RESUMO
Accumulating evidence indicates that skeletal muscle secrets proteins referred to as myokines and that exercise contributes to their regulation. In this study, we propose that chemokine (C-X-C motif) ligand 10 (CXCL10) functions as a novel myokine. Initially, we stimulated differentiated C2C12 myotubes with or without electrical pulse stimulation (EPS) to identify novel myokines. Cytokine array analysis revealed that CXCL10 secretion was significantly reduced by EPS, which was further confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction analysis. Treadmill experiments in mice identified significant reduction of Cxcl10 gene expression in the soleus muscle. Additionally, contraction-dependent p38 MAPK activation appeared to be involved in this reduction. Furthermore, C2C12 conditioned medium obtained after applying EPS could induce survival of MSS31, a vascular endothelial cell model, which was partially attenuated by the addition of recombinant CXCL10. Overall, our findings suggest CXCL10 as a novel exercise-reducible myokine, to control endothelial cell viability.
Assuntos
Quimiocina CXCL10/fisiologia , Teste de Esforço , Fibras Musculares Esqueléticas/fisiologia , Indutores da Angiogênese , Animais , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Sistema de Sinalização das MAP Quinases , Camundongos , Contração Muscular , Condicionamento Físico Animal , Reação em Cadeia da PolimeraseRESUMO
Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose-dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN-sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.
RESUMO
BACKGROUND: Oxidative stress is one of the mechanisms underlying pathogenesis in neurodegenerative diseases such as Alzheimer's disease. Generally, oxidative stress represents cell toxicity; however, we recently found that oxidative stress promotes the expression of growth factor progranulin (PGRN) in HT22 murine hippocampus cells, thereby protecting the HT22 cells. In this study, we attempted to clarify whether a similar system exists in the other neuronal cell model, rat pheochromocytoma (PC12) cells. RESULTS: After confirming that high concentrations of hydrogen peroxide (H2O2; 100-250 µM) initiate PC12 cell death, we analyzed growth factor expressional changes after H2O2 treatment. We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2. Although little expression of the high affinity BDNF receptor tropomyosin-related kinase TrkB was observed in PC12 cells, expression of low affinity neurotrophin receptor, p75NTR, was clearly observed. This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment. CONCLUSIONS: Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Regulação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/agonistas , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso , Estresse Oxidativo , Células PC12 , Progranulinas , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de SinaisRESUMO
Nutrient availability is one of the most important signals regulating cellular fates including cell growth, differentiation, and death. Recent evidence suggests that the NAD(+)-dependent histone deacetylase sirtuin 1 (SIRT1) plays a prominent role in linking changes in nutritional availability with cellular fate regulation. SIRT1 expression is observed in neurons, yet the expressional and functional regulation of this protein is not fully understood. In the present study, we examined whether extracellular glucose concentration affects the expression and localization of SIRT1 in PC12 cells. Further, we examined levels of forkhead box O3a (FoxO3a), which is also controlled by changes in extracellular glucose concentration. We observed the total expression levels of SIRT1 and FoxO3a in PC12 cells were reduced when glucose availability increased via gene expressional control, at least in part. Nuclear localization of SIRT1 and FoxO3a was increased by glucose deprivation. Even though the changes in extracellular glucose concentration regulated SIRT1 and FoxO3a in a similar direction, the effects of nerve growth factor on these two proteins were completely different. Finally, we found the potent SIRT1 inhibitor enhanced glucose deprivation-induced cell death. Therefore, we propose that glucose deprivation-induced SIRT1 expression potentially plays a major role in protecting PC12 cells.
Assuntos
Glucose/deficiência , Neurônios/metabolismo , Sirtuína 1/metabolismo , Animais , Western Blotting , Imunofluorescência , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead , Células PC12 , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (~12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".
Assuntos
Glicogênio/metabolismo , Resistência à Insulina , Interleucina-6/metabolismo , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Regulação para Cima , Animais , Calcineurina/metabolismo , Inibidores de Calcineurina , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/metabolismo , Estimulação Elétrica , Glucose/metabolismo , Hiperglicemia/metabolismo , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Imunossupressores/farmacologia , Insulina/metabolismo , Insulina/farmacologia , Interleucina-6/genética , Cinética , Camundongos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/imunologia , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under these conditions, cAMP treatment increased tyrosine phosphorylation of a 125-kDa protein (p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase (p85 PI3K), which were suggested to mediate potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis revealed p125 to be a rat ortholog of human XB130, which we named PI3K-associated protein (PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and interaction with p85 PI3K leading to increased PI3K activities associated with PI3KAP/XB130, supporting the role of PI3KAP/XB130 in DNA synthesis potentiation. Importantly, PI3KAP/XB130 knockdown attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Furthermore, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. Finally, introduction of PI3KAP/XB130 into NIH3T3 fibroblasts lacking endogenous PI3KAP/XB130 enhanced IGF-I-induced DNA synthesis; however, a mutant Y72F incapable of binding to p85 PI3K did not show this response. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130, which is associated with PI3K, is required for enhancement of IGF mitogenic activities.
Assuntos
Proteínas de Transporte/metabolismo , AMP Cíclico/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Mitógenos/fisiologia , Glândula Tireoide/citologia , Animais , Proteínas de Transporte/genética , Ciclo Celular , Proliferação de Células , Replicação do DNA , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Análise de Sequência de DNA , Tireotropina/fisiologia , Quinases da Família src/metabolismoRESUMO
Progranulin (PGRN) is a growth modulating factor released by a variety of cells. This molecule has gained the attention of the neuroscience community with recent discoveries of multifunctional roles of PGRN in normal brain and neurodegenerative disorders. We focus on novel roles of PGRN as a sex steroid-responsible gene in the developing and adult rodent brain. While the developing brain is feminine by default, hormone exposure, including androgen and estrogen, induces masculinization during the critical period. We have shown that PGRN is a sex steroid-responsible gene that may be involved in masculinization of the perinatal rat brain. We also found that in adult rats PGRN gene expression was up-regulated by estrogen in the hippocampus, suggesting that PGRN may mediate the mitogenic effects of estrogen in the active area of neurogenesis. Since it has been recently reported that mutations in PGRN gene are responsible for a type of frontotemporal lobar degeneration in humans, PGRN appears to be also involved in modulating neurodegeneration. Together, PGRN gene expression is induced by estrogen in both developing and adult brains, and it may play multifunctional roles in the organization of functional masculinization in the developing brain and the maintenance of adult brain function.
Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neurônios/metabolismo , Animais , Sistema Endócrino , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Camundongos , Modelos Biológicos , Doenças Neurodegenerativas/metabolismo , Neurônios/fisiologia , Peptídeos/química , Progranulinas , Ratos , Diferenciação Sexual , Esteroides/metabolismoRESUMO
Physical exercise triggers the release of several cytokines/chemokines from working skeletal muscles, but the underlying mechanism(s) by which skeletal muscles decipher and respond to highly complex contractile stimuli remains largely unknown. In an effort to investigate the regulatory mechanisms of the expressions of two contraction-inducible CXC chemokines, CXCL1/KC and CXCL5/LIX, in contracting skeletal muscle cells, we took advantage of our in vitro exercise model using highly developed contractile C(2)C(12) myotubes, which acquire properties similar to those of in vivo skeletal muscle via manipulation of Ca(2+) transients with electric pulse stimulation (EPS). Production of these CXC chemokines was immediately augmented by EPS-evoked contractile activity in a manner dependent on the activities of JNK and NF-kappaB, but not p38, ERK1/2, or calcineurin. Intriguingly, exposure of myotubes to cyclic mechanical stretch also induced expression of these CXC chemokines; however, a much longer period of stimulation (approximately 12 h) was required, despite rapid JNK phosphorylation. We also demonstrate herein that CXCL1/KC and CXCL5/LIX have the ability to raise intracellular Ca(2+) concentrations via CXCR2-mediated activation of pertussis toxin-sensitive Galpha(i) proteins in C(2)C(12) myoblasts, an action at least partially responsible for their migration and differentiation. Although we revealed a possible negative feedback regulation of their own production in response to the contractile activity in differentiated myotubes, exogenous administration of these CXC chemokines did not acutely influence either insulin-induced Akt phosphorylation or GLUT4 translocation in C(2)C(12) myotubes. Taken together, these data shed light on the fundamental characteristics of contraction-inducible CXC chemokine production and their potential roles in skeletal muscle cells.
Assuntos
Quimiocinas CXC/fisiologia , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Quimiocinas CXC/biossíntese , Estimulação Elétrica , Ensaio de Imunoadsorção Enzimática , Mecanorreceptores/fisiologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/fisiologia , NF-kappa B/metabolismo , Estimulação Física , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C(2)C(12) myotubes by manipulating intracellular Ca(2+) transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser(231) after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from "fast type" to "slow type" was observed in the C(2)C(12) myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C(2)C(12) myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise.
Assuntos
Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/metabolismo , Animais , Linhagem Celular , Quimiocinas/sangue , Quimiocinas/genética , Quimiocinas/metabolismo , Estimulação Elétrica , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Sarcômeros/metabolismoRESUMO
Bioactivities of IGFs in various cells are often potentiated in the presence of other hormones. In previous studies we showed that pretreatment of rat FRTL-5 thyroid cells with TSH or other cAMP-generating agents markedly potentiated DNA synthesis induced by IGF-I. Under these conditions we found that phosphatidylinositol (PI) 3-kinase was activated in response to either cAMP or IGF stimulus, and both activation modes were indispensable for the potentiation of DNA synthesis. The present studies were undertaken to elucidate how cAMP and/or IGF-I stimulus regulated the G1 cyclin-cyclin dependent kinase (CDK)-inhibitor system, and to determine the roles of PI 3-kinase activation by cAMP or IGF-I stimulus in this system. We found that cAMP pretreatment enhanced IGF-I-dependent increases in cyclin D1, due to synergistic increases in mRNA and elevation of translation rates. Furthermore, cAMP pretreatment enhanced IGF-I-induced protein degradation of the CDK inhibitor, p27(Kip1). These changes well explained an increase in cyclin E, leading to marked activation of G1 CDKs, followed by retinoblastoma protein phosphorylation. Our results using a PI 3-kinase inhibitor showed that cAMP-dependent PI 3-kinase activation plays an important role in the increase in cyclin D1 translation. In contrast, IGF-I-dependent PI 3-kinase activation was required for the increase in cyclin D1 mRNA levels and degradation of p27(Kip1). Together, the present study elucidates the role of cAMP and IGF-I in differentially activating PI 3-kinase as a mediator of multiple molecular events. These events converge in the regulation of cyclin D1 and p27(Kip1), leading to cAMP-dependent potentiation of IGF-I-dependent CDK activation and DNA synthesis.
Assuntos
AMP Cíclico/farmacologia , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Northern Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Ciclina D1/genética , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Immunoblotting , Imunoprecipitação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Nutrition availability is one of the major environmental signals influencing cell fate, such as proliferation, differentiation, and apoptosis, often functioning in concert with other humoral factors, including insulin. Herein, we show that low-serum-induced differentiation of C(2)C(12) myocytes is significantly hampered under low glucose (LG; 5 mM) compared with high glucose (HG; 22.5 mM) conditions, concurrently with nuclear accumulation of SIRT1, an NAD(+)-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending on glucose availability, with regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient glucose, whereas insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive upregulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the hampered differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinol-dependent restoration, indicating that insulin can function as a negative as well as a positive myogenic factor depending on glucose availability. Taken together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved, at least in part, through regulation of the cellular contents and localization of SIRT1 and FoxO3a in differentiating C(2)C(12) myocytes.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Mioblastos/fisiologia , Sirtuínas/metabolismo , Animais , Proteínas Sanguíneas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Sinergismo Farmacológico , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirtuína 1 , Sirtuínas/genética , Serina-Treonina Quinases TORRESUMO
cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Oócitos/citologia , Oogênese/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/deficiência , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Insulina/farmacologia , Isoenzimas/metabolismo , Fator Promotor de Maturação/metabolismo , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Fenótipo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , XenopusRESUMO
It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Androstadienos/farmacologia , Animais , Transporte Biológico , Western Blotting , Linhagem Celular , Clonagem Molecular , Desoxiglucose/farmacocinética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/biossíntese , Transportador de Glucose Tipo 4/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Antagonistas da Insulina/farmacologia , Camundongos , Microscopia de Fluorescência , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , WortmaninaRESUMO
Protein tyrosine phosphatase nonreceptor type 13 (PTPN13) is a tyrosine phosphatase with multiple interacting domains that has been implicated previously in the regulation of apoptosis. We provide evidence that PTPN13 plays an important role in the control of the meiotic cell cycle. A cDNA coding for PTPN13 was isolated during the screening for the substrate of protein kinase A expressed in mammalian oocytes. PTPN13 is expressed in both mouse and Xenopus oocytes and is a substrate for protein kinase A in vitro and in vivo. Expression of a truncated constitutively-active PTPN13 in Xenopus oocytes synergizes with progesterone in the induction of germinal vesicle breakdown, the translation of Mos, the phosphorylation of Erk and the dephosphorylation of Cdc2. The phosphatase activity of PTPN13 is required for this synergism. Oocyte injection with specific small interference RNA downregulates the expression of mRNA for PTPN13 and blocks oocyte maturation induced by progesterone, a blockade that can be overcome by Cdc25 overexpression. These findings indicate that PTPN13 is involved in the regulation of the meiotic cell cycle.
Assuntos
Meiose/fisiologia , Oócitos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Camundongos , Dados de Sequência Molecular , Oogênese/fisiologia , Fosforilação , Progesterona/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Proteínas Tirosina Fosfatases/genética , XenopusRESUMO
Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a(-/-) females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a(-/-) oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a(-/-) oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a(-/-) oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a(-/-) mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Infertilidade Feminina , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Infertilidade Feminina/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose/fisiologia , Mesotelina , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Ovário/anatomia & histologia , Ovário/fisiologia , Ovulação/fisiologia , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.