RESUMEN
The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and ß-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and ß-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3ß-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.
Asunto(s)
Bovinos/fisiología , Glucosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Folículo Ovárico/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-O-Metilglucosa/metabolismo , Animales , Desoxiglucosa/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Regulación de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Subunidades de Proteína , Trazadores Radiactivos , Receptores de HFE/genética , Receptores de HFE/metabolismo , Factores de TiempoRESUMEN
Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.
Asunto(s)
Eritrocitos/efectos de los fármacos , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Glucosa/metabolismo , Gosipol/farmacología , 3-O-Metilglucosa/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Citocalasina B/metabolismo , Desoxiglucosa/metabolismo , Relación Dosis-Respuesta a Droga , Eritrocitos/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Gosipol/metabolismo , Células HL-60 , Humanos , Cinética , Modelos Biológicos , Niacinamida/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Espectrometría de Fluorescencia , TransfecciónRESUMEN
Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.
Asunto(s)
Caveolas/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 3/metabolismo , Hexoquinasa/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , 3-O-Metilglucosa/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Caveolas/química , Caveolas/efectos de los fármacos , Caveolina 1/análisis , Caveolina 1/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Desoxiglucosa/metabolismo , Transportador de Glucosa de Tipo 1/análisis , Transportador de Glucosa de Tipo 3/análisis , Hexoquinasa/análisis , Inmunohistoquímica , Cinética , Masculino , Microscopía Fluorescente , Unión Proteica , Ratas , Ratas Sprague-Dawley , Espermátides/química , Espermátides/efectos de los fármacos , Espermatocitos/química , Espermatocitos/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
The facilitative glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in adipocytes and muscles, and the participation of GLUT4 in the pathogenesis of various clinical conditions associated with obesity, visceral fat accumulation and insulin resistance has been proposed. Glucose uptake by some members of the GLUT family, mainly GLUT1, is inhibited by flavonoids, the natural polyphenols present in fruits, vegetables and wine. Therefore it is of interest to establish if these polyphenolic compounds present in the diet, known to be effective antioxidants but also endowed with several other biological activities such as protein-tyrosine kinase inhibition, interfere with GLUT4 function. In the present study, we show that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM. These three flavonoids show a competitive pattern of inhibition, with K(i)=16, 33.5 and 90 microM respectively. In contrast, neither catechin nor gallic acid inhibit methylglucose uptake. To obtain a better understanding of the interaction among GLUT4 and flavonoids, we have derived a GLUT4 three-dimensional molecular comparative model, using structural co-ordinates from a GLUT3 comparative model and a mechanosensitive ion channel [PDB (Protein Data Bank) code 1MSL] solved by X-ray diffraction. On the whole, the experimental evidence and computer simulation data favour a transport inhibition mechanism in which flavonoids and GLUT4 interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signalling inhibition. Furthermore, the results suggest that GLUT transporters are involved in flavonoid incorporation into cells.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Catequina/análogos & derivados , Flavonoides/farmacología , Glucosa/metabolismo , Quercetina/farmacología , 3-O-Metilglucosa/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Catequina/química , Catequina/farmacología , Simulación por Computador , Flavonoides/química , Flavonoides/metabolismo , Transportador de Glucosa de Tipo 4 , Cinética , Modelos Moleculares , Estructura Molecular , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosforilación/efectos de los fármacos , Conformación Proteica , Quercetina/química , Quercetina/metabolismo , Ratas , Termodinámica , Tirosina/metabolismoRESUMEN
A fluorometric method that allows repeatable measurement of sugar transport rates and parameters in single living cells is presented. Intracellular sugar concentrations were estimated in real time from changes in cell volume that occur secondary to permeation of sugars across the plasma membrane. In turn, the cell volume changes were estimated from variations of intracellular calcein fluorescence measured by confocal microscopy. Using HeLa cells, the assay allowed reproducible measurement of the uptake and exit of D-galactose and 3-O-methyl-D-glucose. The rate of zero-trans uptake (i.e. at an intracellular concentration of zero) of galactose at an extracellular concentration of 200 mM was 0.34+/-0.05 mM/s (n=8). Apparent Vmax and Km for galactose exit were 0.32+/-0.05 mM/s (n=9) and 30+/-7.2 mM (n=9), respectively. The apparent affinity of infinite-trans (i.e. at a very high intracellular concentration) uptake of 3-O-methyl-D-glucose was 3.8+/-0.47 mM (n=8). Galactose uptake was 93+/-8% (n=8) inhibited in the presence of 50 microM phloretin, whereas galactose exit was 96+/-6% (n=5) trans-inhibited by 100 mM 4,6-ethylidine-D-glucose. This technique may help to characterize sugar transport in freshly isolated cells, co-cultures and heterogeneous cell explants. It may also allow available cell microinjection technology to be used to study the regulation of sugar transporters' intrinsic activity. In principle, similar approaches might also be applied in functional studies of other transporters for which non-metabolized substrates are available.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Carbohidratos/análisis , 3-O-Metilglucosa/metabolismo , Algoritmos , Transporte Biológico , Tamaño de la Célula , Fluoresceínas , Colorantes Fluorescentes , Fluorometría , Galactosa/metabolismo , Células HeLa , Humanos , Cinética , Microinyecciones , Floretina/farmacologíaAsunto(s)
Células Epiteliales/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Transporte de Monosacáridos/metabolismo , 3-O-Metilglucosa/metabolismo , Células 3T3 , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Clonales , Desoxiglucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Ratones , Concentración Osmolar , Ratas , Sorbitol/farmacología , Estrés Fisiológico , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
3-O-14C-methyl-D-Glucose (3-O-MG) transport and 14C-saccharose incorporation were measured in isolated uterine strips from ovariectomized-estrogenized diabetic rats. Glucose transport was decreased in uterine strips from diabetic rats compared with control animals. PGE1 and PGE2 (10(-7) M) stimulated 3-O-MG transport, PGF2 alpha failed to modify this parameter at the same concentration, while insulin (0.5 U/ml) evoked an improvement 30% greater than PGs. In spite of the negative influence exerted by TXA2 over glucose metabolism in the isolated rat uterus, U46619, 10(-5) M (a TXA2 analogue), and OKY064, 10(-7) M (an inhibitor of TXA2 synthesis), failed to modify basal or insulin-treated hexose transport. Neither additive or synergistic interactions between PGE1 or PGE2 (10(-7) M) and insulin at 0.5 U/ml and 0.25 U/ml were detected. We conclude that the stimulatory action of PGE1 and PGE2 on glucose metabolism that has been previously described by us, involves enhancement of glucose transport.