RESUMO
Furosemide inhibits 3-O-methyl-D-glucose equilibrium flux in isolated adipocytes. The inhibition is saturable with an increasing concentration of furosemide and shows a noncompetitive type of kinetics. Both basal and insulin-stimulated fluxes are equally affected by the inhibition. Hydrochlorothiazide and piretanide also inhibit the flux with a similar potency, whereas bumetanide, a more potent diuretic, is much less potent. To understand the molecular basis of this inhibition, effects of furosemide on the glucose-sensitive cytochaslasin B binding activities of adipocytes were studied. Furosemide inhibits the glucose-sensitive cytochalasin B binding of both microsomal and plasma membrane preparations. For both preparations, the inhibition is time dependent and only slowly reversible, is saturable with an increasing concentration of furosemide, shows a noncompetitive type of kinetics with apparent Ki (the inhibitor concentration that gives the half-maximum effect) of 3.5 and 0.7 mM after 2 and 18 h incubation, respectively, and is essentially identical between the basal and insulin-stimulated adipocytes. The inhibition develops with a first-order rate constant of approximately 0.12/h at 4 degrees C. These results indicate that furosemide inhibits glucose transport in adipocytes by directly inactivating transport carriers of both plasma membranes and microsomal reserve pool. This inactivation of glucose carrier may play a part in the diuretic-induced glucose intolerance frequently observed during diuretic therapy.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Furosemida/farmacologia , Glucose/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Citocalasina B/metabolismo , Cinética , Masculino , Proteínas de Transporte de Monossacarídeos , RatosRESUMO
The present study examines the effects of phytohemagglutinin stimulation of a population of human (h) PBMC enriched in lymphocytes (hPBMC) on D-glucose displaceable cytochalasin B binding sites or medium-affinity sites (M-sites) in relation to glucose transport. Previously we have shown that M-sites are glucose transporters in hPBMC (Mookerjee, B.K., et al. 1981. J. Biol. Chem. 256:1290-1300). Equilibrium exchange of 3-O-methyl D-glucose in unstimulated cells revealed two populations with fast and slow flux rates. Phytohemagglutinin stimulates flux rates by converting part of the slow flux population to the fast flux population. M-sites occur in two distinct pools, one in plasma membrane and the other in microsomal fraction. Phytohemagglutinin treatment increases the plasma membrane pool size of M-sites with a concomitant reduction in the microsomal pool size without affecting the binding affinities or the total number of M-sites/cell. Data presented in this paper demonstrate that there are two pools of glucose transporters in these cells and phytohemagglutinin stimulation induces an energy-dependent net translocation of glucose transporters from an intracellular reserve pool to the plasma membrane, which accounts for greater than 60% of the increment in glucose transport.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Neutrófilos/metabolismo , 3-O-Metilglucose , Citocalasina B/metabolismo , Humanos , Insulina/farmacologia , Cinética , Metilglucosídeos/sangue , Fito-Hemaglutininas/farmacologia , Cianeto de Potássio/farmacologia , Temperatura , Fatores de TempoRESUMO
A large scale, rapid, high-yield purification procedure for Azotobacter vinelandii nitrogenase proteins has been developed. Yields of approx. 600 mg of the FeMo protein (Av1) and approx. 550 mg of the Fe protein Av2 are routinely obtained using a procedure that requires only 28 h. The specific activities of Av1 and Av2, respectively, are 3000 and 2100 nmol H2 evolved/min per mg. These activities are significantly higher higher than those comonly used in reactivity studies. Procedures for the isolation and concentration of large quantities of iron-molybdenum cofactor of nitrogenase are also reported. Techniques for anaerobic protein manipulation, generally applicable to the purification of oxygen sensitive proteins are also described.
Assuntos
Azotobacter/enzimologia , Nitrogenase/isolamento & purificação , Anaerobiose , Métodos , Nitrogenase/análiseRESUMO
The in vitro effects of the sulfonylurea glyburide on insulin binding and action were compared in adipocytes from control and nonketotic streptozocin-induced diabetic rats. Adipose tissue from control and diabetic animals was maintained in the absence or presence of 2 micrograms/ml glyburide for 20 h. Insulin binding and insulin-stimulated glucose transport were examined in adipocytes prepared from this tissue. As expected, insulin binding was increased in adipocytes from diabetic animals. Exposure of tissue to glyburide did not influence insulin binding in either control or diabetic cells. Glucose transport activity of diabetic cells, assessed with 2-deoxyglucose, was decreased 30-40% in both the absence (basal) and presence of insulin compared with controls. Glyburide potentiated insulin's effects in both control (15-20%) and diabetic (30-40%) adipocytes. As a result, glucose transport activity in glyburide-treated diabetic cells was restored to a level similar to that of control cells not exposed to the drug. The mechanism by which glyburide potentiated glucose transport activity was examined with the D-glucose-displaceable cytochalasin B-binding technique to measure glucose-transporter concentration in membranes prepared from control and diabetic adipocytes exposed to the drug. Adipocytes from this model of diabetes are known to have a decreased cellular content of glucose transporters. The concentration of glucose transporters was decreased by 31% in plasma membranes from insulin-treated diabetic cells. There were corresponding decreases in diabetic microsomal and total membrane fractions. There was also a 40% decrease in the translocation of transporters from the microsomes to the plasma membrane in response to insulin in diabetic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Tecido Adiposo/metabolismo , Desoxiaçúcares/metabolismo , Desoxiglucose/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glibureto/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Citocalasina B/metabolismo , Cinética , Masculino , Ratos , Receptor de Insulina/metabolismo , Valores de ReferênciaRESUMO
The early stages of weight loss are associated with a reduction in blood pressure, and the mechanisms mediating this reduction remain unclear. Platelet free calcium levels, [Ca2+]i, have been reported to be elevated in essential hypertension and to decrease with pharmacological treatment of the hypertension. In the current study, 18 obese subjects had measurements of blood pressure, forearm blood flow, forearm vascular resistance, and both basal platelet [Ca2+]i and [Ca2+]i responses to vasopressin during 12 weeks on a very low calorie (3,360 kJ, or 800 kcal) diet. Weight reduction was associated with reduction in mean arterial blood pressure at 3-4 weeks. There were associated reductions in forearm vascular resistance and platelet [Ca2+]i as well as increases in forearm blood flow at 3-4 weeks of the diet. Increased forearm blood flow was correlated with weight loss. Vasopressin-induced platelet [Ca2+]i responses increased, which correlated with the reduction in mean arterial pressure at 7-8 weeks of weight loss. Assuming that platelet [Ca2+]i metabolism reflects vascular smooth muscle cell [Ca2+]i metabolism, the data suggest that blood pressure reduction after weight loss may be related to reduced vascular smooth muscle cell [Ca2+]i. The reason for the increased vasopressin-induced [Ca2+]i after weight reduction is unclear.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Resistência Vascular , Redução de Peso/fisiologia , Adulto , Feminino , Antebraço/irrigação sanguínea , Humanos , Masculino , Pessoa de Meia-Idade , Fluxo Sanguíneo RegionalRESUMO
Propidium iodide uptake as an indicator of cell viability was used to measure antibody-mediated cytotoxicity. In conjunction with analysis using the fluorescence-activated cell sorter, this method is as sensitive as isotope release assay systems and has the advantage of gathering multiparameter data on individual cells. This feature enables the resolution of antibody-binding target cells into a population which is sensitive to complement-mediated killing and another population which is resistant to complement-mediated killing.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Animais , Sobrevivência Celular , Proteínas do Sistema Complemento/imunologia , Citometria de Fluxo/métodos , Leucemia L1210/imunologia , Camundongos , Camundongos Endogâmicos DBA , Propídio , Coloração e RotulagemRESUMO
The cellular mechanism of action of sulfonylureas may vary depending upon the exact nature of the drug, the tissue or cell type in question, and the status of the subject from which it was obtained. In adipocytes from patients with type II non-insulin-dependent diabetes, there is increasing evidence indicating that sulfonylureas ameliorate a post-receptor defect in insulin action by potentiating the insulin-stimulated glucose transport normally seen in these cells. Studies undertaken to elucidate the molecular mechanism of this potentiation investigated the effects of a 48-hour incubation with glyburide (2 micrograms/ml) on the recruitment of glucose carriers from microsomal storage pools to the plasma membrane. With the use of cytochalasin B, a potent competitive inhibitor of glucose transport, glucose-sensitive cytochalasin B binding was studied in basal and insulin-stimulated adipocytes from control and sulfonylurea-treated tissue. The data indicated that sulfonylurea treatment did not affect the total glucose-sensitive cytochalasin B binding capacity of adipocyte membranes. It did, however, increase the insulin-induced recruitment of the glucose carrier from the microsome to the plasma membrane by 27 to 31 percent. This suggests that the molecular mechanism of sulfonylurea-enhanced insulin-stimulated glucose transport is the recruitment of glucose transporters from an intracellular microsomal storage pool to the plasma membrane.
Assuntos
Tecido Adiposo/metabolismo , Glicemia/metabolismo , Glibureto/uso terapêutico , Transporte Biológico/efeitos dos fármacos , Membrana Celular , Citocalasina B/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glibureto/farmacologia , Humanos , Insulina/farmacologia , Cinética , MicrossomosRESUMO
Angiotension converting enzyme (ACE) inhibitors and beta-blockers have been reported to possess disparate effects on insulin sensitivity. The aim of this study was to study the effects of the selective beta-1 blocker bisoprolol and of the ACE inhibitor captopril on cellular insulin action in hypertensive individuals. After washout, 12 mild to moderate essential hypertensives were randomized in a double-blind manner to 5 mg bisoprolol daily or 25 mg captopril twice daily for 8 weeks. Erythrocyte insulin binding and insulin-stimulated tyrosine kinase (TK) activity were measured before and after therapy. Both agents decreased diastolic blood pressure significantly (bisoprolol 96.5+/-0.9 to 87.8+/-3.1 mm Hg; captopril 96.5+/-0.9 to 91.5+/-1.8 mm Hg; P < .05). Fasting plasma glucose, insulin, and insulin/glucose indices remained unchanged after both therapies, as did lipid profiles. Maximal insulin-stimulated TK activity, assessed by phosphorylation of the exogenous substrate poly-Glu80Tyr20, was significantly higher (P < .05) after bisoprolol treatment, but not after captopril treatment, when compared to placebo (bisoprolol 8.5+/-1.8; captopril 7.3+/-1.5; placebo: 6.4+/-1.3 pmol 32P-ATP/fmol bound insulin). However, captopril, but not bisoprolol, increased the sensitivity of the receptor TK activity, as measured by the half-maximal activity concentration (ED50). Specific insulin binding was not affected by these two agents. Thus, both captopril and bisoprolol may have favorable but different effects on TK activity and insulin action at the cellular level.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bisoprolol/farmacologia , Captopril/farmacologia , Hipertensão/tratamento farmacológico , Receptor de Insulina/efeitos dos fármacos , Idoso , Método Duplo-Cego , Humanos , Hipertensão/enzimologia , Resistência à Insulina , Pessoa de Meia-Idade , Receptor de Insulina/metabolismoRESUMO
alpha-Adrenergic blockers have shown favorable metabolic effects. We evaluated the glucose and insulin responses to a glucose load and lipid profiles in 36 diabetic hypertensive patients before and after 8 weeks of doxazosin administration. To evaluate insulin action at the cellular level, erythrocyte insulin binding and tyrosine kinase (TK) activity were measured in 12 of these patients. Systolic and diastolic blood pressures decreased significantly (P < .0001) after 8 weeks of doxazosin therapy. Doxazosin administration significantly reduced the integrated insulin response (area under the curve [AUC]-insulin: 6093 +/- 894 to 5260 +/- 807; P = .04) and the insulin/glucose index (I/G) at 90 and 120 min after a glucose load (at 90 min, 0.230 +/- 0.055 v 0.180 +/- 0.04, P < .05; at 120 min, 0.275 +/- 0.071 v 0.173 +/- 0.036, P < .05). HDL3 level increased from 31.1 +/- 1.5 mg% to 34 +/- 1.6 mg% (P < .05) after doxazosin. Erythrocyte insulin binding and tyrosine kinase activity were not significantly altered after doxazosin. No significant correlation was found between the insulin or glucose responses and the insulin receptor binding or tyrosine kinase activity before and after treatment.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Doxazossina/uso terapêutico , Hipertensão/tratamento farmacológico , Insulina/sangue , Proteínas Tirosina Quinases/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Humanos , Hipertensão/sangue , Hipertensão/complicações , Hipertensão/fisiopatologia , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/efeitos dos fármacos , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismoRESUMO
In an attempt to elucidate the cellular mechanism(s) by which sulfonylureas exert their extrapancreatic hypoglycemic effects, various parameters of insulin action were examined in vitro, using rat adipocytes maintained in a biochemically defined medium. Cells were maintained for 20 hours in the absence or presence of 175 micrograms/mL chlorpropamide and insulin binding, hexose transport, glucose metabolism, and insulin receptor tyrosine kinase activity were compared. Chlorpropamide treatment had no effect on insulin binding, altering neither receptor number nor affinity. However, the sulfonylurea did enhance 2-deoxyglucose transport in both the absence (17%, P less than .01) and presence (20%, P less than .01) of insulin. Furthermore, glucose metabolism as measured by the conversion of glucose (0.2 mmol/L) to CO2 and total lipids was also significantly increased by chlorpropamide treatment in both the absence (30%, P less than .01) and presence (31%, P less than .05) of insulin. Potentiation of insulin-stimulated transport or metabolism was not explained by an increase in the basal state alone because the incremental responses to 40 ng/mL insulin were potentiated by 19% (P less than .01) and 25% (P less than .05), respectively. Activity of the insulin receptor kinase was unchanged as evaluated by autophosphorylation of partially purified receptors, phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. These studies demonstrate that the sulfonylurea, chlorpropamide, stimulates glucose transport and potentiates insulin's effect on this process by acting at a site(s) beyond insulin receptor binding and phosphorylation.
Assuntos
Tecido Adiposo/metabolismo , Clorpropamida/farmacologia , Glucose/metabolismo , Insulina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Masculino , Fosforilação , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
We have shown that preeclampsia is associated with insulin resistance. In the present study, we examined young normal, preeclamptic (PE), and gestational hypertensive (GH) nulliparous African-American women at term to investigate cellular determinants of this resistance and insulin and insulin-like growth factor-I (IGF-I) binding to partially purified erythrocyte receptors and receptor tyrosine kinase activity (TKA). Blood pressure was significantly elevated in PE and GH subjects as compared with controls. Insulin binding was similar in number and affinity in the three groups (femtomoles per microgram). IGF-I binding was increased in PE subjects as compared with either normals or GH subjects (0.2 +/- 0.02, 0.15 +/- 0.01, and 0.14 +/- 0.02 fmol/microgram protein). Insulin receptor TKA was increased in PE subjects as compared with normals when assessed either per microgram protein or per femtomole insulin binding (P < .01). In contrast, IGF-I-potentiated TKA was elevated in PE subjects only when assessed per microgram protein (P < .03). Thus, the increased number of IGF-I receptors in erythrocytes of PE subjects yields a net increase in receptor tyrosine kinase. Also, there is an augmentation of insulin receptor TKA in PE subjects. Together, these two alterations may be a compensatory mechanism for the insulin resistance associated with hypertensive diseases of pregnancy.
Assuntos
Eritrócitos/química , Hipertensão/sangue , Insulina/sangue , Complicações Cardiovasculares na Gravidez/sangue , Proteínas Tirosina Quinases/sangue , Receptor IGF Tipo 1/análise , Adulto , População Negra , Pressão Sanguínea/fisiologia , Separação Celular , Eritrócitos/citologia , Feminino , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Insulina/metabolismo , Resistência à Insulina/fisiologia , Gravidez , Complicações Cardiovasculares na Gravidez/enzimologia , Complicações Cardiovasculares na Gravidez/fisiopatologia , Proteínas Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/metabolismoRESUMO
Binding of ribonucleoproteins to specific regions of mRNA can alter mRNA stability. This level of posttranscriptional regulation has been shown to play a major role in gene expression of eukaryotic cells. This process involves the binding of ribonucleoproteins to specific region(s) of unstable, rapidly degrading mRNAs such as those found in various cytokines, lymphokines, and oncogenes, thereby increasing the mRNA's stability. In many instances the instability of the mRNA has been mapped to an AU-rich motif in the 3' untranslated region. We transcribed RNA molecules containing four reiterations of an AUUUA motif, and demonstrated with RNA- band shift experiments that the AUUUA motif complexes with phosphorylated AUUUA-specific 43-47 kDa mRNA binding protein(s) found in the cytosol of both rat brain and cultured rat astrocytes.
Assuntos
Astrócitos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Insulin and insulin-like growth factor I (IGF-I) play a role in lens cell growth and development. The binding of these hormones to their respective receptors with its concomitant signal transduction is an important step in these cellular processes. Hormone binding to adult chicken lens insulin and IGF-I receptors, partially purified from epithelial and fiber cells, was studied to examine this activity in lens. The associated stimulation of receptor-mediated tyrosine kinase by the hormones was also studied. At an insulin concentration of 0.02 nM, specific binding was similar for epithelial and fiber receptor preparations (Epi = 0.23 +/- 0.03 fmol, Fib = 0.19 +/- 0.02 fmol). Displacement studies revealed that there was also no difference between epithelial and fiber receptor preparations in the concentration of insulin necessary for half maximal displacement of specific [125I]-insulin binding (IC50: Epi = 0.32 nM +/- 0.07 nM, Fib = 0.31 nM +/- 0.05 nM). Comparison of IGF-I (0.02 nM) binding to receptor preparations from epithelial and fiber cells demonstrated that specific binding was similar in the two preparations (Epi = 0.50 +/- 0.05 fmol, Fib = 0.42 +/- 0.05 fmol). Also, there was no difference in the concentration of IGF-I necessary for half maximal displacement of specific [125I]-IGF-I binding (IC50 = Epi: 0.27 +/- 0.05 nM, Fib: 0.28 +/- 0.04 nM). The ability of IGF-I to displace bound [125I]-insulin was also examined. The IC50 for IGF-I binding to the insulin receptors isolated from epithelial and fiber cells was 37.4 +/- 2.4 nM, and 35.4 +/- 2.8 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cristalino/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Ligação Competitiva , Galinhas , Reações Cruzadas , Epitélio/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Especificidade por SubstratoRESUMO
Sulfonylureas potentiate the insulin-induced stimulation of glucose transport in adipocytes. In order to elucidate the molecular mechanism of this potentiation, effects of a long-term (48-h) incubation with glyburide (2 micrograms/ml) on the relative sizes of the plasma membrane, the microsome, and the total membrane pools of the glucose-sensitive cytochalasin B binding were studied for basal and insulin-stimulated adipocytes. The drug treatment potentiated an insulin-induced stimulation of 3-O-methyl-D-glucose flux by 31 to 45%, with little effect on the basal flux. The same drug treatment increased the plasma membrane pool size of the glucose-sensitive cytochalasin B binding in the insulin-stimulated adipocytes with a concomitant decrease in the microsomal pool size. This effect was minimal, if any, in basal adipocytes. The drug treatment did not affect the total glucose-sensitive, cytochalasin B binding capacity of adipocyte membranes. These results indicate that the drug treatment increases the insulin-induced recruitment of the glucose carrier from the microsome to the plasma membrane by 27-31%. It is concluded that potentiation of the insulin-induced stimulation of the hexose transport by sulfonylureas is mainly due to a potentiation of the insulin-induced recruitment of the glucose carrier.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Insulina/farmacologia , Compostos de Sulfonilureia/farmacologia , 3-O-Metilglucose , Tecido Adiposo/metabolismo , Animais , Citocalasina B/metabolismo , Sinergismo Farmacológico , Glibureto/metabolismo , Masculino , Metilglucosídeos/metabolismo , Proteínas de Transporte de Monossacarídeos , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
To further define the cellular alteration(s) involved in the impaired glucose transport associated with chronic uremia, we examined the concentration and translocation of glucose transport systems in adipocytes isolated from partially nephrectomized uremic rats. Uremic animals, compared with matched controls, had increased blood urea nitrogen and serum insulin, whereas serum glucose was unchanged. In agreement with previous work, 125I-insulin binding to its receptor was unaltered and transport of 2-deoxy-D-glucose was decreased in both the absence (basal) and presence of a maximal (7 nM) insulin concentration by 44 and 35%, respectively. To assess the movement and concentration of glucose transport systems in various membrane fractions prepared from basal and insulin-treated (20 nM) uremic fat cells, the technique of D-glucose-inhibitable cytochalasin B binding was utilized. In plasma membranes isolated from these cells the concentration of glucose transporters was decreased by 16 (P less than 0.01) and 30% (P less than 0.005) in basal and insulin-treated cells, respectively. Concomitantly, microsomal membranes prepared from uremic cells treated in the absence and presence of insulin had a 28 (P less than 0.01) and 15% (P less than 0.05) decrease in concentration of glucose transport systems, respectively. Additionally, glucose transporter concentration was significantly decreased by 17% (P less than 0.025) in total membranes prepared from uremic cells. Thus, impairment of glucose transport in uremic fat cells can be attributed to a postbinding defect that, at least in part, results from a decrease in the total concentration of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Resistência à Insulina , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Uremia/metabolismo , Tecido Adiposo/metabolismo , Animais , Membrana Celular/metabolismo , Citocalasina B/farmacologia , Glucose/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Microssomos/metabolismo , Nefrectomia , Ratos , Ratos Endogâmicos , Receptor de Insulina/metabolismo , Valores de ReferênciaRESUMO
The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.
Assuntos
Tecido Adiposo/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Tecido Adiposo/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Citocalasina B/metabolismo , Relação Dose-Resposta à Radiação , Cinética , Masculino , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 X 10(-4) mol/l). Exposure to the drug resulted in a significant enhancement (p less than 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhanced p less than 0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Metformina/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/efeitos dos fármacos , Animais , Glicemia/metabolismo , Hexoses/metabolismo , Masculino , Fosforilação , Ratos , Ratos EndogâmicosRESUMO
Measurements of charge injection from indium tin oxide (ITO) into the organic semiconductor, tetraphenyl diamine doped polycarbonate (PC:TPD), were carried out. The current injected at the contact was measured as a function of the hole mobility in the organic semiconductor, which was varied from 10(-6) to 10(-3) cm (2)/V x s by adjusting the concentration of the hole transport agent, TPD, in the PC host. These experiments reveal that the current injected at the contact is proportional to the hole mobility in the bulk. As a result, the ITO/PC:TPD contact is found to limit current flow in all samples, regardless of the hole mobility in PC:TPD.
RESUMO
Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.