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Diamond-Blackfan anemia (DBA) features hypoplastic anemia and congenital malformations, largely caused by mutations in various ribosomal proteins. The aim of this study was to characterize the spectrum of genetic lesions causing DBA and identify genotypes that correlate with phenotypes of clinical significance. Seventy-four patients with DBA from across Canada were included. Nucleotide-level mutations or large deletions were identified in 10 ribosomal genes in 45 cases. The RPS19 mutation group was associated with higher requirement for chronic treatment for anemia than other DBA groups. Patients with RPS19 mutations, however, were more likely to maintain long-term corticosteroid response without requirement for further chronic transfusions. Conversely, patients with RPL11 mutations were less likely to need chronic treatment. Birth defects, including cardiac, skeletal, hand, cleft lip or palate and genitourinary malformations, also varied among the various genetic groups. Patients with RPS19 mutations had the fewest number of defects, while patients with RPL5 had the greatest number of birth defects. This is the first study to show differences between DBA genetic groups with regards to treatment. Previously unreported differences in the rate and types of birth defects were also identified. These data allow better patient counseling, a more personalized monitoring plan, and may also suggest differential functions of DBA genes on ribosome and extra-ribosomal functions.
Assuntos
Anemia de Diamond-Blackfan/genética , Proteínas Ribossômicas/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/epidemiologia , Anemia de Diamond-Blackfan/patologia , Canadá , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Adulto JovemRESUMO
AIMS/HYPOTHESIS: Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue. METHODS: Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control. RESULTS: Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr(183)/Tyr(185)) of JNK, but no differences were observed in Ser(307) phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr(1361)) and phosphorylation of Akt at (Ser(473) and Thr(308)) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species. CONCLUSIONS/INTERPRETATION: Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.
Assuntos
Resistência à Insulina/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Músculo Esquelético/metabolismo , Animais , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.
Assuntos
Jejum/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Glicogênio/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Músculo Esquelético/metabolismo , Adenilato Quinase/metabolismo , Adulto , Estudos Cross-Over , Glucose/metabolismo , Técnica Clamp de Glucose , Humanos , Insulina/metabolismo , Masculino , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.
Assuntos
Resistência à Insulina/fisiologia , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Musculares , Músculo Esquelético/metabolismo , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Primers do DNA/genética , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4 , Humanos , Técnicas In Vitro , Insulina/farmacologia , Resistência à Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacosRESUMO
The AMP-activated protein kinase (AMPK) is a metabolite sensing serine/threonine kinase that has been termed the master regulator of cellular energy metabolism due to its numerous roles in the regulation of glucose, lipid, and protein metabolism. In this review, we first summarize the current literature on a number of important aspects of AMPK in skeletal muscle. These include the following: (1) the structural components of the three AMPK subunits (i.e. AMPKalpha, beta, and gamma), and their differential localization in response to stimulation in muscle; (2) the biochemical regulation of AMPK by AMP, protein phosphatases, and its three known upstream kinases, LKB1, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and transforming growth factor-beta-activated kinase 1 (TAK1); (3) the pharmacological agents that are currently available for the activation and inhibition of AMPK; (4) the physiological stimuli that activate AMPK in muscle; and (5) the metabolic processes that AMPK regulates in skeletal muscle.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético/fisiologia , Modelos Biológicos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Componentes do Gene , Glucose/metabolismo , Metabolismo dos Lipídeos , MAP Quinase Quinase Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
INTRODUCTION: New strategies for weight loss and weight maintenance in humans are needed. Human brown adipose tissue (BAT) can stimulate energy expenditure and may be a potential therapeutic target for obesity and type 2 diabetes. However, whether exercise training is an efficient stimulus to activate and recruit BAT remains to be explored. This study aimed to evaluate whether regular exercise training affects cold-stimulated BAT metabolism and, if so, whether this was associated with changes in plasma metabolites. METHODS: Healthy sedentary men (n = 11; aged 31 [SD 7] years; body mass index 23 [0.9] kg m-2; VO2 max 39 [7.6] mL min-1 kg-1) participated in a 6-week exercise training intervention. Fasting BAT and neck muscle glucose uptake (GU) were measured using quantitative [18F]fluorodeoxyglucose positron emission tomography-magnetic resonance imaging three times: (1) before training at room temperature and (2) before and (3) after the training period during cold stimulation. Cervico-thoracic BAT mass was measured using MRI signal fat fraction maps. Plasma metabolites were analysed using nuclear magnetic resonance spectroscopy. RESULTS: Cold exposure increased supraclavicular BAT GU by threefold (p < 0.001), energy expenditure by 59% (p < 0.001) and plasma fatty acids (p < 0.01). Exercise training had no effect on cold-induced GU in BAT or neck muscles. Training increased aerobic capacity (p = 0.01) and decreased visceral fat (p = 0.02) and cervico-thoracic BAT mass (p = 0.003). Additionally, training decreased very low-density lipoprotein particle size (p = 0.04), triglycerides within chylomicrons (p = 0.04) and small high-density lipoprotein (p = 0.04). CONCLUSIONS: Although exercise training plays an important role for metabolic health, its beneficial effects on whole body metabolism through physiological adaptations seem to be independent of BAT activation in young, sedentary men.
RESUMO
To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we investigated the effects of glucocorticoids on in vivo tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate IRS-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cortisone treatment of rats resulted in both hyperglycemia and hyperinsulinemia. Anesthetized animals were injected with 10 U/kg insulin via cardiac puncture and, after 2 min, hindlimb muscles were removed, snap-frozen, and homogenized in SDS. Protein tyrosine phosphorylation was studied by immunoblotting with phosphotyrosine antibody. Insulin receptors and substrate IRS-1 were identified and quantified with specific antibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine phosphorylation (69 +/- 4% of control, P < 0.05). The decreased level of receptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6 +/- 5% of control, P < 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content by 50%, but did not significantly alter the total level of IRS-1 tyrosine phosphorylation. The apparent M(r) of IRS-1 was reduced by approximately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that the lower M(r) likely results from lower phosphoserine and/or phosphothreonine content. To investigate the role of hyperinsulinemia in the glucocorticoid response, rats were made insulin-deficient with streptozotocin (100 mg/kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoid-treated skeletal muscle is characterized by: (a) decreased total tyrosine phosphorylation of insulin receptors as a result of a reduction in the pool of receptors undergoing tyrosine phosphorylation; (b) decreased IRS-1 content and reduced serine and/or threonine phosphorylation of IRS-1. Glucocorticoid-induced hyperinsulinemia appears to be essential for the development of these alterations.
Assuntos
Cortisona/farmacologia , Insulina/farmacologia , Músculos/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Immunoblotting , Proteínas Substratos do Receptor de Insulina , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/imunologia , Fosfoproteínas/isolamento & purificação , Fosforilação , Fosfotirosina , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/isolamento & purificação , Estreptozocina/farmacologia , Tirosina/metabolismoRESUMO
Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Exercício Físico/fisiologia , Músculo Esquelético/enzimologia , Adulto , Ativação Enzimática , Feminino , Humanos , Masculino , Proteína Quinase 1 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Músculo Esquelético/fisiologia , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf , Proteínas Quinases S6 Ribossômicas , Transdução de SinaisRESUMO
To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action.
Assuntos
Músculo Esquelético/metabolismo , Obesidade/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor de Insulina/metabolismo , Adulto , Transporte Biológico/efeitos dos fármacos , Biópsia , Glucose/metabolismo , Humanos , Técnicas In Vitro , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Obesidade/patologia , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotirosina , Valores de Referência , Tirosina/análogos & derivadosRESUMO
Physical exercise promotes glucose uptake into skeletal muscle and makes the working muscles more sensitive to insulin. To understand the role of insulin receptor (IR) signaling in these responses, we studied the effects of exercise and insulin on skeletal muscle glucose metabolism and insulin signaling in mice lacking insulin receptors specifically in muscle. Muscle-specific insulin receptor knockout (MIRKO) mice had normal resting 2-deoxy-glucose (2DG) uptake in soleus muscles but had no significant response to insulin. Despite this, MIRKO mice displayed normal exercise-stimulated 2DG uptake and a normal synergistic activation of muscle 2DG uptake with the combination of exercise plus insulin. Glycogen content and glycogen synthase activity in resting muscle were normal in MIRKO mice, and exercise, but not insulin, increased glycogen synthase activity. Insulin, exercise, and the combination of exercise plus insulin did not increase IR tyrosine phosphorylation or phosphatidylinositol 3-kinase activity in MIRKO muscle. In contrast, insulin alone produced a small activation of Akt and glycogen synthase kinase-3 in MIRKO mice, and prior exercise markedly enhanced this insulin effect. In conclusion, normal expression of muscle insulin receptors is not needed for the exercise-mediated increase in glucose uptake and glycogen synthase activity in vivo. The synergistic activation of glucose transport with exercise plus insulin is retained in MIRKO mice, suggesting a phenomenon mediated by nonmuscle cells or by downstream signaling events.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Condicionamento Físico Animal , Proteínas Serina-Treonina Quinases , Receptor de Insulina/metabolismo , Transdução de Sinais , Animais , Transporte Biológico , Desoxiglucose/farmacocinética , Teste de Tolerância a Glucose , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Masculino , Camundongos , Camundongos Knockout , Contração Muscular , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Serina/metabolismo , Fatores de Tempo , Tirosina/metabolismoRESUMO
Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformin's beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/farmacologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Inibidores de Proteínas Quinases , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1RESUMO
The p90 ribosomal S6 kinase (RSK), a cytosolic substrate for the extracellular signal-regulated kinase (ERK), is involved in transcriptional regulation, and one isoform (RSK2) has been implicated in the activation of glycogen synthase by insulin. To determine RSK2 function in vivo, mice lacking a functional rsk2 gene were generated and studied in response to insulin and exercise, two potent stimulators of the ERK cascade in skeletal muscle. RSK2 knockout (KO) mice weigh 10% less and are 14% shorter than wild-type (WT) mice. They also have impaired learning and coordination. Hindlimb skeletal muscles were obtained from mice 10, 15, or 30 min after insulin injection or immediately after strenuous treadmill exercise for 60 min. While insulin and exercise significantly increased ERK phosphorylation in skeletal muscle from both WT and KO mice, the increases were twofold greater in the KO animals. This occurred despite 27% lower ERK2 protein expression in skeletal muscle of KO mice. KO mice had 18% less muscle glycogen in the fasted basal state, and insulin increased glycogen synthase activity more in KO than WT mice. The enhanced insulin-stimulated increases in ERK and glycogen synthase activities in KO mice were not associated with higher insulin receptor or with IRS1 tyrosine phosphorylation or with IRS1 binding to phosphatidylinositol 3-kinase. However, insulin-stimulated serine phosphorylation of Akt was significantly higher in the KO animals. c-fos mRNA was increased similarly in muscle from WT and KO mice in response to insulin (2. 5-fold) and exercise (15-fold). In conclusion, RSK2 likely plays a major role in feedback inhibition of the ERK pathway in skeletal muscle. Furthermore, RSK2 is not required for activation of muscle glycogen synthase by insulin but may indirectly modulate muscle glycogen synthase activity and/or glycogen content by other mechanisms, possibly through regulation of Akt. RSK2 knockout mice may be a good animal model for the study of Coffin-Lowry syndrome.
Assuntos
Deleção de Genes , Glicogênio/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Peso Corporal/genética , Cognição/fisiologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Retroalimentação , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Glicogênio Sintase/metabolismo , Insulina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Fosforilação/efeitos dos fármacos , Condicionamento Físico Animal/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas/deficiência , Proteínas Quinases S6 Ribossômicas/genéticaRESUMO
BACKGROUND: Recent reports suggest that activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), in response to acute changes in cellular energy status in cardiac and skeletal muscles, results in altered substrate utilization. We hypothesized that chronic alterations in myocardial energetics in hypertrophied hearts (left ventricular hypertrophy, LVH) will lead to elevated AMPK activity, which in turn regulates substrate utilization. METHODS AND RESULTS: Using (31)P NMR spectroscopy and biochemical assays, we found that in LVH hearts, adenosine triphosphate (ATP) concentration decreased by 10%, phosphocreatine concentration decreased by 30%, and total creatine concentration was unchanged. Thus, the ratio of phosphocreatine/creatine decreased to one third of controls, and the ratio of AMP/ATP increased to 5 times above controls. These changes were associated with increased alpha(1) and alpha(2) AMPK activity (3.5- and 4.8-fold above controls, respectively). The increase in AMPK alpha(1) activity was accompanied by a 2-fold increase in alpha(1) expression, whereas alpha(2) expression was decreased by 30% in LVH. The basal rate of 2-deoxyglucose uptake increased by 3-fold in LVH, which was associated with an increased amount of glucose transporters present on the plasma membrane. CONCLUSIONS: These results demonstrate for the first time that chronic changes in myocardial energetics in hypertrophied hearts are accompanied by significant elevations in AMPK activity and isoform-specific alterations in AMPK expression. It also raises the possibility that AMPK signaling plays an important role in regulating substrate utilization in hypertrophied hearts.
Assuntos
Hipertrofia Ventricular Esquerda/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Musculares , Miocárdio/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Acil-CoA Desidrogenase , Animais , Transporte Biológico , Carnitina O-Palmitoiltransferase/biossíntese , Carnitina O-Palmitoiltransferase/genética , Ativação Enzimática , Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Hipertrofia Ventricular Esquerda/genética , Técnicas In Vitro , Masculino , Modelos Cardiovasculares , Proteínas de Transporte de Monossacarídeos/metabolismo , Oxirredução , Fosfatos/metabolismo , Pressão , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Endurance exercise training can result in increased rates of insulin-stimulated glucose uptake in skeletal muscle; however, this effect may be lost rapidly once training ceases. To examine a mechanism for these changes, the skeletal-muscle glucose transport system of female rats exercise-trained in wheelcages for 6 wk were studied against a group of untrained female rats. The trained rats were studied immediately following and 2 and 5 days after removal from wheelcages; both trained and untrained rats were studied 30 min after insulin (90 nmol/rat, intraperitoneal) or saline injection. The total number of skeletal-muscle plasma-membrane glucose transporters (R0), total muscle-homogenate and plasma-membrane GLUT4 protein, and rates of plasma-membrane vesicle D-facilitated glucose transport were higher in the exercise-trained rats immediately after exercise training and did not decrease significantly during the 5 days after cessation of training. On the other hand, exercise training did not alter microsomal-membrane total glucose-transporter number or GLUT4 protein, nor did training alter GLUT1 protein in total muscle homogenates nor either membrane fraction. The carrier-turnover number, an estimate of average functional activity of glucose transporters in the plasma membrane, was elevated slightly, but not significantly, in the trained muscle. In both the trained and untrained muscle, insulin administration resulted in translocation of glucose transporters from the microsomal-membrane fraction to the plasma membrane and an increase in the carrier-turnover number.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/fisiologia , Proteínas Musculares , Músculos/química , Músculos/citologia , Condicionamento Físico Animal , 4-Nitrofenilfosfatase/análise , Animais , Glicemia/análise , Western Blotting , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Membrana Celular/química , Membrana Celular/fisiologia , Citrato (si)-Sintase/análise , Citocalasina B/metabolismo , Ingestão de Alimentos/fisiologia , Feminino , Glucose/farmacocinética , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Glicogênio/sangue , Insulina/sangue , Insulina/farmacologia , Insulina/fisiologia , Isomerismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Músculos/fisiologia , Ratos , Ratos Endogâmicos , Frações Subcelulares/químicaRESUMO
Insulin, contraction, and the nitric oxide (NO) donor, sodium nitroprusside (SNP), all increase glucose transport in skeletal muscle. Some reports suggest that NO is a critical mediator of insulin- and/or contraction-stimulated transport. To determine if the mechanism leading to NO-stimulated glucose uptake is similar to the insulin- or contraction-dependent signaling pathways, isolated soleus and extensor digitorum longus (EDL) muscles from rats were treated with various combinations of SNP (maximum 10 mmol/l), insulin (maximum 50 mU/ml), electrical stimulation to produce contractions (maximum 10 min), wortmannin (100 nmol/l), and/or the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) (0.1 mmol/l). The combinations of SNP plus insulin and SNP plus contraction both had fully additive effects on 2-deoxyglucose uptake. Wortmannin completely inhibited insulin-stimulated glucose transport and only slightly inhibited SNP-stimulated 2-deoxyglucose uptake, whereas L-NMMA did not inhibit contraction-stimulated 2-deoxyglucose uptake. SNP significantly increased the activity of the alpha1 catalytic subunit of 5'AMP-activated protein kinase (AMPK), a signaling molecule that has been implicated in mediating glucose transport in fuel-depleted cells. Addition of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (1 mg/ml) to the drinking water of rats for 2 days failed to affect the increase in muscle 2-deoxyglucose uptake in response to treadmill exercise. These data suggest that NO stimulates glucose uptake through a mechanism that is distinct from both the insulin and contraction signaling pathways.
Assuntos
Glucose/metabolismo , Insulina/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Óxido Nítrico/fisiologia , Animais , Desoxiglucose/farmacocinética , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Atividade Motora/fisiologia , Músculo Esquelético/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Ratos , Ratos Sprague-Dawley , ômega-N-Metilarginina/farmacologiaRESUMO
5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent glucose transport, which enables fuel-depleted muscle cells to take up glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of glucose transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of glucose transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both glucose transport and AMPK activity but did not change fuel status. Insulin stimulated glucose transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent glucose transport in skeletal muscle under conditions of metabolic stress.
Assuntos
Monofosfato de Adenosina/farmacologia , Glucose/metabolismo , Proteínas Quinases/metabolismo , Estresse Fisiológico/metabolismo , 2,4-Dinitrofenol/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Creatina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Cinética , Masculino , Contração Muscular , Músculo Esquelético/metabolismo , Concentração Osmolar , Fosfocreatina/metabolismo , Ratos , Ratos Sprague-Dawley , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 pmol/l)-euglycemic clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t1/2] = 11 vs. 34 min) and glycogen synthase activity (t1/2 = 8 vs. 17 min), and the magnitude of increase was two- to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase (IRTK) activity (t1/2 = 50 min), serine phosphorylation of Akt (t1/2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t1/2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t1/2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase activity, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulin-signaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (IRTK as well as insulin receptor substrate 1 and phosphatidylinositol 3-kinase [Wojtaszewski et al., Diabetes 46:1775-1781, 1997]); and 3) prior exercise increases insulin stimulation of both glucose uptake and glycogen synthase activity in the absence of an upregulation of signaling events in human skeletal muscle.
Assuntos
Exercício Físico/fisiologia , Resistência à Insulina/fisiologia , Insulina/fisiologia , Músculo Esquelético/fisiologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Adulto , Sequência de Aminoácidos/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/metabolismo , Técnica Clamp de Glucose , Glicogênio/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Perna (Membro) , Masculino , Músculo Esquelético/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
The intracellular signaling proteins that lead to exercise-stimulated glucose transport in skeletal muscle have not been identified, although it is clear that there are separate signaling mechanisms for exercise- and insulin-stimulated glucose transport. We have hypothesized that the 5'AMP-activated protein kinase (AMPK) functions as a signaling intermediary in exercise-stimulated glucose uptake. This hypothesis was based on recent studies showing the following: 1) muscle contraction increases AMPK activity and 2) perfusion of rat hindlimb skeletal muscles with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that results in increased AMPK activity, increased insulin-stimulated glucose uptake. In the current study, isolated rat epitrochlearis muscles were treated to contract in vitro (via electrical stimulation for 10 min) and/or incubated in the absence or presence of AICAR (2 mmol/l), insulin (1 micromol/l), or wortmannin (100 nmol/l). Both contraction and AICAR significantly increased AMPK activity, while the enzyme was not activated by insulin. AICAR, contraction, and insulin all increased 3-O-methylglucose (3MG) transport by threefold to fivefold above basal. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin completely blocked insulin-stimulated transport, but did not inhibit AICAR- or contraction-stimulated transport. The increase in glucose transport with the combination of maximal AICAR plus maximal insulin treatments was partially additive, suggesting that these stimuli increase glucose transport by different mechanisms. In contrast, there was no additive effect on glucose transport with the combination of AICAR plus contraction. These data suggest that AICAR and contraction stimulate glucose transport by a similar insulin-independent signaling mechanism and are consistent with the hypothesis that AMPK is involved in exercise-stimulated glucose uptake.
Assuntos
Glucose/metabolismo , Complexos Multienzimáticos/fisiologia , Contração Muscular/fisiologia , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Androstadienos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Sinergismo Farmacológico , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Antagonistas da Insulina/farmacologia , Masculino , Músculo Esquelético/enzimologia , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , WortmaninaRESUMO
It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Ativação Enzimática/fisiologia , Frutosefosfatos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Glucose-6-Fosfato/metabolismo , Membro Posterior , Insulina/farmacologia , Membranas Intracelulares/metabolismo , Masculino , Complexos Multienzimáticos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Proteínas Serina-Treonina Quinases , Ratos , Ratos Sprague-Dawley , Ribonucleosídeos/farmacologiaRESUMO
Insulin-stimulated GLUT4 translocation is impaired in people with type 2 diabetes. In contrast, exercise results in a normal increase in GLUT4 translocation and glucose uptake in these patients. Several groups have recently hypothesized that exercise increases glucose uptake via an insulin-independent mechanism mediated by the activation of AMP-activated protein kinase (AMPK). If this hypothesis is correct, people with type 2 diabetes should have normal AMPK activation in response to exercise. Seven subjects with type 2 diabetes and eight matched control subjects exercised on a cycle ergometer for 45 min at 70% of maximum workload. Biopsies of vastus lateralis muscle were taken before exercise, after 20 and 45 min of exercise, and at 30 min postexercise. Blood glucose concentrations decreased from 7.6 to 4.77 mmol/l with 45 min of exercise in the diabetic group and did not change in the control group. Exercise significantly increased AMPK alpha2 activity 2.7-fold over basal at 20 min in both groups and remained elevated throughout the protocol, but there was no effect of exercise on AMPK alpha1 activity. Subjects with type 2 diabetes had similar protein expression of AMPK alpha1, alpha2, and beta1 in muscle compared with control subjects. AMPK alpha2 was shown to represent approximately two-thirds of the total alpha mRNA in the muscle from both groups. In conclusion, people with type 2 diabetes have normal exercise-induced AMPK alpha2 activity and normal expression of the alpha1, alpha2 and beta1 isoforms. Pharmacological activation of AMPK may be an attractive target for the treatment of type 2 diabetes.