RESUMEN
Myopathy is a group of muscle diseases that can be induced or exacerbated by drug-drug interactions (DDIs). We sought to identify clinically important myopathic DDIs and elucidate their underlying mechanisms. Five DDIs were found to increase the risk of myopathy based on analysis of observational data from the Indiana Network of Patient Care. Loratadine interacted with simvastatin (relative risk 95% confidence interval [CI] = [1.39, 2.06]), alprazolam (1.50, 2.31), ropinirole (2.06, 5.00), and omeprazole (1.15, 1.38). Promethazine interacted with tegaserod (1.94, 4.64). In vitro investigation showed that these DDIs were unlikely to result from inhibition of drug metabolism by CYP450 enzymes or from inhibition of hepatic uptake via the membrane transporter OATP1B1/1B3. However, we did observe in vitro synergistic myotoxicity of simvastatin and desloratadine, suggesting a role in loratadine-simvastatin interaction. This interaction was epidemiologically confirmed (odds ratio 95% CI = [2.02, 3.65]) using the data from the US Food and Drug Administration Adverse Event Reporting System.
Asunto(s)
Interacciones Farmacológicas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Músculo Esquelético/efectos de los fármacos , Enfermedades Musculares/inducido químicamente , Investigación Biomédica Traslacional/métodos , Sistemas de Registro de Reacción Adversa a Medicamentos , Animales , Transporte Biológico , Biotransformación , Línea Celular , Inhibidores Enzimáticos del Citocromo P-450/efectos adversos , Inhibidores Enzimáticos del Citocromo P-450/farmacocinética , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/epidemiología , Enfermedades Musculares/metabolismo , Oportunidad Relativa , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Ratas , Medición de Riesgo , Factores de Riesgo , Estados Unidos/epidemiología , United States Food and Drug AdministrationRESUMEN
AIMS/HYPOTHESIS: Diminished cortical filamentous actin (F-actin) has been implicated in skeletal muscle insulin resistance, yet the mechanism(s) is unknown. Here we tested the hypothesis that changes in membrane cholesterol could be a causative factor, as organised F-actin structure emanates from cholesterol-enriched raft microdomains at the plasma membrane. METHODS: Skeletal muscle samples from high-fat-fed animals and insulin-sensitive and insulin-resistant human participants were evaluated. The study also used L6 myotubes to directly determine the impact of fatty acids (FAs) on membrane/cytoskeletal variables and insulin action. RESULTS: High-fat-fed insulin-resistant animals displayed elevated levels of membrane cholesterol and reduced F-actin structure compared with normal chow-fed animals. Moreover, human muscle biopsies revealed an inverse correlation between membrane cholesterol and whole-body glucose disposal. Palmitate-induced insulin-resistant myotubes displayed membrane cholesterol accrual and F-actin loss. Cholesterol lowering protected against the palmitate-induced defects, whereas characteristically measured defects in insulin signalling were not corrected. Conversely, cholesterol loading of L6 myotube membranes provoked a palmitate-like cytoskeletal/GLUT4 derangement. Mechanistically, we observed a palmitate-induced increase in O-linked glycosylation, an end-product of the hexosamine biosynthesis pathway (HBP). Consistent with HBP activity affecting the transcription of various genes, we observed an increase in Hmgcr, a gene that encodes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. In line with increased HBP activity transcriptionally provoking a membrane cholesterol-based insulin-resistant state, HBP inhibition attenuated Hmgcr expression and prevented membrane cholesterol accrual, F-actin loss and GLUT4/glucose transport dysfunction. CONCLUSIONS/INTERPRETATION: Our results suggest a novel cholesterolgenic-based mechanism of FA-induced membrane/cytoskeletal disorder and insulin resistance.
Asunto(s)
Actinas/metabolismo , Colesterol/metabolismo , Glucosa/metabolismo , Adulto , Animales , Transporte Biológico , Biopsia con Aguja/métodos , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Ácidos Grasos/metabolismo , Femenino , Humanos , Insulina/metabolismo , Masculino , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Ácido Palmítico/metabolismo , RatasRESUMEN
Nmp4 proteins are transcription factors that contribute to the expression of type I collagen and many of the matrix metalloproteinase genes. Numerous Nmp4 isoforms have been identified. These proteins, all derived from a single gene, have from five to eight Cys(2)His(2) zinc fingers, the arrangement of which directs specific isoforms to nuclear matrix subdomains. Nmp4 isoforms also have an SH3 binding domain, typical of cytoplasmic docking proteins. Although recent evidence indicates that Nmp4 proteins also reside in the osteoblast cytoplasm, whether they localize to specific organelles or structures is not well defined. The intracellular localization of a protein is a determinant of its function and provides insights into its mechanism of action. As a first step toward determining the functional relationship between the cytoplasmic and nuclear Nmp4 compartments, we mapped their location in the osteoblast cytoplasm. Immunocytochemical analysis of osteoblasts demonstrated that Nmp4 antibodies labeled the mitochondria, colocalized with Golgi protein 58K, and lightly stained the cytoplasm. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these proteins were specific to defined intracellular domains. Consistent with the western analyses, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected previously uncharacterized Nmp4 isoforms. These data necessarily enlarge the known Nmp4 family from nuclear matrix transcription factors to a more widely extended class of intracellular proteins.
Asunto(s)
Líquido Intracelular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo , Células 3T3/química , Células 3T3/metabolismo , Animales , Animales Recién Nacidos , Citoplasma/química , Citoplasma/metabolismo , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Líquido Intracelular/química , Masculino , Ratones , Mitocondrias/química , Mitocondrias/metabolismo , Proteínas Asociadas a Matriz Nuclear/biosíntesis , Osteoblastos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/biosíntesisAsunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Transducción de Señal/fisiología , Adipocitos/metabolismo , Transporte Biológico Activo , Proteínas Portadoras , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4 , Músculo Esquelético/metabolismo , Transporte de ProteínasRESUMEN
It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.
Asunto(s)
Calmodulina/antagonistas & inhibidores , Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinasas , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Ratones , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Sulfonamidas/farmacología , Trifluoperazina/farmacologíaRESUMEN
Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.
Asunto(s)
Adipocitos/metabolismo , Aminopeptidasas/metabolismo , Insulina/farmacología , Fusión de Membrana , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Células 3T3 , Adipocitos/química , Animales , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4 , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/química , Proteínas Recombinantes de Fusión/metabolismo , TemperaturaRESUMEN
Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Insulina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Línea Celular , Clonación Molecular , Cricetinae , Genes Dominantes , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Orgánulos/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Levaduras/genéticaAsunto(s)
Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Compartimento Celular , Endocitosis , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas SNARE , Vesículas Sinápticas/metabolismoRESUMEN
Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.
Asunto(s)
Adipocitos/efectos de los fármacos , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Células 3T3 , Adipocitos/metabolismo , Animales , Unión Competitiva , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Antagonistas de Insulina/farmacología , Proteína Antagonista del Receptor de Interleucina 1 , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Munc18 , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Proteínas SNARE , Sialoglicoproteínas/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTPgammaS-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTPgammaS was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5'-O-2-(thio)diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTPgammaS- and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTPgammaS did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120-130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTPgammaS stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAKbeta, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTPgammaS-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.
Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Androstadienos/farmacología , Animales , Benzoquinonas , Transporte Biológico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Lactamas Macrocíclicas , Ratones , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Rifabutina/análogos & derivados , WortmaninaRESUMEN
Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin- and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130(cas), and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125(FAK) and p130(cas). These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.
Asunto(s)
Adipocitos/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Adipocitos/efectos de los fármacos , Androstadienos/farmacología , Animales , Anticuerpos/farmacología , Transporte Biológico/efectos de los fármacos , Membrana Celular/metabolismo , Desoxiglucosa/metabolismo , Genisteína/farmacología , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Proteínas de la Membrana/biosíntesis , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Concentración Osmolar , Fosfotirosina/inmunología , Fosfotirosina/metabolismo , Proteínas Qa-SNARE , Sorbitol/farmacología , Wortmanina , Xilosa/farmacologíaRESUMEN
Preincubation of rat soleus muscle with 1 and 10 microM monensin for 2 h increased the subsequent basal 2-deoxyglucose uptake by muscle 76 and 121% respectively. Under the same conditions, monensin decreased the insulin-stimulated (1 mU/ml) 2-deoxyglucose uptake by 29 and 37% respectively. The monensin-induced augmentation of basal 2-deoxyglucose uptake was inhibited 92% by cytochalasin B suggesting that the uptake is mediated by glucose transporters. Monensin did not increase the cellular accumulation of L-glucose in muscle indicating that it does not affect the cell membrane integrity. Neither the stimulatory effect of monensin on basal 2-deoxyglucose uptake nor the opposite, inhibitory action of monensin on the insulin-stimulated 2-deoxyglucose uptake were influenced by the removal of Ca2+ from the medium or by dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, suggesting that the actions of monensin are not mediated by calcium. Monensin had no effect on muscle ATP concentration. The monensin-induced augmentation of basal 2-deoxyglucose uptake was neither associated with stimulation of muscle phosphatidylinositol 3-kinase activity nor inhibited by wortmannin, demonstrating that the increase in basal 2-deoxyglucose uptake is not mediated by activation of phosphatidylinositol 3-kinase. The inhibition of insulin-stimulated 2-deoxyglucose uptake by monensin was associated with a 31% decrease in the abundance of insulin receptors in muscles, a 64% decrease in the insulin-induced autophosphorylation of the insulin receptor beta-subunit, and a 44% reduction of the insulin-stimulated phosphatidylinositol 3-kinase activity. Addition of monensin into the phosphatidylinositol 3-kinase reaction had no effect on the activity of the enzyme, demonstrating that the inhibition in monensin-treated muscles is indirect and occurs upstream of phosphatidylinositol 3-kinase. It is concluded that monensin has a dual effect on 2-deoxyglucose uptake by skeletal muscle: it stimulates basal uptake but inhibits the insulin-stimulated uptake. The primary cause of the latter, inhibitory effect of monensin is at the level of the insulin receptor.
Asunto(s)
Desoxiglucosa/metabolismo , Ionóforos/farmacología , Monensina/farmacología , Músculo Esquelético/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor de Insulina/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Androstadienos/farmacología , Animales , Citocalasina B/farmacología , Dantroleno/farmacología , Glucosa/metabolismo , Insulina/farmacología , Antagonistas de Insulina/farmacología , Masculino , Relajantes Musculares Centrales/farmacología , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinasas , Ratas , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Estimulación Química , WortmaninaRESUMEN
Treatment of rats with growth hormone (GH; 1 mg/kg sc) twice daily over 2.5 days did not alter fasting plasma glucose or glucose tolerance but increased fasting plasma insulin levels 65% and peak insulin response to a glucose load 35% over controls, indicating the development of insulin resistance. Studies on partially purified insulin receptors from soleus muscles showed that GH increased the abundance of insulin receptor beta-subunits by 48% as measured by immunoblotting. Despite this increase, GH abolished the increase in autophosphorylation of the insulin receptor beta-subunit in response to physiological hyperinsulinemia and diminished by 28% the response to supraphysiological hyperinsulinemia. Similarly, insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) was decreased 25% by GH, but the abundance of IRS-1 was not affected. Studies on rats pretreated with streptozotocin suggested that the effects of GH are direct and not secondary to GH-induced hyperinsulinemia. GH decreased basal GLUT-1 abundance in the low-density microsome and plasma membrane fractions of epididymal adipocytes by 50 and 42%, respectively, but decreased basal GLUT-4 abundance only in the low-density microsome fraction by 24%. Despite these alterations, the abundance of both transporters in the plasma membrane fraction of adipocytes incubated with 0.1 U insulin/ml was not diminished by GH.
Asunto(s)
Adipocitos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Hormona del Crecimiento/farmacología , Resistencia a la Insulina , Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/biosíntesis , Receptor de Insulina/metabolismo , Adipocitos/efectos de los fármacos , Animales , Glucemia/metabolismo , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina , Secreción de Insulina , Masculino , Músculo Esquelético/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Regulation of glucose uptake by 1- and 3-day denervated soleus (slow-twitch) and plantaris (fast-twitch) muscles in vivo was investigated. One day after denervation, soleus and plantaris muscles exhibited 62 and 65% decreases in insulin-stimulated 2-deoxyglucose uptake, respectively, compared with corresponding control muscles. At this interval, denervated muscles showed no alterations in insulin receptor binding and activity, amount and activity of phosphatidylinositol 3-kinase, and amounts of GLUT-1 and GLUT-4. Three days after denervation, there was no increase in 2-deoxyglucose uptake in response to insulin in soleus muscle, whereas plantaris muscle exhibited a 158% increase in basal and an almost normal absolute increment in insulin-stimulated uptake. Despite these differences, denervated soleus and plantaris muscles exhibited comparable decreases in insulin-stimulated activities of the insulin receptor (approximately 40%) and phosphatidylinositol 3-kinase (approximately 50%) and a pronounced decrease in GLUT-4. An increase in GLUT-1 in plantaris, but not soleus, muscle 3 days after denervation is consistent with augmented basal 2-deoxyglucose uptake in plantaris muscle at this interval. These results demonstrate that, in denervated muscles, there is a clear dissociation between insulin-stimulated 2-deoxyglucose uptake and upstream events involved in insulin-stimulated glucose uptake.
Asunto(s)
Resistencia a la Insulina , Desnervación Muscular , Proteínas Musculares , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Desoxiglucosa/farmacocinética , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Miembro Posterior , Masculino , Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares de Contracción Rápida/enzimología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/enzimología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/enzimología , Fosfatidilinositol 3-Quinasas , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/metabolismo , Factores de TiempoRESUMEN
The effects of sphingomyelinase, phosphorylcholine, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide) and sphingosine on basal and insulin-stimulated cellular accumulation of 2-deoxy-D-glucose in rat soleus muscles were investigated. Preincubation of muscles with sphingomyelinase (100 or 200 m-units/ml) for 1 or 2 h augmented basal 2-deoxyglucose uptake by 29-91%, and that at 0.1 and 1.0 m-unit of insulin/ml 32-82% and 19-25% respectively compared with control muscles studied at the same insulin concentrations. The sphingomyelinase-induced increase in basal and insulin-stimulated 2-deoxyglucose uptake was inhibited by 91% by 70 microM cytochalasin B, suggesting that it involves glucose transporters. Sphingomyelinase had no effect on the cellular accumulation of L-glucose, which is not transported by glucose transporters. The sphingomyelinase-induced increase in 2-deoxyglucose uptake could not be reproduced by preincubating the muscles with 50 microM phosphorylcholine, 50 microM C2-ceramide or 50 microM C6-ceramide. Preincubation of muscles with 50 microM sphingosine augmented basal 2-deoxyglucose transport by 32%, but reduced the response to 0.1 and 1.0 m-unit of insulin/ml by 17 and 27% respectively. The stimulatory effect of sphingomyelinase on basal and insulin-induced 2-deoxyglucose uptake was not influenced by either removal of Ca2+ from the incubation medium or dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum. This demonstrates that Ca2+ does not mediate the action of sphingomyelinase on 2-deoxyglucose uptake. Sphingomyelinase also had no effect on basal and insulin-stimulated activities of insulin receptor tyrosine kinase and phosphatidylinositol 3-kinase. In addition, 1 and 5 microM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, failed to inhibit the sphingomyelinase-induced increase in 2-deoxyglucose uptake. These results suggest that sphingomyelinase does not increase 2-deoxyglucose uptake by stimulating the insulin receptor or the initial steps of the insulin-transduction pathway. The data suggest the possibility that sphingomyelinase increases basal and insulin-stimulated 2-deoxyglucose uptake in skeletal muscle as the result of an unknown post-receptor effect.
Asunto(s)
Desoxiglucosa/farmacocinética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Esfingomielina Fosfodiesterasa/farmacología , Androstadienos/farmacología , Animales , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Insulina/metabolismo , Antagonistas de Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Masculino , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Transducción de Señal , Esfingomielinas/metabolismo , Estimulación Química , WortmaninaRESUMEN
To date there is suggestive evidence that phosphatidylinositol 3-kinase participates in insulin-stimulated glucose transport. However, its involvement in skeletal muscle, a major site of insulin-stimulated glucose disposal, has not been addressed. Therefore, we tested the effects of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated 2-deoxyglucose uptake by rat soleus muscle in vitro: Wortmannin (1 microM) reversibly inhibited insulin-induced 2-deoxyglucose uptake in soleus muscle by 44%. Inclusion of 5 microM wortmannin in the incubation medium completely abolished the insulin-induced increment in 2-deoxyglucose uptake. In conclusion, the insulin-signaling cascade linking insulin-receptor tyrosine kinase activation to glucose uptake in skeletal muscle.