Quercetin-iron chelates are transported via glucose transporters.

Flavonoids are well-known antioxidants and free radical scavengers. Their metal-binding activity suggests that they could be effective protective agents in pathological conditions caused by both extracellular and intracellular oxidative stress linked to metal overload. Quercetin is both a permeant ligand via glucose transport proteins (GLUTs) and a high-affinity inhibitor of GLUT-mediated glucose transport. Chelatable "free iron" at micromolar concentrations in body fluids is a catalyst of hydroxyl radical (OH(•)) production from hydrogen peroxide. A number of flavonoids, e.g., quercetin, luteolin, chrysin, and 3,6-dihydroxyflavone, have been demonstrated to chelate intracellular iron and suppress OH(•) radical production in Madin Darby canine kidney cells. The most effective chelation comes from the flavonone B ring catechol found in both quercetin and luteolin. We show here that quercetin concentrations of <1µM can facilitate chelatable iron shuttling via GLUT1 in either direction across the cell membrane. These siderophoric effects are inhibited by raised quercetin concentrations (>1µM) or GLUT inhibitors, e.g., phloretin or cytochalasin B, and iron efflux is enhanced by impermeant extracellular iron chelators, either desferrioxamine or rutin. This iron shuttling property of quercetin might be usefully harnessed in chelotherapy of iron-overload conditions.

Protection of ischemic hearts by high glucose is mediated, in part, by GLUT-4.

Metabolic interventions that promote glucose use during ischemia have been shown to protect ischemic myocardium and improve functional recovery on reperfusion. We evaluated whether the cardioprotection afforded by high glucose during low-flow ischemia is associated with changes in the sarcolemmal content of glucose transporters, specifically GLUT-4. Isolated rat hearts were paced at 300 beats/min and perfused under normal glucose (5 mM) or high glucose (10 mM) conditions in buffer containing 0.4 mM albumin, 0.4 mM palmitate, and 70 mU/l insulin and subjected to 50 min of low-flow ischemia and 60 min of reperfusion. To determine the importance of insulin-sensitive glucose transporters in mediating cardioprotection, a separate group of hearts were perfused in the presence of cytochalasin B (10 microM), a preferential inhibitor of insulin-sensitive glucose transporters. Ischemic contracture during low-flow ischemia and creatine kinase release on reperfusion was decreased, and the percent recovery of left ventricular function with reperfusion was enhanced in hearts perfused with high glucose (P < 0.03). Hearts perfused with high glucose exhibited increased GLUT-4 protein expression in the sarcolemmal membrane compared with control hearts under baseline conditions, and these changes were additive with low-flow ischemia. In addition, high glucose did not affect the baseline distribution of sarcolemmal GLUT-1 and blunted any changes with low-flow ischemia. These salutary effects were abolished when glucose transporters are blocked with cytochalasin B. These data demonstrate that protection of ischemic myocardium by high glucose is associated with increased sarcolemmal content of the insulin-sensitive GLUT-4 and suggest a target for the protection of jeopardized myocardium.

Glucose transporter asymmetries in the bovine blood-brain barrier.

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.

Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocin-damaged rat brain.

To reveal whether an extract of Ginkgo biloba (EGb761) may affect streptozotocin (STZ)-induced impairments in brain glucose metabolism, autoradiographies of [3H]cytochalasin-B binding to the total population of glucose transporters, [125I]insulin binding to insulin receptors, [3H]glyburide binding to sulfonylurea receptors, and radioactive in situ hybridization for GLUT3 mRNA were carried out in hippocampal brain sections of adult rats that have additionally been divided into good performers (GP) and poor performers (PP) by behavioural tests before the experiments. The STZ-induced increases in hippocampal [3H]cytochalasin-B binding to (total) glucose transporters returned to almost normal values following EGb761 treatment, regardless of the experimental animal group (GP or PP) tested. Similarly, the STZ-mediated enhancements in hippocampal insulin receptor binding of GP rats were partially compensated by the treatment with EGb761. The data suggest beneficial effects of EGb671 on impaired brain glucose metabolism, at least under the experimental conditions used in the study presented.

Normal and Ha-ras-1 oncogene transformed Buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors.

In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha-ras-1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10(-5) M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1-1 cells were not seriously affected; a higher cytochalasin B concentration (> or = 2 x 10(-5) M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10(-6) M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cells, without affecting their counterparts in the transformed BRLHO6T1-1 cells. A 10-fold higher drug concentration (10(-5) M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]-cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1-1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha-ras-1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity.

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.

Studies of metabolism of round spermatids: cytochalasin B binding to cell membrane in relation to glucose transport.

Cytochalasin B inhibited uptake of 2-deoxy-D-glucose in rat spermatids (Ki = 6.3 X 10(-7) M). This inhibition was reversible. The [3H]cytochalasin B was found to bind to spermatid plasma membranes at low drug concentrations. Lineweaver-Burk plots of binding data indicated that there was a class of high affinity sites (Kd = 2.9 X 10(-7) M). The results suggest that the high affinity cytochalasin B binding sites of spermatids are membrane-associated substances and are intimately related to the glucose transport system.

Effects of colchicine and cytochalasin B on distribution of concanavalin A receptors in isolated and cultured guinea pig epidermal cells.

Regulation of the distribution of concanavalin A (Con A)/receptor complexes by the cytoskeletal contracture system was studied in guinea pig epidermal cells in suspension and culture using the fluorescence double staining method. After treatment with 100 micrograms/ml of Con A at 37 degrees C for 30 min lectin/receptor complexes were endocytosed by the less-differentiated cells in suspension and by the adherent cells in 1- and 3-day cultures that represent a growing cell fraction. The same treatment resulted in diffuse surface distribution of the complexes in the well-differentiated cells in suspension. Colchicine (10(-5) and 10(-6) M) inhibited internalization of the complexes with resultant diffuse distribution in 60% of the adherent cells in culture. Cytochalasin B (5 and 10 micrograms/ml) not only inhibited endocytosis but promoted formation of surface patchy clumps of the complexes in suspended, less-differentiated cells and cultured adherent cells. The distribution profile was not influenced by these drug treatments in the well-differentiated cells. SDS polyacrylamide gel electrophoresis and autoradiography of 125I-labelled epidermal membranes revealed several Con A-reactive polypeptides common to the cells at various differentiation steps. The progressive decrease in endocytosis and mobility of Con A/receptor complexes was suggested to occur with differentiation. In the germinative cells the distribution of lectin/receptor complexes seemed to be regulated by microfilaments and microtubules.

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells.

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.