RESUMEN
Obesity, the major cause of the current global epidemic of type 2 diabetes (T2D), induces insulin resistance in peripheral insulin target tissues. Several mechanisms have been identified related to cross-talk between adipose tissue, skeletal muscle and liver. These mechanisms involve both increased free fatty acid release and altered secretion of adipokines from adipose tissue. A major determinant of metabolic health is the ability of subcutaneous adipose tissue (SAT) to store excess fat rather than allowing it to accumulate in ectopic depots including liver (i.e. in nonalcoholic fatty liver disease), muscle and heart, or in epicardial/pericardial and visceral fat depots which promote the metabolic complications of obesity. The ability to recruit and differentiate precursor cells into adipose cells (adipogenesis) in SAT is under genetic regulation and is reduced in high-risk individuals who have first-degree relatives with T2D. Early recruitment of new adipose cells is dependent on the cross-talk between canonical WNT and BMP4 signalling; WNT enhances their undifferentiated and proliferative state whereas BMP4 induces their commitment to the adipogenic lineage. Dysregulation of these signalling pathways is associated with impaired adipogenesis and impaired ability to respond to the need to store excess lipids in SAT. This leads to hypertrophic, dysfunctional and insulin-resistant adipose cells with a reduced content of GLUT4, the major insulin-regulated glucose transporter, which in turn reduces adipose tissue glucose uptake and de novo lipogenesis. We recently identified that reduced GLUT4 and lipogenesis in adipocytes impairs the synthesis of a novel family of lipids secreted by adipose tissue (and potentially other tissues), branched fatty acid esters of hydroxy fatty acids (FAHFAs). FAHFAs have beneficial metabolic effects, including enhancing insulin-stimulated glucose transport and glucose-stimulated GLP1 and insulin secretion, as well as powerful anti-inflammatory effects. FAHFA levels are reduced in subcutaneous adipose tissue in insulin-resistant individuals, and this novel family of lipids may become of future therapeutic use.
Asunto(s)
Adipogénesis/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina/fisiología , Lipogénesis/fisiología , Grasa Subcutánea/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Índice de Masa Corporal , Diferenciación Celular , Proliferación Celular , Diabetes Mellitus Tipo 2/etnología , Ésteres , Ácidos Grasos/metabolismo , Humanos , Metabolismo de los Lípidos , Obesidad/metabolismoRESUMEN
AIMS/HYPOTHESIS: Pregnancy is characterised by temporarily increased insulin resistance. Gestational diabetes occurs when pancreatic beta cell function is unable to compensate for this insulin resistance. Retinol-binding protein 4 (RBP4) could be related to insulin resistance. We hypothesised that RBP4 is elevated in gestational diabetes. METHODS: Serum RBP4, transthyretin and retinol were cross-sectionally measured in 42 women with gestational diabetes and 45 pregnant controls. Of these, 20 women with and 22 without gestational diabetes were included in an additional longitudinal study. RBP4 was determined by enzyme immunometric assay (EIA) and western blot. RESULTS: Women with gestational diabetes had lower RBP4 EIA and western blot levels than controls (median 6.8 [interquartile range, 3.9-14.3] vs 11.3 [7.8-19.9] microg/ml, p < 0.001 and 25.1 [21.7-29.6] vs 26.6 [23.5-32.2] microg/ml, p = 0.026). Transthyretin and the RBP4:transthyretin molar ratio were comparable between the groups. Serum retinol was lower (p < 0.001) and the RBP4 Western blot level: retinol molar ratio was higher in women with gestational diabetes (p = 0.044). RBP4 was not associated with the glucose or homeostasis model assessment of insulin resistance (HOMA-IR), but in gestational diabetes the RBP4:retinol molar ratio correlated with blood glucose and negatively with 2 h post-load insulin. The RBP4:transthyretin ratio correlated with HOMA-IR and fasting insulin in controls. In women with gestational diabetes RBP4 EIA and western blot levels increased after delivery. Retinol increased in both groups, while transthyretin and the RBP4:transthyretin ratio were not altered after parturition. CONCLUSIONS/INTERPRETATION: RBP4 measured by two different techniques is not elevated, but the RBP4:retinol molar ratio is higher and correlates with fasting blood glucose in women with gestational diabetes. Thus, the RBP4:retinol ratio and the RBP4:transthyretin ratio are more informative than RBP4 levels alone when assessing insulin-glucose homeostasis during pregnancy.
Asunto(s)
Diabetes Gestacional/sangre , Homeostasis/fisiología , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Adulto , Glucemia/metabolismo , Western Blotting , Estudios Transversales , Femenino , Humanos , Técnicas para Inmunoenzimas , Insulina/sangre , Resistencia a la Insulina , Estudios Longitudinales , Prealbúmina/metabolismo , Embarazo , Vitamina A/sangreRESUMEN
CONTEXT: High levels of circulating retinol-binding protein 4 (RBP4) and baseline expression of adipogenic genes correlate with subsequent improvement in insulin sensitivity following Thiazolidinedione (TZD) treatment. OBJECTIVE: The aim was to identify baseline characteristics and early changes related to TZD treatment that could predict a good treatment response. DESIGN: Subjects were examined with oral glucose tolerance test, intravenous glucose tolerance test, hyperinsulinaemic euglycaemic clamp, body composition and standard blood sampling at baseline and after 4 and 12 weeks treatment. Subcutaneous adipose tissue biopsies were taken from the abdominal region at baseline, after 3 days and 4 weeks treatment to examine the gene expression profile. SETTING: Research laboratory in a University hospital. PARTICIPANTS: Ten newly diagnosed and previously untreated type 2 diabetic subjects were treated with pioglitazone for 3 months. MAIN OUTCOME MEASURES: Baseline characteristics and early changes related to TZD treatment that could predict the response after 3 months. RESULTS: Pioglitazone improved insulin sensitivity after 4 weeks combined with lower glucose and insulin levels without any change in BMI. It was accompanied by lower circulating resistin and plasminogen activator inhibitor-1 levels rapidly increased levels of circulating total and high molecular weight adiponectin as well as adiponectin and adipocyte fatty acid-binding protein (aP2) mRNA expression in the adipose tissue. High levels of circulating RBP4 at baseline and adipose tissue expression of aP2, proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1alpha) and uncoupling protein 2 (UCP-2) predicted a good treatment response measured as improvement in insulin-stimulated whole-body glucose uptake after 3 months. CONCLUSIONS: Circulating levels of RBP4 as an index of insulin sensitivity and mRNA levels of adipogenic genes correlate with the subsequent improvement in insulin sensitivity following TZD treatment.
Asunto(s)
Proteínas Portadoras/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Canales Iónicos/sangre , Proteínas Mitocondriales/sangre , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Tiazolidinedionas/uso terapéutico , Tejido Adiposo/metabolismo , Composición Corporal , Diabetes Mellitus Tipo 2/sangre , Femenino , Humanos , Resistencia a la Insulina/fisiología , Masculino , Persona de Mediana Edad , Pioglitazona , ARN Mensajero/sangre , Proteínas de Unión al ARN , Resultado del Tratamiento , Proteína Desacopladora 2RESUMEN
Insulin resistance plays a major role in the pathogenesis of type 2 diabetes. Insulin regulates blood glucose levels primarily by promoting glucose uptake from the blood into multiple tissues and by suppressing glucose production from the liver. The glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in muscle and adipose tissue. Decreased GLUT4 expression in adipose tissue is a common feature of many insulin resistant states. GLUT4 expression is preserved in skeletal muscle in many insulin resistant states. However, functional defects in the intracellular trafficking and plasma membrane translocation of GLUT4 result in impaired insulin-stimulated glucose uptake in muscle. Tissue-specific genetic knockout of GLUT4 expression in adipose tissue or muscle of mice has provided new insights into the pathogenesis of insulin resistance. We recently determined that the expression of serum retinol binding protein (RBP4) is induced in adipose tissue as a consequence of decreased GLUT4 expression. We found that RBP4 is elevated in the serum of insulin resistant humans and mice. Furthermore, we found that increasing serum RBP4 levels by transgenic overexpression or by injection of purified RBP4 protein into normal mice causes insulin resistance. Therefore, RBP4 appears to play an important role in mediating adipose tissue communication with other insulin target tissues in insulin resistant states.
Asunto(s)
Comunicación Celular/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Obesidad/genética , Obesidad/metabolismo , Animales , Transporte Biológico/genética , Comunicación Celular/fisiología , Diabetes Mellitus Tipo 2/fisiopatología , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Resistencia a la Insulina/genética , Modelos Biológicos , Obesidad/fisiopatología , Especificidad de Órganos , Proteínas Plasmáticas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Proteínas Plasmáticas de Unión al Retinol/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Levels of retinol binding protein (RBP4) are increased in the serum of insulin-resistant human subjects even before overt diabetes develops. RBP4 levels correlate with insulin resistance, BMI, WHR, dyslipidaemia and hypertension. Improvement of insulin sensitivity with exercise training is associated with reduction in serum RBP4 levels. Therefore serum RBP4 may be useful for early diagnosis of insulin resistance and for monitoring improvements in insulin sensitivity. We sought to determine the performance of assays for this application. SUBJECTS AND METHODS: We compared quantitative western blotting and three commercially available multiwell immunoassays in parallel measurements of RBP4 concentrations in serum from insulin-sensitive subjects and from insulin-resistant subjects with impaired glucose tolerance or type 2 diabetes. RESULTS: The assays yielded different absolute values and magnitudes of elevation of serum RBP4. Western blotting and a sandwich ELISA reported RBP4 concentrations that highly inversely correlated with insulin sensitivity measured by euglycaemic-hyperinsulinaemic clamp. However, western blotting yielded concentrations with a greater dynamic range and less overlap between control and insulin-resistant subjects. Two competitive enzyme-linked immunoassays undervalued serum RBP4 concentrations in insulin-resistant subjects, possibly due to assay saturation. Poor linearity of dilution also limited assay utility. All assays tested exhibited greater immunoreactivity with urinary (C-terminal proteolysed) RBP4 than with full-length RBP4, the predominant form in serum. CONCLUSIONS/INTERPRETATIONS: These findings support the use of quantitative western blotting standardised to full-length RBP4 protein as a 'gold standard' method for measuring serum RBP4 in insulin-resistant states. Other assays should use full-length RBP4 and be extensively cross-validated using other methods.
Asunto(s)
Inmunoensayo/instrumentación , Resistencia a la Insulina , Insulina/metabolismo , Proteínas de Unión al Retinol/análisis , Proteínas de Unión al Retinol/biosíntesis , Glucemia/análisis , Western Blotting , Diabetes Mellitus Tipo 2/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Prueba de Tolerancia a la Glucosa , Humanos , Inmunoensayo/métodos , Insulina/sangre , Síndrome Metabólico/metabolismo , Obesidad/sangre , Obesidad/metabolismo , Reproducibilidad de los Resultados , Proteínas Plasmáticas de Unión al Retinol , Vitamina A/metabolismoRESUMEN
Leptin regulates energy homoeostasis through central and peripheral mechanisms. Initial steps in leptin action include signalling through a cytokine-like receptor which activates the JAK/STAT pathway. We investigated whether the metabolic effects of leptin in muscle could be mediated by the AMP-activated protein kinase (AMP kinase). Through studies involving leptin injection intrahypothalamically or intravenously, as well as incubation of soleus muscle or cultured muscle cells with leptin, we determined that leptin stimulates fatty acid oxidation in skeletal muscle by activating AMP kinase. Leptin exerts this effect directly at the level of muscle and also through the hypothalamic sympathetic nervous system, specifically engaging alpha-adrenergic receptors in muscle. This represents a novel and important pathway mediating leptin's metabolic actions.
Asunto(s)
Ácidos Grasos/metabolismo , Leptina/metabolismo , Complejos Multienzimáticos/fisiología , Músculos/metabolismo , Oxígeno/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Quinasas Activadas por AMP , Animales , Humanos , Hipotálamo/metabolismo , Leptina/fisiología , Modelos Biológicos , Músculo Esquelético/metabolismo , Transducción de Señal , Sistema Nervioso Simpático/metabolismo , Factores de TiempoRESUMEN
Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas/fisiología , Acetil-CoA Carboxilasa/metabolismo , Adiponectina , Animales , Activación Enzimática , Hepatocitos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Oxidación-Reducción , FosforilaciónRESUMEN
This study investigated the metabolic changes with age in the Fischer 344 x Brown Norway rat and its suitability as an animal model of postmaturational insulin resistance. Specifically, we determined whether an age-associated decrease in glucose disposal is associated with diminished whole body insulin responsiveness and/or a decrease in glucose transporter (GLUT-4) protein and mRNA content in medial gastrocnemius muscle of male Fischer 344 x Brown Norway rats of ages 8, 18, and 28 months. Fasting plasma glucose was unchanged with age. There was a significant age effect on visceral adiposity, fasting plasma insulin levels, insulin responsiveness, and GLUT-4 protein content. Insulin responsiveness and GLUT-4 protein were lower in the 18-month-old rats than in the 8-month-old rats. The findings of age-associated increases in visceral adiposity and insulin resistance, and decreases in GLUT-4 in the Fisher 344 x Brown Norway rat, suggest that this rat strain may be an appropriate model for studying the effects of aging on glucose homeostasis.
Asunto(s)
Envejecimiento/metabolismo , Resistencia a la Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Tejido Adiposo/patología , Envejecimiento/genética , Envejecimiento/patología , Animales , Glucemia/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Homeostasis , Insulina/sangre , Masculino , Modelos Animales , Proteínas de Transporte de Monosacáridos/genética , Obesidad/genética , Obesidad/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344RESUMEN
Using cre/loxP gene targeting, transgenic mice with muscle-specific inactivation of the GLUT4 gene (muscle GLUT4 KO) were generated and shown to develop a diabetes phenotype. To determine the mechanism, we examined insulin-stimulated glucose uptake and metabolism during hyperinsulinemic-euglycemic clamp in control and muscle GLUT4 KO mice before and after development of diabetes. Insulin-stimulated whole body glucose uptake was decreased by 55% in muscle GLUT4 KO mice, an effect that could be attributed to a 92% decrease in insulin-stimulated muscle glucose uptake. Surprisingly, insulin's ability to stimulate adipose tissue glucose uptake and suppress hepatic glucose production was significantly impaired in muscle GLUT4 KO mice. To address whether these latter changes were caused by glucose toxicity, we treated muscle GLUT4 KO mice with phloridzin to prevent hyperglycemia and found that insulin-stimulated whole body and skeletal muscle glucose uptake were decreased substantially, whereas insulin-stimulated glucose uptake in adipose tissue and suppression of hepatic glucose production were normal after phloridzin treatment. In conclusion, these findings demonstrate that a primary defect in muscle glucose transport can lead to secondary defects in insulin action in adipose tissue and liver due to glucose toxicity. These secondary defects contribute to insulin resistance and to the development of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Glucosa/toxicidad , Resistencia a la Insulina/genética , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares/genética , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Edad de Inicio , Animales , Depresión Química , Modelos Animales de Enfermedad , Glucosa/farmacocinética , Transportador de Glucosa de Tipo 4 , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/prevención & control , Insulina/administración & dosificación , Insulina/farmacología , Insulina/uso terapéutico , Sistemas de Infusión de Insulina , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas de Transporte de Monosacáridos/deficiencia , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares/deficiencia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Florizina/farmacología , Florizina/uso terapéutico , Estado Prediabético/tratamiento farmacológico , Estado Prediabético/metabolismo , Transporte de Proteínas/efectos de los fármacosRESUMEN
A peer-verified gas chromatographic (GC) method is presented for the weight percent (wt %) determination of acetochlor herbicide in technical and formulated products. During method development, the method was found to be rugged by the Youden Ruggedness Test. Two laboratories with experience in the wt % determination of acetochlor in various matrixes participated in this study. Each laboratory received 10 blind duplicate test samples of the following 5 matrixes: one acetochlor technical and 4, different, emulsifiable concentrate (EC) formulations--Harness EC (MON 5841), Harness Export/Fist (MON 8435), Surpass EC (HF), and Surpass EC (LF). Each participant was asked to make duplicate weighings of each of the test samples and to inject each test sample solution twice. All test samples were analyzed on the same day, and 8 data points (replicates) per matrix were obtained. The test samples were dissolved in acetone that contained dipentyl phthalate as an internal standard. They were analyzed by GC on a 15 m capillary column by using split injection and a flame ionization detector. Acetochlor (wt %) was determined by comparing the ratios of peak area of acetochlor/peak area of dipentyl phthalate internal standard obtained for the test sample and calibration solutions. Repeatability of the method, expressed as the within-laboratory (between replicates) relative standard deviation (RSDr), was found to be 0.09-0.77% for the 5 matrixes. Reproducibility of the method, expressed as the within-test sample relative standard deviation (RSDR), was found to be 0.18-0.78% for the
Asunto(s)
Herbicidas/análisis , Toluidinas/análisis , Algoritmos , Cromatografía de Gases , Estabilidad de Medicamentos , Electroforesis Capilar , Indicadores y Reactivos , Control de Calidad , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39-50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3' kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34-57%, and association of p85alpha with both IRS proteins was reduced by 39-52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.
Asunto(s)
Resistencia a la Insulina/genética , Músculos/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Glucemia/metabolismo , Composición Corporal , Creatina Quinasa/genética , Forma MM de la Creatina-Quinasa , Ácidos Grasos no Esterificados/metabolismo , Humanos , Insulina/sangre , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/genética , Ratones , Ratones Transgénicos , Músculos/efectos de los fármacos , Músculos/metabolismo , Especificidad de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.
Asunto(s)
División Celular/fisiología , Glucosa/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Adenoviridae , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Activación Enzimática/efectos de los fármacos , Vectores Genéticos , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Islotes Pancreáticos , Cinética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Timidina/metabolismo , TransfecciónRESUMEN
UCP3 is a mitochondrial membrane protein expressed in humans selectively in skeletal muscle. To determine the mechanisms by which UCP3 plays a role in regulating glucose metabolism, we expressed human UCP3 in L6 myotubes by adenovirus-mediated gene transfer and in H(9)C(2) cardiomyoblasts by stable transfection with a tetracycline-repressible UCP3 construct. Expression of UCP3 in L6 myotubes increased 2-deoxyglucose uptake 2-fold and cell surface GLUT4 2.3-fold, thereby reaching maximally insulin-stimulated levels in control myotubes. Wortmannin, LY 294002, or the tyrosine kinase inhibitor genistein abolished the effect of UCP3 on glucose uptake, and wortmannin inhibited UCP3-induced GLUT4 cell surface recruitment. UCP3 overexpression increased phosphotyrosine-associated phosphoinositide 3-kinase (PI3K) activity 2.2-fold compared with control cells (p < 0.05). UCP3 overexpression increased lactate release 1.5- to 2-fold above control cells, indicating increased glucose metabolism. In H(9)C(2) cardiomyoblasts stably transfected with UCP3 under control of a tetracycline-repressible promotor, removal of doxycycline resulted in detectable levels of UCP3 at 12 h and 2.2-fold induction at 7 days compared with 12 h. In parallel, glucose transport increased 1.3- and 2-fold at 12 h and 7 days, respectively, and the stimulation was inhibited by wortmannin or genistein. p85 association with membranes was increased 5.5-fold and phosphotyrosine-associated PI3K activity 3.8-fold. In contrast, overexpression of UCP3 in 3T3-L1 adipocytes did not alter glucose uptake, suggesting tissue-specific effects of human UCP3. Thus, UCP3 stimulates glucose transport and GLUT4 translocation to the cell surface in cardiac and skeletal muscle cells by activating a PI3K dependent pathway.
Asunto(s)
Proteínas Portadoras/metabolismo , Desoxiglucosa/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células 3T3 , Adipocitos/citología , Adipocitos/metabolismo , Androstadienos/farmacología , Animales , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Doxiciclina/farmacología , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Glucosa/metabolismo , Humanos , Insulina/farmacología , Canales Iónicos , Lactatos/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales , Músculo Esquelético/citología , Miocardio/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Proteína Desacopladora 3 , WortmaninaRESUMEN
The earliest defect in developing type 2 diabetes is insulin resistance, characterized by decreased glucose transport and metabolism in muscle and adipocytes. The glucose transporter GLUT4 mediates insulin-stimulated glucose uptake in adipocytes and muscle by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in muscle. Because skeletal muscle is the main site of insulin-stimulated glucose uptake, the role of adipose tissue GLUT4 downregulation in the pathogenesis of insulin resistance and diabetes is unclear. To determine the role of adipose GLUT4 in glucose homeostasis, we used Cre/loxP DNA recombination to generate mice with adipose-selective reduction of GLUT4 (G4A-/-). Here we show that these mice have normal growth and adipose mass despite markedly impaired insulin-stimulated glucose uptake in adipocytes. Although GLUT4 expression is preserved in muscle, these mice develop insulin resistance in muscle and liver, manifested by decreased biological responses and impaired activation of phosphoinositide-3-OH kinase. G4A-/- mice develop glucose intolerance and hyperinsulinaemia. Thus, downregulation of GLUT4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes.
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Adipocitos/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Biológico , Cruzamientos Genéticos , Diabetes Mellitus/metabolismo , Regulación hacia Abajo , Femenino , Marcación de Gen , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Resistencia a la Insulina , Masculino , Ratones , Proteínas de Transporte de Monosacáridos/genéticaAsunto(s)
Resistencia a la Insulina/fisiología , Obesidad/fisiopatología , Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Tejido Adiposo/fisiopatología , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Glucosa/metabolismo , Homeostasis , Humanos , Insulina/farmacología , Insulina/fisiología , Ratones , Modelos Biológicos , Transducción de SeñalRESUMEN
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.
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Resistencia a la Insulina/fisiología , Proteínas de Transporte de Monosacáridos/deficiencia , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Músculo Esquelético/metabolismo , Animales , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Cartilla de ADN/genética , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Humanos , Técnicas In Vitro , Insulina/farmacología , Resistencia a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte de Monosacáridos/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/efectos de los fármacosRESUMEN
To determine whether impaired Akt (protein kinase B or rac) activation contributes to insulin resistance in vivo, we examined the expression, phosphorylation, and kinase activities of Akt1 and Akt2 isoforms in insulin target tissues of insulin-resistant obese Zucker rats. In lean rats, insulin (10 U/kg i.v. x 2.5 min) stimulated Akt1 activity 6.2-, 8.8-, and 4.4-fold and Akt2 activity 5.4-, 9.3-, and 1.8-fold in muscle, liver, and adipose tissue, respectively. In obese rats, insulin-stimulated Akt1 activity decreased 30% in muscle and 21% in adipose tissue but increased 37% in liver compared with lean littermates. Insulin-stimulated Akt2 activity decreased 29% in muscle and 37% in liver but increased 24% in adipose tissue. Akt2 protein levels were reduced 56% in muscle and 35% in liver of obese rats, but Akt1 expression was unaltered. Phosphoinositide 3-kinase (PI3K) activity associated with insulin receptor substrate (IRS)-1 or phosphotyrosine was reduced 67-86% in tissues of obese rats because of lower IRS-1 protein levels and reduced insulin receptor and IRS-1 phosphorylation. In adipose tissue of obese rats, in spite of an 86% reduction in insulin-stimulated PI3K activity, activation of Akt2 was increased. Maximal insulin-stimulated (100 nmol/l) glucose transport was reduced 70% in isolated adipocytes, with a rightward shift in the insulin dose response for transport and for Akt1 stimulation but normal sensitivity for Akt2. These findings suggest that PI3K-dependent effects on glucose transport in adipocytes are not mediated primarily by Akt2. Akt1 and Akt2 activations by insulin have a similar time course and are maximal by 2.5 min in adipocytes of both lean and obese rats. We conclude that 1) activation of Akt1 and Akt2 in vivo is much less impaired than activation of PI3K in this insulin-resistant state, and 2) the mechanisms for divergent alterations in insulin action on Akt1 and Akt2 activities in tissues of insulin-resistant obese rats involve tissue- and isoform-specific changes in both expression and activation.
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Insulina/fisiología , Isoenzimas/metabolismo , Obesidad/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tejido Adiposo/metabolismo , Animales , Transporte Biológico , Femenino , Glucosa/metabolismo , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Zucker , Valores de Referencia , Distribución Tisular , Tirosina/metabolismoRESUMEN
AIMS/HYPOTHESIS: The glycogen-associated protein phosphatase-1 (PP1G) is thought to play an important part in the regulation of skeletal muscle glycogen content. We have previously identified an Asp905Tyr polymorphism of the glycogen-associated regulatory subunit of the protein phosphatase 1 (PPP1R3) gene which among healthy subjects was associated with decreased insulin stimulated non-oxidative glucose metabolism, i.e. primary glycogen synthesis. In this study, the functional effect of the polymorphism was examined in vitro. METHODS: Wild type (PPP1R3-Asp905) and mutant (PPP1R3-Tyr905) PPP1R3 were expressed in L6 myotubes using adenovirus-mediated gene transfer. Basal and insulin-stimulated glucose uptake and glycogen synthesis were measured. Furthermore, the sensitivity of glycogen synthesis to a cyclic AMP agonist was measured. RESULTS: Compared with green fluorescent protein-transduced myotubes and non-transduced myotubes, overexpression of PPP1R3-Asp905 and PPP1R3-Tyr905 increased both basal and insulin-stimulated glycogen synthesis approximately twofold. Treatment of both non-transduced and PPP1R3-transduced L6 myotubes with a cAMP agonist decreased both basal and insulin-stimulated glycogen synthesis by about 40%. Overexpression of PPP1R3 did not affect either basal or insulin-stimulated 2-deoxy-D-glucose uptake compared with green fluorescent protein-transduced cells. CONCLUSION/INTERPRETATION: Results obtained from L6 myotubes transduced with PPP1R3-Asp905 or PPP1R3-Tyr905 showed no statistically significant difference. Therefore, the Asp905Tyr variant alone is unlikely to account for the decreased insulin stimulated non-oxidative glucose metabolism observed in the human study reported previously.
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Variación Genética , Músculo Esquelético/metabolismo , Fosfoproteínas Fosfatasas/genética , Adenoviridae , Animales , Ácido Aspártico , Transporte Biológico , Células CHO , Línea Celular , Cricetinae , Desoxiglucosa/metabolismo , Vectores Genéticos , Glucógeno/biosíntesis , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/análisis , Músculo Esquelético/citología , Fosfoproteínas Fosfatasas/biosíntesis , Polimorfismo Genético , Proteína Fosfatasa 1 , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , TirosinaRESUMEN
Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.
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Tejido Adiposo/fisiología , Metabolismo Energético , Resistencia a la Insulina/genética , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas Tirosina Fosfatasas/deficiencia , Animales , Peso Corporal/genética , Proteínas Portadoras/genética , Femenino , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Homeostasis , Hiperinsulinismo/metabolismo , Canales Iónicos , Leptina/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Esquelético/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/genética , Proteínas/genética , ARN Mensajero , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
Obesity and insulin resistance in skeletal muscle are two major factors in the pathogenesis of type 2 diabetes. Mice with muscle-specific inactivation of the insulin receptor gene (MIRKO) are normoglycemic but have increased fat mass. To identify the potential mechanism for this important association, we examined insulin action in specific tissues of MIRKO and control mice under hyperinsulinemic-euglycemic conditions. We found that insulin-stimulated muscle glucose transport and glycogen synthesis were decreased by about 80% in MIRKO mice, whereas insulin-stimulated fat glucose transport was increased threefold in MIRKO mice. These data demonstrate that selective insulin resistance in muscle promotes redistribution of substrates to adipose tissue thereby contributing to increased adiposity and development of the prediabetic syndrome.