RESUMO
We examined the effect of physiological hyperinsulinemia on insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity in skeletal muscle from six lean-to-moderately obese NIDDM patients and six healthy subjects. A rise in serum insulin levels from approximately 60 to approximately 650 pmol/l increased IRS-1 tyrosine phosphorylation sixfold over basal levels in control muscle (P < 0.01), whereas no significant increase was noted in NIDDM muscle. The reduced IRS-1 phosphorylation in the NIDDM muscle was not related to changes in IRS-1 protein content, since IRS-1 protein expression was similar between control and NIDDM subjects (16.0 +/- 1.7 vs. 22.9 +/- 4.0 arbitrary units/mg protein for control and NIDDM, respectively; NS). Physiological hyperinsulinemia increased PI 3-kinase activity in control muscle twofold (P < 0.01), whereas no increase in insulin-stimulated PI 3-kinase activity was noted in the NIDDM muscle. Furthermore, in vitro insulin-stimulated (600 pmol/l) 3-O-methylglucose transport was 40% lower in isolated muscle from NIDDM subjects (P < 0.05). The present findings couple both reduced insulin-stimulated IRS-1 tyrosine phosphorylation and PI 3-kinase activity to the impaired insulin-stimulated glucose transport in skeletal muscle from lean-to-moderately obese NIDDM subjects.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Hiperinsulinismo , Insulina/farmacologia , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , 3-O-Metilglucose/metabolismo , Transporte Biológico/efeitos dos fármacos , Biópsia , Humanos , Proteínas Substratos do Receptor de Insulina , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotirosina/análise , Valores de Referência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To determine whether defects in the insulin signal transduction cascade are present in skeletal muscle from prediabetic individuals, we excised biopsies from eight glucose-intolerant male first-degree relatives of patients with type 2 diabetes (IGT relatives) and nine matched control subjects before and during a euglycemic-hyperinsulinemic clamp. IGT relatives were insulin-resistant in oxidative and nonoxidative pathways for glucose metabolism. In vivo insulin infusion increased skeletal muscle insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (P = 0.01) and phosphatidylinositide 3-kinase (PI 3-kinase) activity (phosphotyrosine and IRS-1 associated) in control subjects (P < 0.02) but not in IGT relatives (NS). The incremental increase in insulin action on IRS-1 tyrosine phosphorylation was lower in IGT relatives versus control subjects (P < 0.05). The incremental defects in signal transduction noted for IRS-1 and PI 3-kinase may be attributed to elevated basal phosphorylation/activity of these parameters, because absolute phosphorylation/activity under insulin-stimulated conditions was similar between IGT relatives and control subjects. Insulin increased Akt serine phosphorylation in control subjects and IGT relatives, with a tendency for reduced phosphorylation in IGT relatives (P = 0.12). In conclusion, aberrant phosphorylation/activity of IRS-1, PI 3-kinase, and Akt is observed in skeletal muscle from relatives of patients with type 2 diabetes with IGT. However, the elevated basal activity of these signaling intermediates and the lack of a strong correlation between these parameters to glucose metabolism suggests that other defects of insulin signal transduction and/or downstream components of glucose metabolism may play a greater role in the development of insulin resistance in skeletal muscle from relatives of patients with type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Adulto , Biópsia , Glicemia/metabolismo , Índice de Massa Corporal , Técnica Clamp de Glucose , Humanos , Insulina/sangue , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.
Assuntos
Hipóxia Celular , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Marcadores de Fotoafinidade , Transporte Biológico , Biotinilação , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestruturaRESUMO
We characterized metabolic and mitogenic signaling pathways in isolated skeletal muscle from well-matched type 2 diabetic and control subjects. Time course studies of the insulin receptor, insulin receptor substrate (IRS)-1/2, and phosphatidylinositol (PI) 3-kinase revealed that signal transduction through this pathway was engaged between 4 and 40 min. Insulin-stimulated (0.6-60 nmol/l) tyrosine phosphorylation of the insulin receptor beta-subunit, mitogen-activated protein (MAP) kinase phosphorylation, and glycogen synthase activity were not altered in type 2 diabetic subjects. In contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and anti-phosphotyrosine-associated PI 3-kinase activity were reduced 40-55% in type 2 diabetic subjects at high insulin concentrations (2.4 and 60 nmol/l, respectively). Impaired glucose transport activity was noted at all insulin concentrations (0.6-60 nmol/l). Aberrant protein expression cannot account for these insulin-signaling defects because expression of insulin receptor, IRS-1, IRS-2, MAP kinase, or glycogen synthase was similar between type 2 diabetic and control subjects. In skeletal muscle from type 2 diabetic subjects, IRS-1 phosphorylation, PI 3-kinase activity, and glucose transport activity were impaired, whereas insulin receptor tyrosine phosphorylation, MAP kinase phosphorylation, and glycogen synthase activity were normal. Impaired insulin signal transduction in skeletal muscle from type 2 diabetic patients may partly account for reduced insulin-stimulated glucose transport; however, additional defects are likely to play a role.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Glucose/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Transporte Biológico , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Sintase/metabolismo , Humanos , Insulina/fisiologia , Proteínas Substratos do Receptor de Insulina , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Receptor de Insulina/metabolismo , Fatores de Tempo , Tirosina/metabolismoRESUMO
AIM: To examine the phenotype of mice that lack the adenosine A(3) receptor (A(3)R). METHODS: We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition, the effect of the adenosine analogue R-N(6)-phenylisopropyl-adenosine (R-PIA) was examined. We also examined heat production and food intake. RESULTS: We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A(3)R knock-out mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A(3)R knock-out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A(3)R knock-out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice. CONCLUSIONS: The mice lacking adenosine A(3) receptors exhibit a surprisingly clear phenotype with changes in diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A(3) receptors in these processes or whether they represent a role in development remains to be elucidated.
Assuntos
Temperatura Corporal/fisiologia , Frequência Cardíaca/fisiologia , Atividade Motora/fisiologia , Fenótipo , Receptor A3 de Adenosina/metabolismo , Adenosina/metabolismo , Animais , Peso Corporal , Ritmo Circadiano/fisiologia , Ingestão de Alimentos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A3 de Adenosina/genética , TelemetriaRESUMO
Type II diabetes is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue and liver and pancreatic beta-cell defects. Since the skeletal muscle accounts for approx. 75% of whole body insulin-stimulated glucose uptake, defects in this tissue play a major role in the impaired glucose homoeostasis in Type II diabetic patients. Thus identifying defective steps in this process may reveal attractive targets for drug development to combat insulin resistance and Type II diabetes. This review will describe the effects of insulin on glucose transport and other metabolic events in skeletal muscle that are mediated by intracellular signalling cascades. Evidence for impaired activation of the insulin receptor signalling cascade and defective glucose transporter 4 translocation in the skeletal muscle from Type II diabetic patients will be presented. Through the identification of the intracellular defects in insulin action that control glucose homoeostasis, a better understanding of the disease pathogenesis can be gained and strategies for intervention may be developed.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Humanos , Resistência à Insulina , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The adipose tissue-derived hormone leptin regulates energy balance and neuroendocrine function. Resistance to the appetite-suppressing effects of leptin is associated with common forms of obesity. Here, we review the mechanisms by which leptin activates intracellular signals and the roles that these signals play in leptin action in vivo. Furthermore, we discuss potential mechanisms of leptin resistance, specifically focusing on data regarding the neuroanatomical locus of leptin resistance and potential mechanisms by which expression of the suppressor of cytokine signaling-3 may impair leptin action.
Assuntos
Leptina/metabolismo , Obesidade/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Humanos , Hipotálamo/anatomia & histologia , Hipotálamo/metabolismo , Rede Nervosa/fisiologia , Receptores para Leptina , Fator de Transcrição STAT3 , Transdução de Sinais/fisiologia , Transativadores/metabolismoRESUMO
AIM/HYPOTHESIS: We examined insulin signal transduction at the level of insulin receptor substrates (IRS) 1 and 2, phosphatidylinositol (PI) 3-kinase and glucose transport in isolated subcutaneous adipocytes from obese and lean women. METHODS: Glucose transport and insulin signalling were investigated in isolated adipocytes from six obese women (BMI 36-43 kg/m(2)) (before and after 11 days of very low calorie diet) and from six lean women (BMI 22-26 kg/m(2)). RESULTS: Insulin sensitivity of glucose transport was reduced in adipocytes from obese women (p<0.05), with further reductions in basal and maximal insulin-stimulated glucose transport after a very low calorie diet (p<0.05). In obese women, IRS-1 associated PI 3-kinase activity was markedly impaired (p<0.05), whereas, IRS-2 associated PI 3-kinase activity was normal. IRS-1 associated PI 3-kinase activity remained blunted after a very low calorie diet, whereas IRS-2 associated PI 3-kinase activity was increased. GLUT4 protein was reduced by 37% in obese versus lean subjects (p<0.05), and decreased further after a very low calorie diet (from 19+/-4 to 14+/-4 arbitrary units; p<0.05). CONCLUSION/INTERPRETATION: IRS-1 signalling to PI 3-kinase is a site of insulin resistance in adipocytes from obese women, whereas insulin action on IRS-2 is normal. Thus, IRS-1 and IRS-2 undergo differential regulation in adipocytes from obese insulin resistant subjects. Finally, a very low calorie diet is associated with a further impairment in glucose transport in adipose tissue. The defect in glucose transport after a very low calorie diet occurs independent of further defects in insulin signalling at the level of the PI 3-kinase.
Assuntos
Adipócitos/metabolismo , Ingestão de Energia , Glucose/metabolismo , Insulina/metabolismo , Obesidade/dietoterapia , Obesidade/metabolismo , Transdução de Sinais/fisiologia , Adipócitos/química , Adipócitos/patologia , Adulto , Transporte Biológico/fisiologia , Extratos Celulares/química , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Concentração Osmolar , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismoRESUMO
Complete spinal cord lesion leads to profound metabolic abnormalities and striking changes in muscle morphology. Here we assess the effects of electrically stimulated leg cycling (ESLC) on whole body insulin sensitivity, skeletal muscle glucose metabolism, and muscle fiber morphology in five tetraplegic subjects with complete C5-C7 lesions. Physical training (seven ESLC sessions/wk for 8 wk) increased whole body insulin-stimulated glucose uptake by 33+/-13%, concomitant with a 2.1-fold increase in insulin-stimulated (100 microU/ml) 3-O-methylglucose transport in isolated vastus lateralis muscle. Physical training led to a marked increase in protein expression of GLUT4 (378+/-85%), glycogen synthase (526+/-146%), and hexokinase II (204+/-47%) in vastus lateralis muscle, whereas phosphofructokinase expression (282+/-97%) was not significantly changed. Hexokinase II activity was significantly increased, whereas activity of phosphofructokinase, glycogen synthase, and citrate synthase was not changed after training. Muscle fiber type distribution and fiber area were markedly altered compared to able-bodied subjects before ESLC training, with no change noted in either parameter after ECSL training. In conclusion, muscle contraction improves insulin action on whole body and cellular glucose uptake in cervical cord-injured persons through a major increase in protein expression of key genes involved in the regulation of glucose metabolism. Furthermore, improvements in insulin action on glucose metabolism are independent of changes in muscle fiber type distribution.
Assuntos
Exercício Físico , Glucose/metabolismo , Homeostase , Proteínas Musculares , Quadriplegia/metabolismo , Traumatismos da Medula Espinal/metabolismo , 3-O-Metilglucose/metabolismo , Transporte Biológico , Terapia por Estimulação Elétrica , Técnica Clamp de Glucose , Transportador de Glucose Tipo 4 , Glicogênio/análise , Glicogênio Sintase/biossíntese , Hexoquinase/biossíntese , Humanos , Insulina/farmacologia , Perna (Membro) , Masculino , Proteínas de Transporte de Monossacarídeos/biossíntese , Fibras Musculares Esqueléticas/ultraestrutura , Quadriplegia/terapia , Traumatismos da Medula Espinal/terapiaRESUMO
AIM/HYPOTHESIS: Insulin receptor substrate (IRS) proteins play important roles in insulin action and pancreatic beta-cell function. At least four mammalian IRS molecules have been identified. Although genes and cDNAs encoding human IRS-1, IRS-2, and IRS-4 have been cloned, IRS-3 has been identified only in rodents. Thus, we have attempted to clone the human IRS-3 gene. METHODS: Insulin-stimulated rat or human adipocytes were subjected to Western blot analysis to assess IRS-3 tyrosine phosphorylation. Human liver and adipose cDNA libraries were screened in an effort to clone IRS-3 cDNA. A PCR-based approach was designed to amplify IRS-3 cDNA. Reverse transcription PCR was carried out using mRNA from adipose tissue, liver, and skeletal muscle as templates in combination with an in silico screen using mouse IRS-1, IRS-2 and IRS-3 in a tblastn search of the draft public human genome. RESULTS: In human adipocytes we did not detect a M(r) 60 000 phosphoprotein corresponding to IRS-3, whereas in rat adipocytes IRS-3 protein and insulin-stimulated tyrosine phosphorylation was readily observed. None of the molecular approaches provided evidence for a functional IRS-3gene in human tissue. Two deletions in human IRS-3 gene were identified using bioinformatics. The human IRS-3 gene product is predicted to lack a phosphotyrosine binding domain and also the sequence corresponding amino acid 353-407 of murine IRS-3. The contiguous sequence of genomic DNA between these two homologous regions does not have the coding information for human IRS-3. CONCLUSION/INTERPRETATION: In silico screening of the human IRS-3 genome region, combined with further biological and molecular validation, provides evidence against a functional IRS-3 in humans.
Assuntos
Fosfoproteínas/deficiência , Fosfoproteínas/genética , Adipócitos/metabolismo , Adulto , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Biologia Computacional , Feminino , Deleção de Genes , Biblioteca Gênica , Genoma Humano , Humanos , Proteínas Substratos do Receptor de Insulina , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfoproteínas/fisiologia , Fosforilação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/metabolismoRESUMO
The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one-leg cycle ergometry at approximately 70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal. Activation of the different MAP kinase pathways by physical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.
Assuntos
Exercício Físico/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Glicemia/análise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática , Teste de Esforço , Quinase 2 de Adesão Focal , Regulação da Expressão Gênica/fisiologia , Humanos , Insulina/sangue , Insulina/farmacologia , Lactatos/sangue , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Contração Muscular/fisiologia , Proteínas Musculares/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Surgical trauma induces peripheral insulin resistance; however, the cellular mechanism has not been fully elucidated. We examined the effects of surgical trauma on insulin receptor signaling and glucose transport in skeletal muscle, a tissue that plays a predominant role in maintaining glucose homeostasis. Surgical trauma was induced by intestinal resection in the rat. Receptor phosphorylation was not altered with surgical trauma. Phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase association was increased by 60 and 82% compared with fasted and fed controls, respectively (P < 0. 05). Similar results were observed for insulin receptor substrate-1-associated PI 3-kinase activity. Insulin-stimulated protein kinase B (Akt kinase) phosphorylation was increased by 2.2-fold after surgical trauma (P < 0.05). The hyperphosphorylation of Akt is likely to reflect amplification of PI 3-kinase after insulin stimulation. Submaximal rates of insulin-stimulated 3-O-methylglucose transport were reduced in trauma vs. fasted rats by 51 and 38% for 100 and 200 microU/ml of insulin, respectively (P < 0.05). In conclusion, insulin resistance in skeletal muscle after surgical trauma is associated with reduced glucose transport but not with impaired insulin signaling to PI 3-kinase or its downstream target, Akt. The surgical trauma model presented in this report provides a useful tool to further elucidate the molecular mechanism(s) underlying the development of insulin resistance after surgical trauma.