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1.
Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases. 相似文献
2.
F Koumanov C Henry C Ghezzi JP Mathieu C Morin M Vidal J de Leiris M Comet D Fagret 《Canadian Metallurgical Quarterly》1997,24(6):519-525
Two anomeric analogues of glucose labelled with 123 iodine in position 6, proposed as tracers of glucose transport in vivo, have been synthesized: alpha- and beta-methyl-6-deoxy-6-iodo-D-glucopyranoside (alpha MDIG and beta MDIG). The aim of this study was to determine whether these molecules interact with the glucose transporter and whether they could be used as tracers of glucose transport in vivo. The biodistribution of alpha MDIG and beta MDIG was studied in the mouse in vivo. To determine if these two anomers enter the cell via the glucose transporter, their uptake was measured in isolated perfused rat hearts, in human erythrocytes in suspension, and in cardiomyocytes of neonatal rat in culture. Both alpha MDIG and beta MDIG had similar repartitions in the mouse: myocardial uptake averaged 7% of the injected dose/g of organ at 2 min postinjection and alpha MDIG competed with D-glucose to enter the cells. Insulin produced a 123% increase of its uptake in isolated perfused rat hearts and a 100% increase in cardiomyocytes of neonatal rat in culture. alpha MDIG uptake was lowered in the presence of glucose transport inhibitors in each experimental model. An interaction between beta MDIG and glucose transporters was observed only in human erythrocytes in suspension. Only alpha MDIG interacts with the glucose transporter, and thus could be used to estimate glucose transport in vivo. 相似文献
3.
E Anichini A Zamperini M Chevanne R Caldini M Pucci G Fibbi M Del Rosso 《Canadian Metallurgical Quarterly》1997,36(11):3076-3083
The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC. 相似文献
4.
C Caro S Colby-Germinario B Brenner M Oliveira MA Wainberg RJ Germinario 《Canadian Metallurgical Quarterly》1998,30(9):1031-1038
In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells. 相似文献
5.
E Spooncer L Fairbairn GJ Cowling TM Dexter AD Whetton PJ Owen-Lynch 《Canadian Metallurgical Quarterly》1994,8(4):620-630
A temperature sensitive abl protein tyrosine kinase gene was transferred into a multipotent haemopoietic stem cell line, and the primary biological effects of expression of the gene were examined at the permissive and non-permissive temperatures. Unlike previous studies in factor-dependent cell lines, we found that expression of the functional abl protein tyrosine kinase did not lead to growth autonomy. Furthermore, the cells were still able to undergo terminal myeloid differentiation. However, expression of the functional gene did lead to a delay in maturation with a concomitant increase in cell production, had a modest effect in terms of delayed apoptosis particularly when the cells were maintained at a high cell density, and slightly increased the response to sub-optimal concentrations of IL-3. In many respects, therefore, the effects of abl protein tyrosine kinase in these cells mimics the effect of bcr/abl in primary haemopoietic cells where growth factor independence and an aberrant differentiation profile are relatively late events in clonal evolution and are not intermediate consequences of activation of the abl gene. 相似文献
6.
L Kozma K Baltensperger J Klarlund A Porras E Santos MP Czech 《Canadian Metallurgical Quarterly》1993,90(10):4460-4464
Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway. 相似文献
7.
Y Uehara 《Canadian Metallurgical Quarterly》1993,20(12):1885-1891
Accumulating evidence indicates that the activation of cellular oncogenes is a cause of some human cancers. ErbB-1, erbB-2 and abl oncogenes encoding tyrosine kinases, ras oncogenes encoding GTP binding proteins and myc oncogenes whose functions are not well understood are some examples. Therefore, agents which inhibit the activity of these oncogene products may provide new means to overcome certain human tumors. Herbimycin A and tyrphostins have been found and developed as inhibitors of tyrosine kinases and the effectiveness of these agents against tumors of Ph1-positive leukemia (CML, ALL) or squamous cell carcinomas has been reported. Although specific inhibitors of ras or myc oncogene products have not yet been described, recent studies on the processing of Ras proteins toward the cell membrane provide a strategy to search for inhibitors of ras functions. 相似文献
8.
To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration. 相似文献
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11.
The concentration of D-mannose in serum is 20-50 micron, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K(uptake) of about 30-70 micron and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2-deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mM D-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis. 相似文献
12.
KA Smith ML Agarwal MV Chernov OB Chernova Y Deguchi Y Ishizaka TE Patterson MF Poupon GR Stark 《Canadian Metallurgical Quarterly》1995,347(1319):49-56
Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage. 相似文献
13.
To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells. 相似文献
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15.
The most frequently observed mutations in ras oncogenes in solid human tumors are GC-->AT transitions at the 3' G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3' G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki- and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3' G compared to the 5' G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3' G and the 5' G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5' G and 3' G residues had an effect on the extent of dT misincorporation opposite each other. The 5' G was shown to have a stimulatory effect on dT misincorporation opposite the 3' G, while the 3' G was shown to have an inhibitory effect on dT misincorporation opposite the 5' G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3' G and 5' G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of human ras oncogenes, by causing GC-->AT transitions preferentially at the 3' G of the GG doublet in codon 12 of these oncogenes. 相似文献
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17.
SJ Vannucci RR Clark E Koehler-Stec K Li CB Smith P Davies F Maher IA Simpson 《Canadian Metallurgical Quarterly》1998,20(4-5):369-379
Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat. 相似文献
18.
We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells. Several types of samples gave higher reconstituted activity using liposomes of egg lipids rather than soybean lipids. Diacylglycerol, proposed to activate transporters directly as part of the mechanism of insulin action, increased the intrinsic activity of heart transporters by only 25%, but increased the size of the reconstituted liposomes by 90%. The dipeptide Cbz-Gly-Phe-NH2 inhibited GLUT4 with a Ki of 0.2 mM, compared to 2.5 mM for GLUT1, which explains its preferential inhibition of insulin-stimulated glucose transport in adipocytes. Verapamil, which inhibits insulin- and hypoxia-stimulated glucose transport in muscle, had no effect on reconstituted transporters. Heart transporters had a higher Km for glucose uptake (13.4) than did GLUT1 (1.6 mM), in agreement with a recent study of GLUT1 and GLUT4 expressed in yeast and reconstituted in liposomes. Transporters reconstituted from heart and adipocytes were 40-70% inactivated by external trypsin, suggesting the presence of trypsin-sensitive sites on the cytoplasmic domain of GLUT4. NaCl and KCl both reduced reconstituted transport activity, but KCl had a much smaller effect on the size of the liposomes. 相似文献
19.
GLUT-2 differs from other members of the facilitated glucose transporter family because it transports a wider range of substrates and exhibits a higher Km for transport of glucose analogs such as 2-deoxyglucose (2-DOG). In order to investigate the structural determinants of the unique substrate specificity and kinetic function of GLUT-2, recombinant adenoviruses were used to express native, mutant, and chimeric glucose transporters in the kidney cell line CV-1, yielding the following key observations. (1) A chimera consisting of GLUT-1 with the C-terminal tail of GLUT-2 had a Km for 2-DOG of 9.9 +/- 1.5 that was intermediate between that of native GLUT-1 (3.7 +/- 0.4) and native GLUT-2 (26.3 +/- 3.3). In contrast to the effect of the GLUT-2 C terminus on Km for 2-DOG, this substitution did not confer enhanced uptake of three alternative substrates (fructose, arabinose, or streptozotocin) which are transported efficiently by native GLUT-2 but not by GLUT-1. (2) A chimera consisting of GLUT-2 with the N-terminal 87 amino acids of GLUT-1 exhibited no change in Km for 2-DOG relative to native GLUT-2 but exhibited a significant reduction in capacity for transport of the three alternative substrates. (3) Mutation of asparagine 62 in GLUT-2 to glutamine produced a transporter lacking its N-linked oligosaccharide that exhibited a 2.5-fold increase in Km for 2-DOG but equally efficient transport of the three alternative substrates relative to native GLUT-2. These data provide insight into structural domains that affect substrate specificity in facilitated glucose transporters and demonstrate that they are distinct from elements involved in glucose transport kinetics. 相似文献
20.
The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity. 相似文献