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The translocation of glucose transporter-4 (GLUT4) to the cell surface is a complex multistep process that involves movement of GLUT4 vesicles from a reservoir compartment, and docking and fusion of the vesicles with the plasma membrane. It has recently been proposed that a p38 mitogen-activated protein kinase (MAPK)-dependent step may lead to intrinsic activation of the transporters exposed at the cell surface. In contrast to data obtained in muscle and adipocyte cell lines, we found that no insulin activation of p38 MAPK occurred in rat adipose cells. However, the p38 MAPK inhibitor SB203580 consistently inhibited transport activity after preincubation with the adipose cells. These apparently contradictory findings led us to hypothesize that the inhibitor may have a direct effect on the transport catalytic activity of GLUT4 that was independent of inhibition of the kinase. Kinetic analysis of 3-O-methyl-d-glucose transport activity revealed that SB203580 was a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but was a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport. This pattern of inhibition of GLUT4 was also observed with cytochalasin B. The pattern of inhibition is consistent with interaction at the endofacial surface, but not the exofacial surface of the transporter. Occupation of the endofacial substrate site reduces maximum velocity under zero-trans conditions, because return of the substrate site to the outside is blocked, and no substrate is present inside to displace the inhibitor. Under equilibrium-exchange conditions, internal substrate competitively displaces the inhibitor, and the transport K(m) is increased.

Two radiopharmaceuticals, Tc-99m-MIBI (MIBI) and Tc-99m-Tetrofosmin (Tfos), are currently used for in vivo non-invasive monitoring of the MultiDrug Resistant (MDR) status of tumours. As gliomas are highly multidrug resistant, it is expected that the tracers would be poorly retained in those cells, but the in vivo and in vitro studies to date have shown that Tfos was highly retained in malignant gliomas. The high degree of malignancy of tumour cells is linked to alterations of physiological parameters as plasma membrane potential and intracellular pH. In order to elucidate the contribution of those parameters to Tfos and MIBI uptakes in malignant gliomas, we used several glioma cell lines--G111, G5, G152, and 42 MG-BA. These cells showed to be chemoresistant with a high level of expression and activity of the Multidrug Resistant associated Protein 1 (MRP1). They also had an alkaline intracellular pH (pHi) related to the Na+/H+ antiporter (NHE-1) expression and depolarised plasma membranes (-45 to -55 mV). In spite of their chemoresistance, we have found a high accumulation of both radiotracers in gliomas, more important for Tfos than MIBI, related to the presence and activity of NHE-1. In conjunction, the uptakes of the tracers were only partially dependent upon the plasma membrane potential of the glioma cell lines, again Tfos uptake being less dependent on this parameter than MIBI uptake. In conclusion, the evidence accumulated in this study suggests that Tfos could be a suitable glioma marker in vivo.

Abnormalities in intracellular pH regulation have been proposed to be important in type 2 diabetes and the associated cardiomyopathy and hypertension. We have therefore investigated the dependence of insulin-stimulated glucose transport on cytosolic pH in cardiomyocytes. Insulin treatment of cardiomyocytes resulted in a marked alkalinization of the cytoplasm as measured using carboxy-semi-napthorhodofluor-1. The alkalinizing effect of insulin was blocked by treatment with either cariporide (which inhibits the Na+/H+ exchanger) or by bafilomycin A1 (which inhibits H+-ATPase activity). After treatments with cariporide or bafilomycin A1, insulin stimulation of insulin receptor and insulin receptor substrate-1 phosphorylation and Akt activity were normal. In contrast, glucose transport activity and the levels of functional GLUT4 at the plasma membrane (detected using an exofacial photolabel) were reduced by approximately 50%. Immunocytochemical analysis revealed that insulin treatment caused a translocation of the GLUT4 from perinuclear structures and increased its co-localization with cell surface syntaxin 4. However, neither cariporide nor bafilomycin A1 treatment reduced the translocation of immunodetectable GLUT4 to the sarcolemma region of the cell. It is therefore hypothesized that insulin-stimulated cytosol alkalinization facilitates the final stages of translocation and incorporation of fully functional GLUT4 at the surface-limiting membrane.

We have investigated the effect of glutathione (GSH) depletion on the chemosensitivity of human malignant glioma cell lines: G111, G5 and G152. All the cell lines showed a multidrug resistant (MDR) phenotype associated with MRP1 expression, high intracellular levels of GSH, and depolarized plasma membranes. Tc-99M-Sestamibi (MIBI) and Tc-99M-Tetrofosmin (Tfos) were used for monitoring the MDR mechanisms. Modulation of GSH content was performed with butoxysulfoximide (BSO) pre-treatment alone or in combination with GSH ethyl ester. MIBI and Tfos accumulation in the cells was inversely correlated to the GSH content, a higher accumulation was found after BSO pre-treatment and addition of GSH ethyl ester reversed this process. BSO could therefore play a role as a chemosensitizing drug and thus help to overcome MDR. However, higher accumulation of MIBI and Tfos was observed even in the sensitive cells suggesting another effect of BSO on the cell physiological characteristics. No sign of apoptosis has been found indicating a possible direct effect on the plasma membrane fluidity and permeability. MIBI and Tfos don't follow the expected behavior of a MDR probe in the glioma cells and given the particular morpho-physiological characteristics of these types of tumors, Tfos could be rather used as a marker of the tumor growth and proliferation.

We describe here the cloning, expression and characterisation of centaurin-alpha2 from a rat adipocyte cDNA library. The centaurin-alpha2 cDNA contains an open reading frame, which codes for a protein of 376 amino acids with predicted mass of 43.5 kDa. Centaurin-alpha2 shares 51-59% identity with centaurin-alpha1 proteins and has the same domain organisation, consisting of a predicted N-terminal ArfGAP domain followed by two successive pleckstrin homology domains. Despite the sequence similarity, there are a number of notable differences between the previously characterised centaurin-alpha1 proteins and the newly described centaurin-alpha2: (i) in vitro lipid binding experiments with centaurin-alpha2 do not reveal the same selectivity for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate that has been shown for centaurin-alpha; (ii) unlike centaurin-alpha1 which is expressed mainly in the brain, centaurin-alpha2 has a broad tissue distribution, being particularly abundant in fat, heart and skeletal muscle; (iii) in contrast to centaurin-alpha1 which is found in both membrane and cytosolic fractions, endogenous centaurin-alpha2 is exclusively present in the dense membrane fractions of cell extracts, suggesting a constitutive membrane association. Insulin stimulation, which stimulates phosphatidylinositol 3,4,5-trisphosphate production, does not alter the subcellular distribution of centaurin-alpha2 between adipocyte membrane fractions. This observation is consistent with the lack of specificity of centaurin-alpha2 for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate.

New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1, 3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359+/-10 and 273+/-28 microM for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 microg of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by approximately 10-fold or approximately 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.

A glucose analogue labelled with iodine-123 in position 6 has been synthesized: [123I]-6-deoxy-6-iodo-D-glucose (6DIG). The aim of this study was to examine its biological behaviour in order to assess whether it could be used to evaluate glucose transport with SPECT. To establish whether 6DIG enters the cells using the glucose transporter, four biological models have been used: human erythrocytes in suspension, neonatal rat cardiomyocytes in culture, isolated perfused rat hearts, and biodistribution in mice. 6DIG competed with D-glucose to enter the cells and its entry was increased by insulin and inhibited in the presence of cytochalasin B. The biological behaviour of 6DIG was similar to that of 3-O-methyl-D-glucose. 6DIG is a tracer of glucose transport which is very promising for clinical studies.