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1.
FASEB J ; 36(3): e22211, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35195922

RESUMO

Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation are not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389, and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent on muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, the skeletal muscle could be a target for managing metabolic dysfunction in cancer.


Assuntos
Carcinoma Pulmonar de Lewis/metabolismo , Glucose/metabolismo , Secreção de Insulina , Músculo Esquelético/metabolismo , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo
2.
J Cachexia Sarcopenia Muscle ; 10(6): 1241-1257, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31402604

RESUMO

BACKGROUND: Skeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor ß (TGF-ß) superfamily, including activin A, which causes atrophy. TGF-ß superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined. METHODS: To elucidate if TGF-ß superfamily ligands regulate insulin action, we used an adeno-associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2-deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB). RESULTS: Fst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase-E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin-stimulated protein synthesis, but no correlation was observed between the Fst288-driven hypertrophy and the increase in insulin-stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin-resistant diet-induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (-24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations. CONCLUSIONS: We here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase-E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post-RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF-ß superfamily ligands to improve insulin action and Fst's relevance to muscle wasting-associated insulin-resistant conditions in mice and humans.


Assuntos
Folistatina/sangue , Folistatina/genética , Atrofia Muscular/metabolismo , Obesidade/cirurgia , Adulto , Animais , Dependovirus , Feminino , Derivação Gástrica , Vetores Genéticos/farmacologia , Células HEK293 , Humanos , Subunidades beta de Inibinas/antagonistas & inibidores , Subunidades beta de Inibinas/sangue , Resistência à Insulina , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Obesidade/sangue , Parvovirinae/genética , Ratos , Transdução de Sinais
3.
Exp Physiol ; 104(5): 704-714, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30710396

RESUMO

NEW FINDINGS: What is the central question of this study? Resolving the mechanism(s) leading to glucose transporter 4 (GLUT4) translocation to the muscle surface membrane has great therapeutic potential. However, the measurement of GLUT4 translocation is technically challenging. Here, we asked whether electroporation of GLUT4-7myc-GFP into skeletal muscle could be used as a tool to study GLUT4 translocation in vivo. What is the main finding and its importance? By acutely inducing GLUT4-7myc-GFP expression in skeletal muscle, we verified that in vivo exercise and AICAR stimulation increased the GLUT4 presence in the sarcolemma measured as myc signal. Importantly, the increased myc signal in the sarcolemma was not accompanied by major visual changes in the distribution of the GFP signal. ABSTRACT: Insulin and exercise lead to translocation of the glucose transporter 4 (GLUT4) to the surface membrane of skeletal muscle fibres. This process is pivotal for facilitating glucose uptake into skeletal muscle. To study this, a robust assay is needed to measure the translocation of GLUT4 in adult skeletal muscle directly. Here, we aimed to validate a simple GLUT4 translocation assay using a genetically encoded biosensor in mouse skeletal muscle. We transfected GLUT4-7myc-GFP into mouse muscle to study live GLUT4 movement and to evaluate GLUT4 insertion in the muscle surface membrane after in vivo running exercise and pharmacological activation of AMP-activated protein kinase (AMPK). Transfection led to expression of GLUT4-7myc-GFP that was dynamic in live flexor digitorum brevis fibres and which, upon insulin stimulation, exposed the myc epitope extracellularly. Running exercise, in addition to AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide, induced ∼125 and ∼100% increase, respectively, in extracellularly exposure of GLUT4 in the surface membrane of tibialis anterior muscle. Interestingly, the clear increase in surface-exposed GLUT4 content induced by insulin, exercise or AMPK activation was not accompanied by any discernible reorganization of the GLUT4-GFP signal. In conclusion, we provide a detailed description of an easy-to-use translocation assay to study GLUT4 accumulation at the surface membrane induced by exercise and exercise-mimicking stimuli. Notably, our analyses revealed that increased GLUT4 surface membrane accumulation was not accompanied by a discernible change in the GLUT4 localization pattern.


Assuntos
Transportador de Glucose Tipo 4/metabolismo , Proteínas de Fluorescência Verde , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Eletroporação , Ativação Enzimática , Feminino , Transportador de Glucose Tipo 4/genética , Insulina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Esforço Físico/fisiologia , Transporte Proteico , Ribonucleotídeos/farmacologia , Corrida , Transfecção
4.
Mol Metab ; 17: 112-121, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30193785

RESUMO

OBJECTIVE: Chronic ad libitum low protein-high carbohydrate diet (LPHC) increases health- and life-span in mice. A periodized (p) LPHC regimen would be a more practical long-term human lifestyle intervention, but the metabolic benefits of pLPHC are not known. Also, the interactions between LPHC diet and exercise training have not been investigated. Presently, we aimed to provide proof-of-concept data in mice of the efficacy of pLPHC and to explore the potential interactions with concurrent exercise training. METHODS: A detailed phenotypic and molecular characterization of mice undergoing different durations of 14 d LPHC (5 E% protein)/14 d control diet cycles for up to 4 months with or without concurrent access to activity wheels allowing voluntary exercise training. RESULTS: pLPHC conferred metabolic benefits similar to chronic LPHC, including increased FGF21 and adaptive thermogenesis, obesity-protection despite increased total energy intake and improved insulin sensitivity. The improved insulin sensitivity showed large fluctuations between diet periods and was lost within 14 days of switching back to control diet. Parallel exercise training improved weight maintenance but impaired the FGF21 response to pLPHC whereas repeated pLPHC cycles progressively augmented this response. Both the FGF21 suppression by exercise and potentiation by repeated cycles correlated tightly with Nupr1 mRNA in liver, suggesting dependence on liver integrated stress response. CONCLUSION: These results suggest that pLPHC may be a viable strategy to promote human health but also highlight the transient nature of the benefits and that the interaction with other lifestyle-interventions such as exercise training warrants consideration.


Assuntos
Dieta Rica em Proteínas e Pobre em Carboidratos/métodos , Carboidratos da Dieta/metabolismo , Proteínas Alimentares/metabolismo , Animais , Peso Corporal , Dieta , Ingestão de Energia , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Glucose/metabolismo , Resistência à Insulina , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Estudo de Prova de Conceito
5.
Physiol Rep ; 6(13): e13798, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29998629

RESUMO

A low-protein high carbohydrate (LPHC) diet and a high-protein low carbohydrate (HPLC) diet have been reported to positively and negatively regulate whole-body glucose tolerance and insulin sensitivity, respectively. Skeletal muscle is quantitatively the most important tissue clearing glucose in the postprandial state, but it is unclear if LPHC and HPLC diets directly influence insulin action in skeletal muscle. To test this, mice were placed on control chow diet, LPHC and HPLC diets for 13.5 weeks at which time the submaximal insulin-stimulated glucose transport and insulin signaling were evaluated in ex vivo incubated oxidative soleus and glycolytic EDL muscle. At the whole-body level, the diets had the anticipated effects, with LPHC diet improving glucose tolerance and insulin-sensitivity whereas HPLC diet had the opposite effect. However, neither insulin-stimulated Akt/TBC1D4 signaling and glucose transport ex vivo, nor cell signaling in vivo were altered by the diets. These data imply that skeletal muscle insulin sensitivity does not contribute to the whole-body effects of LPHC and HPLC diets on glucose metabolism.


Assuntos
Dieta da Carga de Carboidratos , Dieta Rica em Proteínas e Pobre em Carboidratos , Insulina/metabolismo , Músculo Esquelético/metabolismo , Animais , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
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