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INTRODUCTION: Oral semaglutide is a novel tablet formulation of the human glucagon-like peptide-1 analogue semaglutide. In two trials, the effects of prior food ingestion (food effect), post-dose fasting period and water volume with dosing (dosing conditions) on oral semaglutide pharmacokinetics were investigated. METHODS: Subjects received once-daily oral semaglutide for 10 days. In the food-effect trial, 78 healthy subjects were randomised 1:1:1 to fed (meal 30 min pre-dose; 240 mL water with dosing), fasting (overnight until 4 h post-dose; 240 mL) or reference (fasting overnight until 30 min post-dose; 120 mL) arms. In the dosing conditions trial, 161 healthy men were randomised into eight dosing groups (overnight fasted with 50/120 mL water and 15/30/60/120 min post-dose fasting). Semaglutide plasma concentrations were measured frequently until 504 h after the 10th dose. RESULTS: In the food-effect trial, limited or no measurable semaglutide exposure was observed in the fed arm, while all subjects in the fasting arm had measurable semaglutide exposure. Area under the semaglutide concentration-time curve (AUC0-24h,semaglutide,day10) and maximum semaglutide concentration (Cmax,semaglutide,day10) were numerically greater by approximately 40% for the fasting versus reference arm (p = 0.082 and p = 0.080, respectively). In the dosing conditions trial, AUC0-24h,semaglutide,day10 and Cmax,semaglutide,day10 were not different between water volumes (p = 0.541 and p = 0.676), but increased with longer post-dose fasting (p < 0.001). CONCLUSION: Administration of oral semaglutide in the fasting state with up to 120 mL water and at least 30 min post-dose fasting results in clinically relevant semaglutide exposure. These dosing conditions have been used in the oral semaglutide phase 3 trials and are part of the approved label. TRIAL REGISTRATION: ClinicalTrials.gov identifiers NCT02172313, NCT01572753.
RESUMO
Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Peptídeos Semelhantes ao Glucagon/farmacologia , Absorção Intestinal , Estômago/fisiologia , Administração Oral , Adolescente , Adulto , Idoso , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/ultraestrutura , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos Semelhantes ao Glucagon/administração & dosagem , Peptídeos Semelhantes ao Glucagon/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Ratos , Estômago/efeitos dos fármacos , Fatores de Tempo , Adulto JovemRESUMO
Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca(2+) as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca(2+) release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca(2+) centric paradigm.
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The 5'-AMP-activated protein kinase (AMPK) is considered "a metabolic master-switch" in skeletal muscle reducing ATP- consuming processes whilst stimulating ATP regeneration. Within recent years, AMPK has also been proposed as a potential target to attenuate insulin resistance, although the exact role of AMPK is not well understood. Here we hypothesized that mice lacking α2AMPK activity in muscle would be more susceptible to develop insulin resistance associated with ageing alone or in combination with high fat diet. Young (â¼4 month) or old (â¼18 month) wild type and muscle specific α2AMPK kinase-dead mice on chow diet as well as old mice on 17 weeks of high fat diet were studied for whole body glucose homeostasis (OGTT, ITT and HOMA-IR), insulin signaling and insulin-stimulated glucose uptake in muscle. We demonstrate that high fat diet in old mice results in impaired glucose homeostasis and insulin stimulated glucose uptake in both the soleus and extensor digitorum longus muscle, coinciding with reduced insulin signaling at the level of Akt (pSer473 and pThr308), TBC1D1 (pThr590) and TBC1D4 (pThr642). In contrast to our hypothesis, the impact of ageing and high fat diet on insulin action was not worsened in mice lacking functional α2AMPK in muscle. It is concluded that α2AMPK deficiency in mouse skeletal muscle does not cause muscle insulin resistance in young and old mice and does not exacerbate obesity-induced insulin resistance in old mice suggesting that decreased α2AMPK activity does not increase susceptibility for insulin resistance in skeletal muscle.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/metabolismo , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Insulina/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Área Sob a Curva , Glicemia , Composição Corporal , Proteínas Ativadoras de GTPase/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/metabolismo , Hexoquinase/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD⺠concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Atividade Motora , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Transporte Biológico , Coenzima A/metabolismo , Regulação para Baixo , Proteínas Ativadoras de GTPase , Regulação da Expressão Gênica , Glucose/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , NAD/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxirredução , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Distribuição AleatóriaRESUMO
In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.
Assuntos
Glucose/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Células Cultivadas , Estimulação Elétrica , Teste de Esforço , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Músculo Esquelético/citologia , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Ribonucleotídeos/farmacologia , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
AIM: The signaling pathways involved in the regulation of cardiac GLUT4 translocation/glucose uptake and CD36 translocation/long-chain fatty acid uptake are not fully understood. We compared in heart/muscle-specific PKC-λ knockout mice the roles of atypical PKCs (PKC-ζ and PKC-λ) in regulating cardiac glucose and fatty acid uptake. RESULTS: Neither insulin-stimulated nor AMPK-mediated glucose and fatty acid uptake were inhibited upon genetic PKC-λ ablation in cardiomyocytes. In contrast, myristoylated PKC-ζ pseudosubstrate inhibited both insulin-stimulated and AMPK-mediated glucose and fatty acid uptake by >80% in both wild-type and PKC-λ-knockout cardiomyocytes. In PKC-λ knockout cardiomyocytes, PKC-ζ is the sole remaining atypical PKC isoform, and its expression level is not different from wild-type cardiomyocytes, in which it contributes to 29% and 17% of total atypical PKC expression and phosphorylation, respectively. CONCLUSION: Taken together, atypical PKCs are necessary for insulin-stimulated and AMPK-mediated glucose uptake into the heart, as well as for insulin-stimulated and AMPK-mediated fatty acid uptake. However, the residual PKC-ζ activity in PKC-λ-knockout cardiomyocytes is sufficient to allow optimal stimulation of glucose and fatty acid uptake, indicating that atypical PKCs are necessary but not rate-limiting in the regulation of cardiac substrate uptake and that PKC-λ and PKC-ζ have interchangeable functions in these processes.
RESUMO
AMP-activated protein kinase (AMPK) ß1 or ß2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, α2ß2γ3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK α2, ß2, and γ3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK ß1 and ß2 isoforms in skeletal muscle (ß1ß2M-KO). ß1ß2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young ß1ß2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle α, ß, or γ subunits.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Proteínas Quinases Ativadas por AMP/genética , Animais , DNA Mitocondrial/genética , Feminino , Glucose/farmacocinética , Hipoglicemiantes/farmacologia , Immunoblotting , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Contração Muscular , Músculo Esquelético/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Exercise-induced phosphorylation of FXYD1 is a potential important regulator of Na(+)-K(+)-pump activity. It was investigated whether skeletal muscle contractions induce phosphorylation of FXYD1 and whether protein kinase Cα (PKCα) activity is a prerequisite for this possible mechanism. In part 1, human muscle biopsies were obtained at rest, after 30 s of high-intensity exercise (166 ± 31% of Vo(2max)) and after a subsequent 20 min of moderate-intensity exercise (79 ± 8% of Vo(2max)). In general, FXYD1 phosphorylation was increased compared with rest both after 30 s (P < 0.05) and 20 min (P < 0.001), and more so after 20 min compared with 30 s (P < 0.05). Specifically, FXYD1 ser63, ser68, and combined ser68 and thr69 phosphorylation were 26-45% higher (P < 0.05) after 20 min of exercise than at rest. In part 2, FXYD1 phosphorylation was investigated in electrically stimulated soleus and EDL muscles from PKCα knockout (KO) and wild-type (WT) mice. Contractile activity caused FXYD1 ser68 phosphorylation to be increased (P < 0.001) in WT soleus muscles but to be reduced (P < 0.001) in WT extensor digitorum longus. In contrast, contractile activity did not affect FXYD1 ser68 phosphorylation in the KO mice. In conclusion, exercise induces FXYD1 phosphorylation at multiple sites in human skeletal muscle. In mouse muscles, contraction-induced changes in FXYD1 ser68 phosphorylation are fiber-type specific and dependent on PKCα activity.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C-alfa/metabolismo , Adulto , Animais , Exercício Físico/fisiologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiologia , Fosfoproteínas/genética , Fosforilação , Proteína Quinase C-alfa/genéticaRESUMO
Some studies suggest that the 5'-AMP-activated protein kinase (AMPK) is important in regulating muscle glucose uptake in response to intense electrically stimulated contractions. However, it is unknown whether AMPK regulates muscle glucose uptake during in vivo exercise. We studied this in male and female mice overexpressing kinase-dead AMPKalpha2 (AMPK-KD) in skeletal and heart muscles. Wild-type and AMPK-KD mice were exercised at the same absolute intensity and the same relative intensity (30 and 70% of individual maximal running speed) to correct for reduced exercise capacity of the AMPK-KD mouse. Muscle glucose clearance was measured using 2-deoxy-[(3)H]glucose as tracer. In wild-type mice, glucose clearance was increased at 30 and 70% of maximal running speed by 40 and 350% in the quadriceps muscle and by 120 and 380% in gastrocnemius muscle, respectively. Glucose clearance was not lower in AMPK-KD muscles compared with wild-type regardless of whether animals were exercised at the same relative or the same absolute intensity. In agreement, surface membrane content of the glucose transporter GLUT4 was increased similarly in AMPK-KD and wild-type muscle in response to running. We also measured signaling of alternative exercise-sensitive pathways that might be compensatorily increased in AMPK-KD muscles. However, increases in phosphorylation of CaMKII, Trisk95, p38 MAPK, and ERK1/2 were not higher in AMPK-KD than in WT muscle. Collectively, these findings suggest that AMPKalpha2 signaling is not essential in regulating glucose uptake in mouse skeletal muscle during treadmill exercise and that other mechanisms play a central role.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/fisiologia , Glucose/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cateterismo , Feminino , Transportador de Glucose Tipo 4/metabolismo , Glicogênio/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/biossíntese , Músculo Esquelético/efeitos dos fármacos , Fosfocreatina/metabolismo , Fosforilação , Ribonucleotídeos/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50-100%, whereas both Gö compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKCalpha was estimated to account for approximately 97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCalpha knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction-induced glucose uptake were similar in muscles lacking PKCalpha and in the wild type. It can be concluded that PKCalpha, representing approximately 97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.
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Glucose/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteína Quinase C-alfa/genética , Animais , Transporte Biológico , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Maleimidas , Camundongos , Camundongos Knockout , Contração Muscular/genética , Naftalenos/farmacologia , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismoRESUMO
This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action compared with a high-carbohydrate diet [65 energy-% (CHO)]. After 4 days of isocaloric diet on two occasions (Fat/CHO), 12 male subjects performed 1 h of one-legged knee extensor exercise (approximately 80% peak workload). Four hours after exercise, insulin-stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin-stimulated glucose uptake was improved (approximately 70%, P<0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (approximately 20%, P<0.05) in MCoA content 4 h after exercise that correlated (r=0.65, P<0.001) with improved insulin-stimulated glucose uptake. Four days of Fat diet resulted in an increased content of intramyocellular triacylglycerol (P<0.01) but did not influence muscle MCoA content or whole body insulin-stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin-stimulated glucose uptake appeared to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle.
Assuntos
Exercício Físico/fisiologia , Glucose/metabolismo , Insulina/farmacologia , Malonil Coenzima A/metabolismo , Músculo Esquelético/metabolismo , Adulto , Algoritmos , Dieta Aterogênica , Carboidratos da Dieta/farmacologia , Gorduras na Dieta/farmacologia , Regulação para Baixo/fisiologia , Glucose/farmacocinética , Técnica Clamp de Glucose , Humanos , Masculino , Músculo Esquelético/efeitos dos fármacos , Respiração/efeitos dos fármacos , Descanso/fisiologia , Adulto JovemRESUMO
Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesized that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) phosphorylation by signalling cascades downstream of rises in intracellular [Ca(2+)] and decreased energy charge via AMP-activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis correlated more closely with changes in eEF2 than 4EBP1 phosphorylation. Using a combination of Ca(2+) release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca(2+) and energy turnover-related mechanisms. Furthermore, eEF2 kinase (eEF2K) inhibition completely blunted increases in eEF2 phosphorylation and partially blunted (i.e. 30-40%) the suppression of protein synthesis during contractions. The 3- to 5-fold increase in skeletal muscle eEF2 phosphorylation during contractions in situ was rapid and sustained and restricted to working muscle. The increase in eEF2 phosphorylation and eEF2K activation were downstream of Ca(2+)-calmodulin (CaM) but not other putative activating factors such as a fall in intracellular pH or phosphorylation by protein kinases. Furthermore, blunted protein synthesis and 4EBP1 dephosphorylation were unrelated to AMPK activity during contractions, which was exemplified by normal blunting of protein synthesis during contractions in muscles overexpressing kinase-dead AMPK. In summary, in fast-twitch skeletal muscle, the inhibition of eEF2 activity by phosphorylation downstream of Ca(2+)-CaM-eEF2K signalling partially contributes to the suppression of protein synthesis during exercise/contractions.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Calmodulina/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Contração Muscular , Proteínas Musculares/biossíntese , Músculo Esquelético/enzimologia , Fator 2 de Elongação de Peptídeos/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Regulação para Baixo , Estimulação Elétrica , Metabolismo Energético , Fatores de Iniciação em Eucariotos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Musculares de Contração Rápida/enzimologia , Músculo Esquelético/inervação , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We investigated if acute endurance-type exercise interacts with insulin-stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one-legged exercise, insulin-induced glucose uptake was approximately 80% higher (N = 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic-hyperinsulinaemic clamp (100 microU ml(-1)). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)-1 and IRS-2 associated phosphatidylinositol (PI)-3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser(473) and Thr(308) in skeletal muscle. Interestingly, in response to prior exercise IRS-2-associated PI-3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular-regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr(410) phosphorylation. However, when adding phosphatidylinositol-3,4,5-triphosphate (PIP3), the signalling product of PI-3 kinase, to basal muscle homogenates, aPKC was more potently activated (P = 0.01) in previously exercised muscle. Collectively, this study shows that endurance-type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance-type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin-stimulated glucose uptake after exercise.
Assuntos
Exercício Físico/fisiologia , Insulina/fisiologia , Músculo Esquelético/fisiologia , Fosfatos de Fosfatidilinositol/fisiologia , Proteína Quinase C/metabolismo , Adulto , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Técnica Clamp de Glucose , Humanos , Hiperinsulinismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lactatos/sangue , Masculino , Elongação Traducional da Cadeia Peptídica , Fosfoproteínas/fisiologia , Transdução de SinaisRESUMO
Atypical protein kinase C (aPKC) and extracellular signal-regulated kinase (ERK) are emerging as important signalling molecules in the regulation of metabolism and gene expression in skeletal muscle. Exercise is known to increase activity of aPKC and ERK in skeletal muscle but the effect of exercise intensity hereon has not been studied. Furthermore, the relationship between activity and phosphorylation of the two enzymes during exercise is unknown. Nine healthy young men exercised for 30 min on a bicycle ergometer on two occasions. One occasion consisted of three consecutive 10 min bouts of 35, 60 and 85% of peak pulmonary oxygen uptake V(O(2 peak)) and the second of one 30 min bout at 35% of V(O(2 peak)). Both trials also included 30 min recovery. Muscle biopsies were obtained from the vastus lateralis muscle before and after each exercise bout. Exercise increased muscle aPKC activity at 35% V(O(2 peak)), whereupon no further increase was observed at higher exercise intensities. Activation of aPKC was not accompanied by increased phosphorylation of aPKC Thr(410/403). ERK1/2 activity increased in a similar pattern to aPKC, reaching maximal activity at 35% V(O(2 peak)), whereas ERK1 Thr(202)/Tyr(204) and ERK2 Thr(183)/Tyr(185) phosphorylation increased with increasing exercise intensity. Thus, aPKC and ERK1/2 activity in muscle during exercise did not correspond to phosphorylation of sites on aPKC or ERK1/2, respectively, which are considered important for their activation. It is concluded that assessment of aPKC and ERK1/2 activity in muscle using phosphospecific antibodies did not reflect direct activity measurements on immunoprecipitated enzyme in vitro. Thus, estimation of enzyme activity during exercise by use of phosphospecific antibodies should not be performed uncritically. In addition, increase in muscle activity of aPKC or ERK1/2 during exercise is not closely related to energy demands of the muscle but may serve other regulatory or permissive functions in muscle.