin (mTOR) in Complex 1 (mTORC1) is regulated in part by the Ras-related GTP-binding proteins (Rag GTPases). Rag GTPases form a heterodimeric complex consisting of either RagA or RagB associated with either RagC or RagD and act to localize mTORC1 to the lysosomal membrane. Until recently, RagA and RagB were thought to be functionally redundant, as were RagC and RagD. However, recent research suggests that the various isoforms differentially activate mTORC1. Here, the mRNA expression and protein abundance of the Rag GTPases was compared across male rat skeletal muscle, heart, liver, kidney, and brain. Whereas mRNA expression of RagA was higher than RagB in nearly all tissues studied, RagB protein abundance was higher than RagA in all tissues besides skeletal muscle. RagC mRNA expression was more abundant or equal to RagD mRNA, and RagD protein was more abundant than RagC protein in all tissues. Moreover, the proportion of RagB in the short isoform was greater than the long in liver, whereas the opposite was true in brain. These results serve to further elucidate Rag GTPase expression and offer potential explanations for the differential responses to amino acids that are observed in different tissues.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Transdução de Sinais , Masculino , Ratos , Animais , Transdução de Sinais/fisiologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Aminoácidos/metabolismo , RNA Mensageiro/genéticaRESUMO
Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Animais , Meios de Cultura Livres de Soro , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fator de Iniciação Eucariótico 4G/metabolismo , Jejum/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fator de Crescimento Insulin-Like I/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr(229) and Thr(389) in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr(229) by PDK1 is a prerequisite for phosphorylation of Thr(389) by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation.