RESUMEN
This study measures hexose monophosphate (HMP) shunt activity, glycolytic rate, and glucose transport in PMN and lymphocytes of patients with glycogen storage disease (GSD) type Ib as compared with controls and with GSD Ia patients. HMP shunt activity and glycolysis were significantly lower in intact PMN cells of GSD Ib patients as compared with GSD Ia patients and with controls. These activities were above normal levels in disrupted GSD Ib PMN. HMP shunt activity and glycolytic rates in lymphocytes were similar in all three groups studied. The rate of 2-deoxyglucose transport into GSD Ib PMN was 30% of that into cells of normal controls. In GSD Ib lymphocytes or in GSD Ia PMN and lymphocytes transport was normal. The striking limitation of glucose transport across the cell membrane of the PMN of GSD Ib patients may account for the impairment of leukocyte function that is characteristic of GSD Ib, but not found in GSD Ia patients.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Enfermedad del Almacenamiento de Glucógeno Tipo I/sangre , Neutrófilos/metabolismo , Adenosina Trifosfato/análisis , Adolescente , Preescolar , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Glucólisis , Hexoquinasa/metabolismo , Humanos , Lactatos/biosíntesis , Ácido Láctico , Fragilidad Osmótica , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa/metabolismo , FosforilaciónRESUMEN
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 , FosforilaciónRESUMEN
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/metabolismoRESUMEN
A 59-year-old man had proximal weakness and wasting that started in early childhood. EMG was "myopathic," serum CK activity was increased, and muscle biopsy showed accumulations of glycogen. Biochemical studies revealed elevated glycogen concentration and absence of myophosphorylase activity. This unusual presentation of a long-standing, painless, and quite static weakness due to myophosphorylase deficiency represents another example of clinical heterogeneity.
Asunto(s)
Atrofia Muscular/enzimología , Fosforilasas/deficiencia , Humanos , Masculino , Persona de Mediana Edad , Músculos/enzimología , Músculos/patología , Atrofia Muscular/congénito , Atrofia Muscular/patologíaRESUMEN
A 35-year-old man had severe exercise intolerance and cramps. Venous blood lactate did not rise after ischemic exercise, and electromyographically silent contracture of hand muscles appeared. Histochemistry and electronmicroscopy of a muscle biopsy revealed subsarcolemmal and intermyofibrillar accumulation of glycogen. Biochemical studies showed moderately increased amount of glycogen. Total phosphorylase activity was normal, but the active form "a" was 27% of normal. Phosphorylase kinase activity was 12% of the normal value and was normal in leukocytes and erythrocytes.
Asunto(s)
Enfermedades Musculares/enzimología , Fosforilasa b/deficiencia , Fosforilasas/deficiencia , Adulto , Humanos , Masculino , Calambre Muscular/enzimología , Esfuerzo FísicoRESUMEN
Glycogen content and six major enzymatic activities involved in glycogen metabolism were analysed in chorionic villi (CV). Glycogen levels were found to be lower than those known to exist in liver and muscle. Activities of alpha-glucosidase, amylo-1,6-glucosidase, phosphorylase b and phosphorylase kinase were detectable by standard methods. The enzymatic activities of glucose-6-phosphatase and phosphorylase a were undetectable. These findings suggest that CV biopsies can be useful for first-trimester diagnosis of glycogen storage disease types II, III and VI, but not for type I (glucose-6-phosphatase deficiency).
Asunto(s)
Vellosidades Coriónicas/enzimología , Glucógeno/metabolismo , Vellosidades Coriónicas/ultraestructura , Femenino , Glucosa-6-Fosfatasa/análisis , Sistema de la Enzima Desramificadora del Glucógeno/análisis , Enfermedad del Almacenamiento de Glucógeno/diagnóstico , Humanos , Cariotipificación , Fosforilasas/análisis , Embarazo , Diagnóstico Prenatal , alfa-Glucosidasas/análisisRESUMEN
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-DawleyRESUMEN
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-DawleyRESUMEN
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 BiológicosRESUMEN
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 , RatasRESUMEN
A study of 2-deoxyglucose transport into polymorphonuclear leukocytes (PMN) was performed in three patients with glycogen storage disease (GSD) type Ib. The rate of 2-deoxyglucose transport into GSD Ib PMN was 30% of that of cells of normal controls. In GSD Ib lymphocytes, transport was normal. Km for 2-deoxyglucose in the PMN of one patient was within the normal range. The reduced transport was not due to the elevation in Km for 2-deoxyglucose nor to the decreased rate of phosphorylation of 2-deoxyglucose. The striking limitation of glucose transport across the cell membrane may account for the impairment of leukocyte function which is characteristic of GSD Ib.
Asunto(s)
Desoxiazúcares/sangre , Desoxiglucosa/sangre , Enfermedad del Almacenamiento de Glucógeno/sangre , Neutrófilos/metabolismo , Fosfotransferasas/deficiencia , Antiportadores , Transporte Biológico , Humanos , Cinética , Proteínas de Transporte de Monosacáridos , FosforilaciónRESUMEN
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/metabolismoRESUMEN
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 , RatonesRESUMEN
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/metabolismoRESUMEN
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-aktRESUMEN
Eighteen patients with alpha-glucosidase deficiency have been diagnosed in Israel during the last 15 years. All patients were Palestinian Arabs, with the exception of two siblings from a Jewish Iraqi family. Clinically all patients had the infantile type (Pompe's disease), except one who had the juvenile type. Muscle glycogen content varied from 4 to 17% wet weight. Muscle alpha-glucosidase activity was zero in 10 of 17 patients examined. Among the seven patients in whom residual activity was present, the highest value was 18% of normal. Leukocyte alpha-glucosidase activity was highly variable, making this tissue unfit for enzymatic diagnosis of the disease. A marked heterogeneity was found in pH profiles of muscle and leukocyte alpha-glucosidase activity. A high prevalence of the disease in the Arab population was noted. In spite of a high rate of consanguinity, only a small number of autosomal recessively inherited diseases have been shown to be unusually prevalent in the Arab population. In view of the serious prognosis of this disease, prenatal diagnosis should be offered to affected families.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/epidemiología , Enfermedad del Almacenamiento de Glucógeno/epidemiología , Etnicidad , Glucano 1,4-alfa-Glucosidasa/análisis , Enfermedad del Almacenamiento de Glucógeno Tipo II/sangre , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Israel , Leucocitos/enzimología , Músculos/enzimología , Fenotipo , Pronóstico , alfa-Glucosidasas/análisisRESUMEN
Maple syrup urine disease (MSUD) was diagnosed in four Bedouin infants as a result of the lack of branched-chain keto amino acids (BCKA) decarboxylase activity in leukocytes and skin fibroblasts. Second trimester amniocenteses were performed in five at risk pregnancies for measurement of BCKA decarboxylase. The activity measured in amniocytes was undetectable in two of them. Pregnancy was terminated only in one case and the second continued to term. The diagnosis of MSUD was confirmed in tissues of one aborted fetus, and in leukocytes of the liveborn infant. The other three pregnancies, in which normal enzymatic activity was found in the amniotic cells, were carried to term, and BCKA decarboxylase activity was evident in the leukocytes.
Asunto(s)
Etnicidad , Enfermedad de la Orina de Jarabe de Arce/genética , Diagnóstico Prenatal , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Consanguinidad , Femenino , Fibroblastos/enzimología , Humanos , Lactante , Cetona Oxidorreductasas/metabolismo , Leucocitos/enzimología , Masculino , Enfermedad de la Orina de Jarabe de Arce/diagnóstico , Complejos Multienzimáticos/metabolismo , LinajeRESUMEN
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 & inhibidoresRESUMEN
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/farmacologíaRESUMEN
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.