RESUMEN

AIMS/HYPOTHESIS: Induction of stress kinases leading to serine hyperphosphorylation of IRS1 may link oxidative stress to insulin resistance. The aim of this study was to investigate the roles of the phosphorylated serine residues Ser307 and Ser632, two sites implicated in the inhibition of IRS1 function in insulin signalling. MATERIALS AND METHODS: Fao hepatoma cells were exposed to an H(2)O(2)-generating system, and antibodies against the two phosphorylated serine residues were used for immunoprecipitation, immunoblot and immunofluorescence analyses. RESULTS: Exposure to approximately 50 mumol/l H(2)O(2) for 2 h resulted in IRS1 phosphorylation on both Ser307 and Ser632, concomitant with activation of inhibitor kappa kinase beta (IKKbeta) and c-Jun kinase (JNK). Immunoprecipitation studies revealed that the maximum overlap between phospho (p) Ser307-IRS1 and pSer632-IRS1 was 20%, and confocal microscopy suggested distinct localisations of IRS1 molecules phosphorylated on either site. Although pSer307-IRS1 showed decreased insulin-induced tyrosine phosphorylation and interaction with phosphatidylinositol 3-kinase (PI3K) in response to insulin, pSer632-IRS1 molecules were normally tyrosine-phosphorylated and exhibited typical associated PI3K activity. Salicylic acid and SP600125 partially inhibited IKKbeta and JNK, respectively, which indicated distinct roles for these two kinases in the phosphorylation of IRS1 at the two serine sites. Protection against oxidation-mediated impairment in insulin-induced phosphorylation of protein kinase B/Akt and in glycogen synthesis was achieved only by combining salicylic acid and SP600125. CONCLUSIONS/INTERPRETATION: These results suggest that pSer307-IRS1 and pSer632-IRS1 may define two minimally overlapping pools of IRS1 in response to oxidative stress, contributing differentially to insulin resistance. A combination of stress kinase inhibitors is required to protect against insulin resistance and IRS1 hyperphosphorylation induced by oxidative stress.

Asunto(s)

Resistencia a la Insulina/fisiología , Estrés Oxidativo/fisiología , Fosfoproteínas/metabolismo , Fosfoserina/metabolismo , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Proteínas Sustrato del Receptor de Insulina , Neoplasias Hepáticas , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas

RESUMEN

AIMS/HYPOTHESIS: Chronic exposure of 3T3-L1 adipocytes to the HIV protease inhibitor nelfinavir induces insulin resistance, recapitulating key metabolic alterations of adipose tissue in the lipodystrophy syndrome induced by these agents. Our goal was to identify the defect in the insulin signal transduction cascade leading to nelfinavir-induced insulin resistance. METHODS: Fully differentiated 3T3-L1 adipocytes were exposed to 30 micro mol/l nelfinavir for 18 h, after which the amount, the phosphorylation and the localisation of key proteins in the insulin signalling cascade were evaluated. RESULTS: Insulin-induced interaction of phosphatidylinositol 3'-kinase (PI 3-kinase) with IRS proteins was normal in cells treated with nelfinavir, as was IRS-1-associated PI 3-kinase activity. Yet insulin-induced phosphorylation of Akt/protein kinase B (PKB), p70S6 kinase and extracellular signal-regulated kinase 1/2 was significantly impaired. This could not be attributed to increased protein phosphatase 2A activity or to increased expression of phosphoinositide phosphatases (SHIP2 or PTEN). However, insulin failed to induce translocation of the PI 3-kinase effectors Akt/PKB and protein kinase C-zeta (PKC-zeta) to plasma membrane fractions of nelfinavir-treated adipocytes. CONCLUSIONS/INTERPRETATION: We therefore conclude that nelfinavir induces a defect in the insulin signalling cascade downstream of the activation of PI 3-kinase. This defect manifests itself by impaired insulin-mediated recruitment of Akt/PKB and PKC-zeta to the plasma membrane.

Asunto(s)

Membrana Celular/metabolismo , Resistencia a la Insulina , Nelfinavir/efectos adversos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Células 3T3-L1 , Animales , Membrana Celular/patología , Desoxiglucosa/antagonistas & inhibidores , Desoxiglucosa/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Glucosa/metabolismo , Japón , Ratones , Fosfatidilinositoles/química , Fosfatidilinositoles/genética , Fosfatidilinositoles/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Fosfatasa 2 , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas 70-kDa/química , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo

RESUMEN

AIM/HYPOTHESIS: Oxidative stress was shown to selectively induce impaired metabolic response to insulin, raising the possible involvement of alterations in Insulin-Receptor-Substrate (IRS) proteins. This study was conducted to assess whether oxidative stress induced IRS protein degradation and enhanced serine phosphorylation, and to assess their functional importance. METHODS: 3T3-L1 adipocytes and rat hepatoma cells (FAO) were exposed to micro-molar H(2)O(2) by adding glucose oxidase to the culture medium, and IRS1 content, its serine phosphorylation and downstream metabolic insulin effects were measured. RESULTS: Cells exposed to oxidative stress exhibited decreased IRS1 (but not IRS2) content, and increased serine phosphorylation of both proteins. Total protein ubiquitination was increased in oxidized cells, but not in cells exposed to prolonged insulin treatment. Yet, lactacystin and MG132, two unrelated proteasome inhibitors, prevented IRS1 degradation induced by prolonged insulin but not by oxidative stress. The PI 3-kinase inhibitor LY294002 and the mTOR inhibitor rapamycin, but not the MEK1 inhibitor PD98059, could prevent IRS1 changes in oxidized cells. Rapamycin, which protected against IRS1 degradation and serine phosphorylation was not associated with improved response to acute insulin stimulation. Moreover, the antioxidant alpha lipoic acid, while protecting against oxidative stress-induced insulin resistance in 3T3-L1 adipocytes, could not prevent IRS1 degradation and serine phosphorylation. CONCLUSION/INTERPRETATION: Oxidative stress induces serine phosphorylation of IRS1 and increases its degradation by a proteasome-independent pathway; yet, these changes do not correlate with the induction of impaired metabolic response to insulin.

Asunto(s)

Peróxido de Hidrógeno/farmacología , Resistencia a la Insulina/fisiología , Estrés Oxidativo/fisiología , Fosfoproteínas/metabolismo , Fosfoserina/metabolismo , Células 3T3 , Animales , Antioxidantes/farmacología , Biomarcadores , Cicloheximida/farmacología , Cisteína Endopeptidasas/metabolismo , Glucosa Oxidasa/metabolismo , Proteínas Sustrato del Receptor de Insulina , Ratones , Complejos Multienzimáticos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Serina/metabolismo , Ácido Tióctico/farmacología , Ubiquitina/metabolismo

RESUMEN

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is a common event in both insulin and platelet-derived growth factor (PDGF) signalling, but only insulin activates this enzyme in the high-speed pellet (HSP), and induces GLUT4 translocation. Recently, we have demonstrated that exposure of 3T3-L1 adipocytes to oxidative stress impairs insulin-stimulated GLUT4 translocation and glucose transport, associated with impaired PI 3-kinase translocation and activation in the HSP [Tirosh, Potashnik, Bashan and Rudich (1999) J. Biol. Chem. 274, 10595-10602]. In this study the effect of a 2 h exposure to approximately 30 microM H(2)O(2) on insulin versus PDGF-BB signalling and metabolic effects was compared. PDGF-stimulated p85-associated PI 3-kinase activity in total cell lysates, as well as co-precipitation of the PDGF receptor, were unaffected by oxidative stress. Additionally, the increase in p85 association with the plasma-membrane lawns by PDGF remained intact following oxidation, whereas the insulin effect was decreased. PDGF significantly increased protein kinase B (PKB) activity in early differentiated cells, and that of p70 S6-kinase in both early and fully differentiated 3T3-L1 adipocytes. Following oxidation the effect of PDGF on PKB and p70 S6-kinase activation remained intact, whereas significant inhibition of insulin-stimulated activation of those enzymes was observed. In accordance, in both early and fully differentiated cells, oxidative stress completely blunted insulin- but not PDGF-stimulated protein synthesis. In conclusion, oxidative stress impairs insulin, but not PDGF, signalling and metabolic actions in both early and fully differentiated 3T3-L1 adipocytes. This emphasizes compartment-specific activation of PI 3-kinase as an oxidation-sensitive step specifically leading to insulin resistance.

Asunto(s)

Adipocitos/metabolismo , Insulina/metabolismo , Estrés Oxidativo/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Células 3T3 , Adipocitos/enzimología , Animales , Activación Enzimática , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas/metabolismo , Fracciones Subcelulares/metabolismo

RESUMEN

AIMS/HYPOTHESIS: Oxidative stress has been shown to impair insulin-stimulated glucose transporter 4 translocation in 3T3-L1 adipocytes. This study explores the potential of the antioxidant lipoic acid to protect the cells against the induction of insulin resistance when given before exposure to oxidative stress. METHODS: 3T3-L1 were exposed for 16 h to lipoic acid after which cells were exposed for 2 h to continuous production of H2O2 by adding glucose oxidase to the culture medium. RESULTS: These conditions resulted in a 50-70% reduction in insulin-stimulated glucose transport activity associated with a decrease in reduced glutathione content from 37.4 +/- 3.1 to 26.4 +/- 4.9 nmol/mg protein, (p < 0.005). Lipoic acid pretreatment increased insulin-stimulated glucose transport following oxidative stress, reaching 84.8 +/- 4.4% of the control, associated with an increase in reduced glutathione content. Oxidation impaired the 4.89 +/- 0.36-fold insulin-stimulated increase in glucose transporter 4 content in plasma membrane lawns of control cells. Lipoic acid pretreatment was, however, associated with preserved insulin-induced glucose transporter 4 translocation in cells exposed to oxidation, yielding 80% of its content in controls. Although tyrosine phosphorylation patterns were not affected by lipoic acid pretreatment, insulin-stimulated protein kinase B/Akt serine 473 phosphorylation and activity were considerably impaired by oxidation but protected by lipoic acid pretreatment. A protective effect was not observed with either troglitazone, its isolated vitamin E moiety, or with vitamin C. CONCLUSION/INTERPRETATION: This study shows the ability of lipoic acid to provide partial protection against the impaired insulin-stimulated glucose transporter 4 translocation and protein kinase B/Akt activation induced by oxidative stress, potentially by its capacity to maintain intracellular redox state.

Asunto(s)

Adipocitos/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Proteínas Musculares , Estrés Oxidativo/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Tiazolidinedionas , Ácido Tióctico/farmacología , Células 3T3 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Ácido Ascórbico/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Cromanos/farmacología , Glucosa Oxidasa/farmacología , Transportador de Glucosa de Tipo 4 , Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Hipoglucemiantes/farmacología , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Tiazoles/farmacología , Troglitazona , Vitamina E/farmacología

RESUMEN

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562-1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to approximately 25 microM H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBalpha and PKBgamma. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.

Asunto(s)

Adipocitos/metabolismo , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Transporte Biológico , Compartimento Celular , Transportador de Glucosa de Tipo 4 , Humanos , Proteínas Sustrato del Receptor de Insulina , Ratones , Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt

RESUMEN

Lipoic acid (LA) is a unique antioxidant that increases peripheral glucose utilization in diabetic patients. This study was conducted to investigate whether the inhibition of glucose production could be an additional mechanism for the action of LA. Intravenous (i.v.) LA injection (100 or 60 mg/kg body weight) to fasting nondiabetic or streptozotocin (STZ)-induced diabetic rats caused a rapid reduction in blood glucose with no effect on circulating insulin levels. In vivo conversion of fructose to glucose was not inhibited by LA, whereas the gluconeogenesis flux from alanine was completely prevented. Reduced liver pyruvate carboxylase (PC) activity in vivo is suggested by the finding that LA induced a decrease in liver coenzyme A (CoA) content (44% and 28% reduction in nondiabetic and diabetic rats, respectively, compared with vehicle-treated animals) and liver acetyl CoA content (80% and 67% reduction in nondiabetic and diabetic rats, respectively). A reduction in plasma free carnitine (42% and 22% in nondiabetic and diabetic rats, respectively) was observed in LA-treated animals, and acylcarnitine levels were increased twofold. This could be attributed to elevated levels of C16 and C18 acylcarnitine, without a detectable accumulation of lipoylcarnitine. Under such conditions, a significant increase in the plasma free fatty acid (FFA) concentration (204% in nondiabetic and 151% in diabetic animals) with no elevation in beta-hydroxybutyrate levels was noted. In conclusion, this study suggests that short-term administration of LA at high dosage to normal and diabetic rats causes an inhibition of gluconeogenesis secondary to an interference with hepatic fatty acid oxidation. This may render LA an antihyperglycemic agent for the treatment of diabetic subjects, who display glucose overproduction as a major metabolic abnormality.

Asunto(s)

Diabetes Mellitus Experimental/sangre , Ayuno/sangre , Hipoglucemia/inducido químicamente , Ácido Tióctico/farmacología , Ácido 3-Hidroxibutírico/sangre , Alanina/sangre , Animales , Glucemia/metabolismo , Carnitina/sangre , Ácidos Grasos no Esterificados/sangre , Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Hipoglucemia/sangre , Hígado/enzimología , Hígado/metabolismo , Masculino , Ácido Pirúvico/sangre , Ratas , Ratas Sprague-Dawley

RESUMEN

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

Asunto(s)

Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Estrés Oxidativo , Células 3T3 , Adipocitos/ultraestructura , Animales , Transporte Biológico , Membrana Celular/metabolismo , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Peróxido de Hidrógeno/farmacología , Proteínas Sustrato del Receptor de Insulina , Ratones , Microsomas/enzimología , Concentración Osmolar , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación

RESUMEN

Alpha lipoic acid (lipoate [LA]), a cofactor of alpha-ketodehydrogenase, exhibits unique antioxidant properties. Recent studies suggest a direct effect of LA on glucose metabolism in both human and experimental diabetes. This study examines the possibility that LA positively affects glucose homeostasis in streptozotocin (STZ)-induced diabetic rats by altering skeletal muscle glucose utilization. Blood glucose concentration in STZ-diabetic rats following 10 days of intraperitoneal (i.p.) injection of LA 30 mg/kg was reduced compared with that in vehicle-treated diabetic rats (495 +/- 131 v 641 +/- 125 mg/dL in fed state, P = .003, and 189 +/- 48 v 341 +/- 36 mg/dL after 12-hour fast, P = .001). No effect of LA on plasma insulin was observed. Gastrocnemius muscle crude membrane GLUT4 protein was elevated both in control and in diabetic rats treated with LA by 1.5- and 2.8-fold, respectively, without significant changes in GLUT4 mRNA levels. Gastrocnemius lactic acid was increased in diabetic rats (19.9 +/- 5.5 v 10.4 +/- 2.8 mumol/g muscle, P < .05 v nondiabetic rats), and was normal in LA-treated diabetic rats (9.1 +/- 5.0 mumol/g muscle). Insulin-stimulated 2-deoxyglucose (2 DG) uptake into isolated soleus muscle was reduced in diabetic rats compared with the control group (474 +/- 15 v 568 +/- 52 pmol/mg muscle 30 min, respectively, P = .05). LA treatment prevented this reduction, resulting in insulin-stimulated glucose uptake comparable to that of nondiabetic animals. These results suggest that daily LA treatment may reduce blood glucose concentrations in STZ-diabetic rats by enhancing muscle GLUT4 protein content and by increasing muscle glucose utilization.

Asunto(s)

Glucemia/análisis , Diabetes Mellitus Experimental/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Ácido Tióctico/farmacología , Animales , Desoxiglucosa/farmacocinética , Transportador de Glucosa de Tipo 4 , Técnicas In Vitro , Insulina/farmacología , Ácido Láctico/metabolismo , Masculino , Proteínas de Transporte de Monosacáridos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Increased oxidant stress has been suggested to occur in diabetes and to contribute to the development of late diabetic complications. Whether oxidant stress plays a role in the development or progression of insulin resistance is not known. In this study we hypothesized that exposing 3T3-L1 adipocytes to prolonged micromolar concentrations of H2O2 would reduce their acute metabolic responses to insulin stimulation. 3T3-L1 adipocytes exposed to 25 mU/ml glucose oxidase (GO) for 18 h exhibited a threefold increase in basal 2-deoxyglucose (2-DG) uptake activity. However, net increase in 2-DG uptake activity after acute insulin (100 nM) stimulation was 355 +/- 56 pmol.mg protein-1.min-1 in control vs. 198 +/- 41 pmol.mg protein-1.min-1 in GO-pretreated cells (P < 0.05). Basal lipogenesis activity was significantly enhanced by GO, but acute insulin stimulation resulted in significantly reduced lipogenesis activity (29 +/- 4 vs. 11 +/- 1 nmol glucose/well for control and 50 mU/ml GO, respectively, P = 0.001). Glycogen synthase alpha activity was reduced by GO (78 +/- 1 vs. 43 +/- 2 pmol UDP-glucose.mg protein-1.min-1, P = 0.03), whereas insulin stimulation of glycogen synthase was reduced, exhibiting a right shift in the insulin dose-response curve. These effects of GO were associated with increased GLUT-1 and reduced GLUT-4 protein and mRNA content. In conclusion, our data suggest that oxidant stress alters glucose transporters expression and insulin-stimulated metabolism in 3T3-L1 adipocytes.

Asunto(s)

Adipocitos/fisiología , Resistencia a la Insulina , Estrés Oxidativo , Células 3T3 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Desoxiglucosa/metabolismo , Relación Dosis-Respuesta a Droga , Glucosa Oxidasa/farmacología , Glucógeno Sintasa/metabolismo , Peróxido de Hidrógeno/farmacología , Insulina/farmacología , Isoenzimas/metabolismo , Lípidos/biosíntesis , Ratones

RESUMEN

Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2. and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 microM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kappa B. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 +/- 0.4-fold and 2.2 +/- 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well.

Asunto(s)

Glucosa/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células 3T3 , Adipocitos/metabolismo , Animales , Transporte Biológico , Línea Celular , Desoxiglucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Peróxido de Hidrógeno/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/biosíntesis , FN-kappa B/metabolismo , Oxidantes/metabolismo , Unión Proteica , ARN Mensajero/biosíntesis , Superóxidos/metabolismo

RESUMEN

Exposure of L6 myotubes to prolonged low grade oxidative stress results in increased Glut1 expression at both the protein and mRNA levels, leading to elevated glucose transport activity. To further understand the cellular mechanisms responsible for this adaptive response, the Glut1 transcription rate and mRNA stability were assessed. Nuclear run-on assays revealed 2.0- and 2.4-fold increases in Glut1 transcription rates in glucose oxidase- and xanthine/xanthine oxidase-pretreated cells, respectively. Glut1 mRNA stability was increased with both treatments compared with the control (t1/2 = 7.8 +/- 1.3, 6.0 +/- 2.0, and 2.4 +/- 0.5 h, respectively). The serum-responsive element and AP-1 (but not the cAMP-responsive element) showed increased binding capacity following oxidative stress. Both activation of AP-1 binding and elevation of Glut1 mRNA were prevented by cycloheximide. The involvement of enhancer 1 of the Glut1 gene was demonstrated using transfected 293 cells. Induction of Glut1 mRNA in response to oxidative stress differed from its activation by chronic insulin exposure as demonstrated by the ability of rapamycin to inhibit the latter without an effect on the former. In conclusion, oxidative stress increases the Glut1 transcription rate by mechanisms that may involve activation of AP-1 binding to enhancer 1 of the Glut1 gene.

Asunto(s)

Proteínas de Transporte de Monosacáridos/genética , Músculos/metabolismo , Estrés Oxidativo , Activación Transcripcional , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Transportador de Glucosa de Tipo 1 , Ratones , Polienos/farmacología , Inhibidores de Proteínas Quinasas , ARN Mensajero/metabolismo , Sirolimus , Factor de Transcripción AP-1/metabolismo

RESUMEN

Possible association between the degree of iron load and glucose metabolism has been postulated by both in vivo and in vitro studies. Because skeletal muscle plays a major role in whole body glucose utilization, we evaluated the effect of iron chelators deferoxamine (DFO) and bipyridyl (Bip) on glucose metabolism and transport in cultured L6 muscle cells. Bip (0.1 mM) or DFO (0.5 mM) added for 24 h to the culture medium increased glucose consumption, lactate production, and [14C]glucose incorporation into glycogen by approximately twofold. 2-Deoxy-glucose uptake by L6 myotubes increased time dependently, reaching a 5-fold and 2.5-fold increase after 12 h for Bip and DFO, respectively. Insulin induced a 2.5-fold increase in glucose uptake in untreated cells, which was additive to the chelator's effect. Iron chelator-induced glucose transport stimulation was inhibited by cycloheximide (2.5 micrograms/ml), indicating dependence on de novo protein synthesis. Increases in GLUT-1 protein and mRNA concentration, without changes in GLUT-4, were found to be responsible for iron chelator effects. We conclude that L6 cells adapt to reduction in iron availability by increasing glucose utilization through an enhanced expression of GLUT-1, without losing their physiological response to insulin.

Asunto(s)

Glucosa/metabolismo , Quelantes del Hierro/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculos/metabolismo , 2,2'-Dipiridil/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Deferoxamina/farmacología , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Proteínas de Transporte de Monosacáridos/genética , Músculos/citología , ARN Mensajero/metabolismo , Ratas

RESUMEN

Decreased 2-deoxyglucose (2-DOG) uptake is well described in the neutrophils of patients with glycogen storage disease type 1b (GSD 1b). We report a patient with GSD 1b who presented with a normal antenatal and perinatal 2-deoxyglucose uptake that showed a slow regression during the first months of life. These indicate limitations of 2-deoxyglucose uptake in the diagnosis of GSD 1b. While it appears that low uptake rate below 0.25 nmol/min in 10(6) cells is of significance, normal uptake does not rule out the presence of the disease. It seems that antenatal diagnosis of GSD 1b cannot be made by measurement of 2-deoxyglucose uptake in the fetal neutrophils.

Asunto(s)

Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Envejecimiento/metabolismo , Desoxiglucosa , Enfermedad del Almacenamiento de Glucógeno Tipo I/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Humanos , Técnicas In Vitro , Lactante , Recién Nacido , Hígado/enzimología , Hígado/metabolismo , Masculino , Neutrófilos/metabolismo

RESUMEN

To better understand the link between lysosomal cystine accumulation and the renal impairment seen in cystinosis, we have studied the effect of cystine loading in vivo, on renal function of rats, and in brush-border membrane vesicles (BBMV) prepared from the kidney cortex of the treated rats. Intraperitoneal injection of cystine dimethyl ester (CDME) (400 mumol, twice a day, for 5 days) led to an increased urine volume and excretion of glucose, phosphate, and protein. Kinetic analysis of alpha-methylglucoside initial flux in BBMV showed reduction in maximal transport capacity (Vmax, from 10.1 +/- 1.3 to 8.5 +/- 0.7 nmol.min-1.mg protein-1; P < 0.01) with no change in Michaelis constant (Km, 4.80 +/- 0.08 and 4.90 +/- 0.05 mM). The number of phlorizin binding sites declined (from 6.5 +/- 0.7 to 4.1 +/- 0.4 pmol/mg protein; P < 0.01) with no significant change in the affinity for phlorizin (0.64 +/- 0.08 and 0.59 +/- 0.06 microM). In the cortex homogenate, cystine concentration, which was undetectable in controls, increased to 0.97 +/- 0.09 nmol 1/2 cystine/mg protein. Two hours after CDME administration, ATP content declined to approximately 50% of control values. This decline was transient, and ATP content was recovered to control values 5 h after CDME administration. The treatment did not affect ouabain-sensitive adenosinetriphosphatase activity (40.0 +/- 3.9 and 38.6 +/- 4.7 nmol Pi.mg protein-1.min-1) or the number and affinity of ouabain binding sites (Bmax = 1.48 +/- 0.25 and 1.44 +/- 0.18 pmol/mg, and Kd = 0.68 +/- 0.09 and 0.72 +/- 0.12 microM, respectively). (ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Cistina/análogos & derivados , Síndrome de Fanconi/inducido químicamente , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Cistina/metabolismo , Cistina/farmacología , Síndrome de Fanconi/fisiopatología , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Masculino , Proteínas de Transporte de Monosacáridos/metabolismo , Florizina/metabolismo , Ratas , Ratas Endogámicas , Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores

RESUMEN

Patients with glycogen storage disease (GSD) 1b suffer from recurrent bacterial infections related to neutropenia and impairment of neutrophil functions. One of these functions is the oxidative burst activity which is initiated by NADPH oxidase and depends on the availability of glucose. This activity was markedly reduced in the patient's intact neutrophils when either N-formyl-methionyl-leucyl-phenylalanine (fMLP), or phorbol myristate acetate were used as stimulants. In disrupted GSD 1b polymorphonuclear leucocytes (PMNs), in the presence of exogenous NADPH, this activity was within the normal range. Degranulation, which is calcium dependent but glucose independent, was not significantly different in neutrophils from the patients as compared to controls. Resting cytosolic calcium concentration was indistinguishable from controls. Activation with 10(-7) M fMLP, in the presence or absence of glucose, triggered a prompt and rapid elevation of cytosolic calcium both in the control and the patients' cells. We have previously shown that hexose monophosphate (HMP) shunt activity and glycolytic rate were found to be lower by 70% in intact PMN cells of the patients compared with controls. These activities were normal in disrupted neutrophils. The uptake of the non-metabolized glucose analogues 2-deoxyglucose (2-DOG) and 3-O-Methylglucose (3-OMG) into PMN of GSD 1b patients was studied. 2-DOG is phosphorylated within the cells, thus its uptake rate reflects hexose transport at low concentrations, as long as phosphorylation is not rate limiting. Under those conditions (5 microM 2-DOG) transport was found to be similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Neutrófilos/metabolismo , 3-O-Metilglucosa , Calcio/metabolismo , Desoxiglucosa/farmacocinética , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Enfermedad del Almacenamiento de Glucógeno Tipo I/fisiopatología , Humanos , Metilglucósidos/farmacocinética , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Neutropenia/etiología , Fosforilación/efectos de los fármacos , Fosfotransferasas/metabolismo , Estallido Respiratorio , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Cistina/análogos & derivados , Riñón/metabolismo , Sodio/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Línea Celular , Cistina/farmacología , Células Epiteliales , Epitelio/metabolismo , Glucosa/metabolismo , Membranas Intracelulares/metabolismo , Riñón/citología , Potasio/metabolismo , Rubidio/metabolismo , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

Neutrophil functions and glucose metabolism are known to be impaired in glycogen storage disease (GSD) Ib patients. The uptake of nonmetabolizing glucose analogues into polymorphonuclear leukocytes (PMN) of GSD Ib patients was studied. 2-Deoxyglucose (2-DOG) and 3-O-methylglucose are transported across the cell membrane by facilitated diffusion mediated by the glucose transporter. Because 2-DOG is phosphorylated within the cell, its uptake rate reflects hexose transport as long as phosphorylation is not rate-limiting. These conditions prevail only at low 2-DOG concentrations. Transport of 5 microM DOG into GSD Ib patient PMN was found to be similar to controls (4.3 +/- 0.5 and 4.65 +/- 1.77 pmol/min X 10(6), respectively). In contrast, 2-DOG uptake at high concentrations (2 mM) decreased by 70% in patient PMN compared with control cells (0.17 +/- 0.06 and 0.51 +/- 0.11 nmol/min X 10(6), for patients and controls, respectively). Transport of 3-O-methylglucose (a glucose analogue that does not undergo intracellular phosphorylation) was not different in patient PMN compared with controls (1.86 +/- 0.53 and 2.19 +/- 0.30 nmol/min X 10(6), respectively). Hexose monophosphate shunt activity in PMN of GSD Ib patients at a glucose concentration of 2 mM was 43% of control values, whereas at 10 microM it was similar to controls. Taken together, these results suggest that the defect in glucose uptake and metabolism found in GSD Ib patient PMN is due to an impairment in hexose phosphorylation rather than in a reduction in the transmembrane glucose transport activity.

Asunto(s)

Enfermedad del Almacenamiento de Glucógeno Tipo I/sangre , Hexosas/sangre , Neutrófilos/metabolismo , 3-O-Metilglucosa , Adolescente , Transporte Biológico Activo , Preescolar , Desoxiglucosa/sangre , Enfermedad del Almacenamiento de Glucógeno Tipo I/clasificación , Humanos , Técnicas In Vitro , Lactante , Cinética , Metilglucósidos/sangre , Vía de Pentosa Fosfato , Fosforilación

Asunto(s)

Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Hexosas/metabolismo , Neutrófilos/metabolismo , 3-O-Metilglucosa , Adolescente , Adulto , Transporte Biológico , Niño , Preescolar , Desoxiglucosa/metabolismo , Humanos , Lactante , Metilglucósidos/metabolismo , Vía de Pentosa Fosfato , Fosforilación

RESUMEN

The established renal cell line LLC-PK1 was used as a model to investigate the mechanism underlying kidney malfunction observed in cystinosis patients. In this disease lysosomal accumulation of cystine impairs kidney function, and glycosuria is an early clinical manifestation. The linkage between lysosomal accumulation of cystine and impairment of kidney function is still unclear, and no animal model is available. In an attempt to gain a better insight into this relationship, we studied the effects of lysosomal loading with cystine on the survival and functions of normal noncystinotic renal epithelial cells (LLC-PK1), nonrenal fibroblasts (NIH-3T3), and cystinotic fibroblasts (GM2837). Incubation of the cells with cystine dimethylester (CDME) resulted in time- and dose-dependent accumulation of cystine, with 80% of the cystine in the lysosomal fraction. The lysosomal concentration of cystine increased in the three cell lines after 3 hours of incubation and declined significantly after 48 hours in the normal, but not cystinotic, cells. The accumulation of cystine in the lysosomes caused dose- and time-dependent cell mortality, assessed by measuring the activity of the cytosolic enzyme, lactic dehydrogenase, in the medium. Survival of fibroblasts and renal cells was similar in all three cell lines. The concentrating capacity (the ratio fo the intra- and extracellular concentrations) of the nonmetabolized sugar analog, alpha methyl glucoside (AMG), was used to assess the function of the kidney cells. The sugar concentrating capacity of LLC-PK1 cells was reduced after incubation with CDME in a dose- and time-dependent manner. Since there was no change in sugar efflux between the untreated and treated cells, we conclude that an impairment of the uptake of AMG is responsible for the reduction in the sugar-concentrating capacity in LLC-PK1 cells. In the absence of a genetically impaired animal model, LLC-PK1 cells treated with CDME can be used to investigate the cellular mechanisms responsible for the impairment of kidney function in cystinotic patients.

Asunto(s)

Cistinosis/fisiopatología , Riñón/citología , Alanina/metabolismo , Aminoácidos/metabolismo , Línea Celular , Células Cultivadas , Cistina/metabolismo , Glucosa/metabolismo , Humanos , Riñón/metabolismo , Leucocitos/metabolismo , Lisosomas/metabolismo , Modelos Biológicos