RESUMO
Depression is a debilitating condition with a profound impact on quality of life for millions of people worldwide. Physical exercise is used as a treatment strategy for many patients, but the mechanisms that underlie its beneficial effects remain unknown. Here, we describe a mechanism by which skeletal muscle PGC-1α1 induced by exercise training changes kynurenine metabolism and protects from stress-induced depression. Activation of the PGC-1α1-PPARα/δ pathway increases skeletal muscle expression of kynurenine aminotransferases, thus enhancing the conversion of kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression and renders skeletal muscle-specific PGC-1α1 transgenic mice resistant to depression induced by chronic mild stress or direct kynurenine administration. This study opens therapeutic avenues for the treatment of depression by targeting the PGC-1α1-PPAR axis in skeletal muscle, without the need to cross the blood-brain barrier.
Assuntos
Depressão/prevenção & controle , Cinurenina/metabolismo , Músculo Esquelético/enzimologia , Estresse Psicológico/complicações , Fatores de Transcrição/metabolismo , Animais , Barreira Hematoencefálica , Depressão/metabolismo , Perfilação da Expressão Gênica , Humanos , Ácido Cinurênico , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Condicionamento Físico Animal , Condicionamento Físico Humano , Transaminases/metabolismo , Fatores de Transcrição/genéticaRESUMO
As a master regulator of metabolism, AMP-activated protein kinase (AMPK) is activated upon energy and glucose shortage but suppressed upon overnutrition. Exaggerated negative regulation of AMPK signaling by nutrient overload plays a crucial role in metabolic diseases. However, the mechanism underlying the negative regulation is poorly understood. Here, we demonstrate that high glucose represses AMPK signaling via MG53 (also called TRIM72) E3-ubiquitin-ligase-mediated AMPKα degradation and deactivation. Specifically, high-glucose-stimulated reactive oxygen species (ROS) signals AKT to phosphorylate AMPKα at S485/491, which facilitates the recruitment of MG53 and the subsequent ubiquitination and degradation of AMPKα. In addition, high glucose deactivates AMPK by ROS-dependent suppression of phosphorylation of AMPKα at T172. These findings not only delineate the mechanism underlying the impairment of AMPK signaling in overnutrition-related diseases but also highlight the significance of keeping the yin-yang balance of AMPK signaling in the maintenance of metabolic homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus/enzimologia , Glucose/farmacologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/efeitos dos fármacos , Obesidade/enzimologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus/sangue , Diabetes Mellitus/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Macaca mulatta , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Obesidade/sangue , Obesidade/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , UbiquitinaçãoAssuntos
Proteínas Quinases Ativadas por AMP , Fígado , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Camundongos , Fígado/metabolismo , Fígado/enzimologia , Músculo Esquelético/metabolismo , Músculo Esquelético/enzimologia , Metabolismo EnergéticoAssuntos
Proteínas Quinases Ativadas por AMP , Fígado , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Fígado/metabolismo , Fígado/enzimologia , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/enzimologia , Metabolismo Energético , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genéticaRESUMO
The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Autofagia , Ativação Enzimática , Exercício Físico , Glucose/metabolismo , Humanos , Masculino , Camundongos , Modelos Animais , Músculo Esquelético/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
UPF1 is an RNA helicase that orchestrates nonsense-mediated decay and other RNA surveillance pathways. While UPF1 is best known for its basal cytoprotective role in degrading aberrant RNAs, UPF1 also degrades specific, normally occurring mRNAs to regulate diverse cellular processes. Here we describe a role for UPF1 in regulated protein decay, wherein UPF1 acts as an E3 ubiquitin ligase to repress human skeletal muscle differentiation. Suppressing UPF1 accelerates myogenesis, while ectopically increasing UPF1 levels slows myogenesis. UPF1 promotes the decay of MYOD protein, a transcription factor that is a master regulator of myogenesis, while leaving MYOD mRNA stability unaffected. UPF1 acts as an E3 ligase via its RING domain to promote MYOD protein ubiquitination and degradation. Our data characterize a regulatory role for UPF1 in myogenesis, and they demonstrate that UPF1 provides a mechanistic link between the RNA and protein decay machineries in human cells.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético/enzimologia , Mioblastos Esqueléticos/enzimologia , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Masculino , Músculo Esquelético/citologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Domínios Proteicos , Proteólise , RNA Helicases , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Transativadores/química , Transativadores/genética , Transcrição Gênica , Transfecção , UbiquitinaçãoRESUMO
Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320â kDa band containing nNOS α-isoforms, while 250 and 300â kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.
Assuntos
Músculo Esquelético , Óxido Nítrico Sintase Tipo I , Animais , Masculino , Camundongos , Éxons , Isoenzimas/metabolismo , Isoenzimas/genética , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo I/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.
Assuntos
Respiração Celular , Potencial da Membrana Mitocondrial , Mitocôndrias Musculares , Animais , Camundongos , Aspartato Aminotransferase Mitocondrial/metabolismo , Aspartato Aminotransferase Mitocondrial/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Succinato Desidrogenase/metabolismo , Succinato Desidrogenase/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismoRESUMO
The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.
Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Reativadores da Colinesterase , Músculo Esquelético , Organofosfatos , Oximas , Animais , Oximas/farmacologia , Oximas/química , Ratos , Acetilcolinesterase/metabolismo , Acetilcolinesterase/sangue , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/toxicidade , Organofosfatos/toxicidade , Masculino , Reativadores da Colinesterase/farmacologia , Reativadores da Colinesterase/química , Compostos de Piridínio/farmacologia , Ratos Sprague-DawleyRESUMO
Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Culina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Proteínas de Transporte/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas Culina/genética , Complicações do Diabetes/enzimologia , Complicações do Diabetes/genética , Complicações do Diabetes/patologia , Retroalimentação Fisiológica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Atrofia Muscular/enzimologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteólise , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismoRESUMO
The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.
Assuntos
Comportamento Animal , Ataxia Cerebelar/enzimologia , Cerebelo/enzimologia , Proteínas Mitocondriais/deficiência , Músculo Esquelético/enzimologia , Ubiquinona/deficiência , Animais , Células COS , Ataxia Cerebelar/genética , Ataxia Cerebelar/fisiopatologia , Ataxia Cerebelar/psicologia , Cerebelo/fisiopatologia , Cerebelo/ultraestrutura , Chlorocebus aethiops , Modelos Animais de Doenças , Tolerância ao Exercício , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Atividade Motora , Força Muscular , Músculo Esquelético/fisiopatologia , Fenótipo , Ligação Proteica , Conformação Proteica , Proteômica/métodos , Reconhecimento Psicológico , Teste de Desempenho do Rota-Rod , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Convulsões/enzimologia , Convulsões/genética , Convulsões/fisiopatologia , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção , Ubiquinona/química , Ubiquinona/genéticaRESUMO
INTRODUCTION/AIMS: Rapid-stretch nerve injuries represent a substantial treatment challenge. No study has examined motor neuron connection after rapid-stretch injury. Our objective in this study was to characterize the electrophysiological properties of graded rapid-stretch nerve injury and assess motor neuron health using retrograde labeling and muscle adenosine triphosphatase (ATPase) histology. METHODS: Male C57BL/6 mice (n = 6 per group) were rapid-stretch injured at four levels of severity: sham injury, stretch within elastic modulus, inelastic deformation, and stretch rupture. Serial compound muscle action potential (CMAP) and motor unit number estimation (MUNE) measurements were made for 48 days, followed by retrograde labeling and muscle ATPase histology. RESULTS: Elastic injuries showed no durable abnormalities. Inelastic injury demonstrated profound initial reduction in CMAP and MUNE (P < .036) on day 2, with partial recovery by day 14 after injury (CMAP: 40% baseline, P = .003; MUNE: 55% baseline, P = .033). However, at the experimental endpoint, CMAP had recovered to baseline with only limited improvement in MUNE. Inelastic injury led to reduced retrograde-labeled neurons and grouped fiber type histology. Rupture injury had severe and nonrecovering electrophysiological impairment, dramatically reducing labeled neurons (P = .005), and atrophic or type 1 muscle fibers. There was an excellent correlation between MUNE and retrograde-labeled tibial motor neurons across injury severities (R2 = 0.96). DISCUSSION: There was no significant electrophysiological derangement in low-severity injuries but there was recoverable conduction block in inelastic injury with slow recovery, potentially due to collateral sprouting. Rupture injuries yielded permanent failure of injured axons to reinnervate. These results provide insight into the pathophysiology of clinical injuries and recovery.
Assuntos
Traumatismos dos Nervos Periféricos , Ruptura , Animais , Masculino , Camundongos , Potenciais de Ação/fisiologia , Adenosina Trifosfatases/análise , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Módulo de Elasticidade , Ruptura/fisiopatologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Neurônios Motores/patologiaRESUMO
Sarcolipin (SLN) is a small regulatory protein that inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. When bound to SERCA, SLN reduces the apparent Ca2+ affinity of SERCA and uncouples SERCA Ca2+ transport from its ATP consumption. As such, SLN plays a direct role in altering skeletal muscle relaxation and energy expenditure. Interestingly, the expression of SLN is dynamic during times of muscle adaptation, in that large increases in SLN content are found in response to development, atrophy, overload, and disease. Several groups have suggested that increases in SLN, especially in dystrophic muscle, are deleterious as it may reduce muscle function and exacerbate already abhorrent intracellular Ca2+ levels. However, there is also significant evidence to show that increased SLN content is a beneficial adaptive mechanism that protects the SERCA pump and activates Ca2+ signaling and adaptive remodeling during times of cell stress. In this review, we first discuss the role for SLN in healthy muscle during both development and overload, where SLN has been shown to activate Ca2+ signaling to promote mitochondrial biogenesis, fiber-type shifts, and muscle hypertrophy. Then, with respect to muscle disease, we summarize the discrepancies in the literature as to whether SLN upregulation is adaptive or maladaptive in nature. This review is the first to offer the concept of SLN hormesis in muscle disease, wherein both too much and too little SLN are detrimental to muscle health. Finally, the underlying mechanisms which activate SLN upregulation are discussed, specifically acknowledging a potential positive feedback loop between SLN and Ca2+ signaling molecules.
Assuntos
Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimologia , Atrofia Muscular/enzimologia , Distrofias Musculares/enzimologia , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Sinalização do Cálcio , Humanos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologiaRESUMO
[Figure: see text].
Assuntos
Heme Oxigenase-1/metabolismo , Inflamassomos/metabolismo , Isquemia/enzimologia , Lisossomos/enzimologia , Macrófagos/enzimologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Células Cultivadas , Bases de Dados Genéticas , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Membro Posterior , Humanos , Inflamassomos/genética , Mediadores da Inflamação/metabolismo , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neovascularização Fisiológica , Proteólise , Recuperação de Função Fisiológica , Fluxo Sanguíneo RegionalRESUMO
OBJECTIVE: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. CONCLUSIONS: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.
Assuntos
Heme Oxigenase-1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/enzimologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Hipóxia Celular , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Glucose/metabolismo , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/genética , Membro Posterior , Isquemia/genética , Isquemia/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Knockout , Músculo Esquelético/patologia , Necrose , Estabilidade Proteica , Fluxo Sanguíneo Regional , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) transports Ca2+ in muscle. Impaired SERCA activity may contribute to diabetic myopathy. Sirtuin (SIRT) 3 regulates muscle metabolism and function; however, it is unknown if SIRT3 regulates muscle SERCA activity or acetylation. We determined if SIRT3 overexpression enhances SERCA activity in mouse gastrocnemius muscle and if SIRT3 overexpression preserves gastrocnemius SERCA activity in a model of type 2 diabetes, induced by high fat - high sucrose (HFHS) feeding. We also determined if the acetylation status of SERCA proteins in mouse gastrocnemius is altered by SIRT3 overexpression or HFHS feeding. Wild-type (WT) and SIRT3 transgenic (SIRT3TG) mice, overexpressing SIRT3 in skeletal muscle, were fed a standard or HFHS diet for 4 months. SIRT3TG and WT mice developed obesity and glucose intolerance after 4 months of HFHS feeding. SERCA Vmax was higher in gastrocnemius of SIRT3TG mice compared with WT mice. HFHS-fed mice had lower SERCA1a protein levels and lower SERCA Vmax in their gastrocnemius than control-fed mice. The decrease in SERCA Vmax in gastrocnemius muscle due to HFHS feeding was attenuated by SIRT3 overexpression in HFHS-fed SIRT3TG mice. SERCA1a and SERCA2a acetylation in mouse gastrocnemius was not altered by genotype or diet. These findings suggest SIRT3 overexpression improves SERCA function in mouse skeletal muscle.
Assuntos
Diabetes Mellitus Tipo 2 , Músculo Esquelético/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Sirtuína 3 , Animais , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Estresse do Retículo Endoplasmático , Camundongos , Retículo Sarcoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Sacarose/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients' quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiopatologia , Quinase de Cadeia Leve de Miosina/genética , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Injeções Intramusculares , Camundongos Endogâmicos C57BL , TetaniaRESUMO
Glucose 6-P dehydrogenase (G6PD) is the first rate-limiting enzyme in pentose phosphate pathway (PPP), and it is proverbial that G6PD is absent in skeletal muscle. However, how and why G6PD is down-regulated during skeletal muscle development is unclear. In this study, we confirmed the expression of G6PD was down-regulated during myogenesis in vitro and in vivo. G6PD was absolutely silent in adult skeletal muscle. Histone H3 acetylation and DNA methylation act together on the expression of G6PD. Neither knock-down of G6PD nor over-expression of G6PD affects myogenic differentiation. Knock-down of G6PD significantly promotes the sensitivity and response of skeletal muscle cells to insulin; over-expression of G6PD significantly injures the sensitivity and response of skeletal muscle cells to insulin. High-fat diet treatment impairs insulin signaling by up-regulating G6PD, and knock-down of G6PD rescues the impaired insulin signaling and glucose uptake caused by high-fat diet treatment. Taken together, this study explored the importance of G6PD deficiency during myogenic differentiation, which provides new sight to treat insulin resistance and type-2 diabetes.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Glucosefosfato Desidrogenase , Insulina , Músculo Esquelético , Adulto , Glucose/metabolismo , Glucose 1-Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Humanos , Insulina/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismoRESUMO
Patients with pulmonary emphysema often develop locomotor muscle dysfunction, which is independently associated with disability and higher mortality in that population. Muscle dysfunction entails reduced force generation capacity, which partially depends on fibers' oxidative potential, yet very little mechanistic research has focused on muscle respiration in pulmonary emphysema. Using a recently established animal model of pulmonary emphysema-driven skeletal muscle dysfunction, we found downregulation of SDHC (succinate dehydrogenase subunit C) in association with lower oxygen consumption and fatigue tolerance in locomotor muscles. Reduced SDH activity has been previously observed in muscles from patients with pulmonary emphysema, and we found that SDHC is required to support respiration in cultured muscle cells. Moreover, in vivo gain of SDH function in emphysema animals' muscles resulted in better oxygen consumption rate and fatigue tolerance. These changes correlated with a larger number of relatively more oxidative type 2-A and 2X fibers and a reduced amount of 2B fibers. Our data suggest that SDHC is a key regulator of respiration and fatigability in pulmonary emphysema-driven skeletal muscles, which could be impactful in developing strategies aimed at attenuating this comorbidity.
Assuntos
Fadiga/enzimologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/enzimologia , Consumo de Oxigênio , Enfisema Pulmonar/enzimologia , Animais , Modelos Animais de Doenças , Fadiga/genética , Fadiga/patologia , Fadiga/fisiopatologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologiaRESUMO
UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.