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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.
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BACKGROUND: phosphorylation of AS160 and TBC1D1 plays an important role for GLUT4 mobilization to the cell surface. The phosphorylation of AS160 and TBC1D1 in humans in response to acute exercise is not fully characterized. OBJECTIVE: to study AS160 and TBC1D1 phosphorylation in human skeletal muscle after aerobic exercise followed by a hyperinsulinemic euglycemic clamp. DESIGN: eight healthy men were studied on two occasions: 1) in the resting state and 2) in the hours after a 1-h bout of ergometer cycling. A hyperinsulinemic euglycemic clamp was initiated 240 min after exercise and in a time-matched nonexercised control condition. We obtained muscle biopsies 30 min after exercise and in a time-matched nonexercised control condition (t = 30) and after 30 min of insulin stimulation (t = 270) and investigated site-specific phosphorylation of AS160 and TBC1D1. RESULTS: phosphorylation on AS160 and TBC1D1 was increased 30 min after the exercise bout, whereas phosphorylation of the putative upstream kinases, Akt and AMPK, was unchanged compared with resting control condition. Exercise augmented insulin-stimulated phosphorylation on AS160 at Ser(341) and Ser(704) 270 min after exercise. No additional exercise effects were observed on insulin-stimulated phosphorylation of Thr(642) and Ser(588) on AS160 or Ser(237) and Thr(596) on TBC1D1. CONCLUSIONS: AS160 and TBC1D1 phosphorylations were evident 30 min after exercise without simultaneously increased Akt and AMPK phosphorylation. Unlike TBC1D1, insulin-stimulated site-specific AS160 phosphorylation is modified by prior exercise, but these sites do not include Thr(642) and Ser(588). Together, these data provide new insights into phosphorylation of key regulators of glucose transport in human skeletal muscle.
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Ejercicio Físico/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Músculo Esquelético/metabolismo , Fosforilación/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Transporte Biológico/fisiología , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Masculino , Contracción Muscular/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Descanso/fisiologíaRESUMEN
INTRODUCTION: Individuals with obesity and type 2 diabetes (T2D) are typically insulin resistant, exhibiting impaired skeletal muscle glucose uptake. Animal and cell culture experiments have shown that site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 and TBC1D1 is critical for GLUT4 translocation facilitating glucose uptake, but their regulation in human skeletal muscle is not well understood. METHODS: Here, lean, obese and T2D subjects underwent a euglycemic-hyperinsulinemic clamp, and vastus lateralis muscle biopsies were obtained before, and at 30 and 180 min post insulin infusion. RESULTS: Obese and T2D subjects had higher body mass indexes and fasting insulin concentrations, and T2D subjects showed insulin resistance. Consistent with the clamp findings, T2D subjects had impaired insulin-stimulated phosphorylation of AS160 Thr(642), a site previously shown to be important in glucose uptake in rodents. Interestingly, insulin-stimulated phosphorylation of TBC1D1 Thr(590), a site shown to be regulated by insulin in rodents, was only increased in T2D subjects, although the functional significance of this difference is unknown. CONCLUSION: These data show that insulin differentially regulates AS160 and TBC1D1 phosphorylation in human skeletal muscle. Impaired insulin-stimulated glucose uptake in T2D subjects is accompanied by dysregulation of AS160 and TBC1D1 phosphorylation in skeletal muscle, suggesting that these proteins may regulate glucose uptake in humans.
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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.
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Resistencia a la Insulina/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Animales , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
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.
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Ayuno/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Glucógeno/metabolismo , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Músculo Esquelético/metabolismo , Adenilato Quinasa/metabolismo , Adulto , Estudios Cruzados , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Humanos , Insulina/metabolismo , Masculino , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
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Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético/fisiología , Modelos Biológicos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Componentes del Gen , Glucosa/metabolismo , Metabolismo de los Lípidos , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Mice deficient in c-jun-NH(2)-terminal kinase 1 (JNK1) exhibit decreased fasting blood glucose and insulin levels, and protection against obesity-induced insulin resistance, suggesting increased glucose disposal into skeletal muscle. Thus, we assessed whether JNK1 deficiency enhances muscle glucose metabolism. Ex vivo insulin or contraction-induced muscle [(3)H]2-deoxyglucose uptake was not altered in JNK1 knockout mice, demonstrating that JNK1 does not regulate blood glucose levels via direct alterations in muscle. In vivo muscle [(3)H]2-deoxyglucose uptake in response to a glucose injection was also not enhanced by JNK1 deficiency, demonstrating that a circulating factor was not required to observe altered muscle glucose uptake in the knockout mice. JNK1 deficiency did not affect muscle glycogen levels or the protein expression of key molecules involved in glucose metabolism. This study is the first to directly demonstrate that enhanced skeletal muscle glucose metabolism does not underlie the beneficial effects of JNK1 deficiency in lean mice.
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Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Delgadez/metabolismo , Animales , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/genéticaRESUMEN
The AMP-activated protein kinase (AMPK) is an enzyme that is activated in situations where there are changes in the cellular energy status such as muscle contraction and hypoxia. AMPK can also be pharmacologically activated by the compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the antidiabetic agent metformin. Several studies support the hypothesis that AMPK plays an important role in the stimulation of muscle glucose uptake by these physiological and pharmacological stimuli. In isolated rat muscles, activation of AMPK is associated with increases in glucose uptake through an insulin-independent mechanism. Studies done in rodents have shown that the activation of AMPK by AICAR is accompanied by decreases in blood glucose concentrations, in part due to enhanced muscle glucose uptake. Similar to exercise, AICAR not only directly stimulates glucose uptake into the skeletal muscle, but also enhances insulin sensitivity. The activation of AMPK and associated increases in muscle glucose uptake are affected by factors such as glycogen content, exercise training and fibre type. The effects of AMPK on muscle glucose uptake makes this protein a promising pharmacological target for the treatment of type 2 diabetes.
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Aminoimidazol Carboxamida/análogos & derivados , Glucosa/metabolismo , Complejos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/metabolismo , Animales , Ejercicio Físico/fisiología , Glucógeno/metabolismo , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Metformina/farmacología , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/efectos de los fármacos , Estado Nutricional/fisiología , Ratas , Ribonucleótidos/metabolismoRESUMEN
Physical exercise increases muscle glucose uptake, enhances insulin sensitivity and leads to fatty acid oxidation in muscle. The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is strongly activated during muscle contraction due to acute decreases in ATP/AMP and phosphocreatine/creatine ratios. Accumulating evidence suggests that AMPK plays an important role in mediating these metabolic processes. Furthermore, AMPK has been implicated in regulating gene transcription and therefore may play a role in some of the cellular adaptations to training exercise. There is also evidence that changes in AMPK activity result in altered cellular glycogen content, suggesting that this enzyme regulates glycogen metabolism. Recent studies have shown that the magnitude of AMPK activation and associated metabolic responses are affected by factors such as glycogen content, exercise training and fibre type. In summary, AMPK regulates several metabolic pathways during acute exercise and modifies the expression of many genes involved in the adaptive changes to exercise training.
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Ejercicio Físico , Complejos Multienzimáticos/fisiología , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Quinasas Activadas por AMP , Animales , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Glucógeno/metabolismo , Humanos , Insulina/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , RatasRESUMEN
The question is no longer whether diet and exercise can benefit the individual with type 2 diabetes. Rather, the type and duration of exercise the magnitude of the effects on glycemic control, insulin sensitivity, and on risk factors for cardiovascular disease must be considered in determining the feasibility and acceptability of an intervention program. It is now clear that regular physical exercise is important in both the prevention and treatment of type 2 diabetes. The benefits of exercise are many and include increased energy expenditure, which, combined with dietary restriction, leads to decreased body fat, increased insulin sensitivity, improved long-term glycemic control, improved lipid profiles, lower blood pressure, and increased cardiovascular fitness. Persons with type 2 diabetes often find it difficult to exercise and are at increased risk for injury or exacerbation of underlying diseases or diabetic complications. Therefore, before starting an exercise program, all patients with type 2 diabetes should have a complete history and physical examination, with particular attention to evaluation of cardiovascular disease, medications that may affect glycemic control during or after exercise, and diabetic complications including retinopathy, nephropathy, and neuropathy. Exercise programs should be designed to start slowly, build up gradually, and emphasize moderately intense exercise performed at least three times a week and preferably five to seven times a week for best results.
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Diabetes Mellitus Tipo 2/etiología , Dieta , Ejercicio Físico/fisiología , Adulto , Anciano , Glucemia/fisiología , Presión Sanguínea , Diabetes Mellitus Tipo 2/prevención & control , Femenino , Humanos , Resistencia a la Insulina/fisiología , Lípidos/sangre , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/fisiologíaRESUMEN
The AMP-activated protein kinase (AMPK) plays an important role in fuel metabolism in exercising skeletal muscle and possibly in the islet cell with respect to insulin secretion. Some of these effects are due to AMPK-mediated regulation of cellular malonyl-CoA content, ascribed to the ability of AMPK to phosphorylate and inactivate acetyl-CoA carboxylase (ACC), reducing malonyl-CoA formation. It has been suggested that AMPK may also regulate malonyl-CoA content by activation of malonyl-CoA decarboxylase (MCD). We have investigated the potential regulation of MCD by AMPK in exercising skeletal muscle, in an islet cell line, and in vitro. Three rat fast-twitch muscle types were studied using two different contraction methods or after exposure to the AMPK activator AICAR. Although all muscle treatments resulted in activation of AMPK and phosphorylation of ACC, no stimulus had any effect on MCD activity. In 832/13 INS-1 rat islet cells, two treatments that result in the activation of AMPK, namely low glucose and AICAR, also had no discernable effect on MCD activity. Last, AMPK did not phosphorylate in vitro either recombinant MCD or MCD immunoprecipitated from skeletal muscle or heart. We conclude that MCD is not a substrate for AMPK in fast-twitch muscle or the 832/13 INS-1 islet cell line and that the principal mechanism by which AMPK regulates malonyl-CoA content is through its regulation of ACC.
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Carboxiliasas/metabolismo , Islotes Pancreáticos/metabolismo , Complejos Multienzimáticos/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Activación Enzimática , Técnicas In Vitro , Islotes Pancreáticos/citología , Contracción Muscular/fisiología , Fosforilación , Ratas , Nervio Ciático , Especificidad por SustratoRESUMEN
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.
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Aminoimidazol Carboxamida/análogos & derivados , Hipoglucemiantes/farmacología , Metformina/farmacología , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/farmacología , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/enzimología , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Inhibidores de Proteínas Quinasas , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ribonucleótidos/farmacología , Proteína 1 de Unión a los Elementos Reguladores de EsterolesRESUMEN
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.
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Hipertrofia Ventricular Izquierda/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Musculares , Miocardio/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Acil-CoA Deshidrogenasa , Animales , Transporte Biológico , Carnitina O-Palmitoiltransferasa/biosíntesis , Carnitina O-Palmitoiltransferasa/genética , Activación Enzimática , Ácido Graso Desaturasas/biosíntesis , Ácido Graso Desaturasas/genética , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Hipertrofia Ventricular Izquierda/genética , Técnicas In Vitro , Masculino , Modelos Cardiovasculares , Proteínas de Transporte de Monosacáridos/metabolismo , Oxidación-Reducción , Fosfatos/metabolismo , Presión , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.
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Proteínas Portadoras/metabolismo , Glucógeno Sintasa/metabolismo , Actividad Motora/fisiología , Músculo Esquelético/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Condicionamiento Físico Animal/fisiología , Esfuerzo Físico/fisiología , Animales , Transporte Biológico , Proteínas Portadoras/genética , Estimulación Eléctrica , Activación Enzimática , Tolerancia al Ejercicio/fisiología , Glucosa/metabolismo , Glucógeno/metabolismo , Glucógeno Fosforilasa/metabolismo , Ratones , Ratones Mutantes , Contracción Muscular/fisiología , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1RESUMEN
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.
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Diabetes Mellitus Tipo 2/fisiopatología , Ejercicio Físico/fisiología , Complejos Multienzimáticos/metabolismo , Proteínas Musculares , Músculo Esquelético/fisiopatología , Esfuerzo Físico/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Transportador de Glucosa de Tipo 4 , Hemoglobina Glucada/análisis , Glucógeno/metabolismo , Humanos , Cinética , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/metabolismo , Complejos Multienzimáticos/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Valores de Referencia , Descanso/fisiología , Transcripción GenéticaRESUMEN
The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK alpha 1 and AMPK alpha 2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-beta-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3-O-methyl-D-glucose (3-MG) uptake. There were dose-dependent increases in AMPK alpha 2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPK alpha1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPK alpha 2 activity and 3-MG uptake but had little effect on AMPK alpha 1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPK alpha 1 and -alpha 2 activity and 3-MG uptake. Although the AMPK alpha 1 and -alpha 2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPK alpha 2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.
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Glucosa/farmacocinética , Complejos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , 3-O-Metilglucosa/farmacocinética , Proteínas Quinasas Activadas por AMP , Animales , Inhibidores Enzimáticos/farmacología , Glucosa/antagonistas & inhibidores , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Actividad Motora/fisiología , Complejos Multienzimáticos/antagonistas & inhibidores , Contracción Muscular/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Glucosa/metabolismo , Insulina/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Óxido Nítrico/fisiología , Animales , Desoxiglucosa/farmacocinética , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Masculino , Actividad Motora/fisiología , Músculo Esquelético/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Nitroprusiato/farmacología , Ratas , Ratas Sprague-Dawley , omega-N-Metilarginina/farmacologíaRESUMEN
Physical exercise and contraction increase c-Jun NH2-terminal kinase (JNK) activity in rat and human skeletal muscle, and eccentric contractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects of isometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus (EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats were subjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20-25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretch had a much more profound effect on the stress-activated protein kinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation of JNK occurred within the muscle fibers. These studies suggest that the stretch component of a muscle contraction may be a major contributor to the increases in JNK activity and p38 phosphorylation observed after exercise in vivo.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Reflejo de Estiramiento/fisiología , Animales , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
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.
Asunto(s)
Eliminación de Gen , Glucógeno/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Animales , Peso Corporal/genética , Cognición/fisiología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Retroalimentación , Regulación Enzimológica de la Expresión Génica , Marcación de Gen , Glucógeno Sintasa/metabolismo , Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Fosforilación/efectos de los fármacos , Condicionamiento Físico Animal/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas/deficiencia , Proteínas Quinasas S6 Ribosómicas/genéticaRESUMEN
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.