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G D Holman

Publications and source records attributed to G D Holman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cell-surface biotinylation of GLUT4 using bis-mannose photolabels.

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.

Adipocytes↗
PubMed1998

Serine-294 and threonine-295 in the exofacial loop domain between helices 7 and 8 of glucose transporters (GLUT) are involved in the conformational alterations during the transport process.

The role of a conserved polar motif (STS) in the exofacial loop between helices 7 and 8 of GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose-transport activity, cytochalasin B binding and photolabelling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis-(d-mannosyloxy)-2-propylamine (ATB-BMPA) were assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Replacement of Ser-294 with Ala or Thr suppressed transport activity and cytochalasin B binding. ATB-BMPA photolabelling was normal in S294A mutants, and even increased in S294T mutants. Replacement of Thr-295 with Ala suppressed transport activity and cytochalasin B binding, whereas ATB-BMPA photolabelling was normal; substitution of Ser failed to alter the investigated parameters. Similarly, exchanging Ser-296 for Ala generated a normally functioning protein. The data suggest that Ser-294 and Thr-295 are involved in the conformational change in GLUT during the transport process, and that their substitution may arrest the transporter in an outward-facing conformation.

Alanine↗
PubMed1997

Effect of insulin on GLUT4 cell surface content and turnover rate in human skeletal muscle as measured by the exofacial bis-mannose photolabeling technique.

Insulin-stimulated glucose transport across the skeletal muscle cell membrane is a major regulatory step in postprandial glucose disposal. To estimate the total molar concentration of GLUT4 as well as the turnover rate of GLUT4 in human vastus lateralis muscles at the cell surface in the basal state and after insulin exposure, we have applied the sensitive exofacial bis-mannose photolabeling technique on in vitro incubated human skeletal muscle strips from healthy subjects. In addition, we have measured 3-O-methylglucose transport in other muscle strips prepared from the same surgically removed human skeletal muscle biopsies to compare glucose transport with cell surface level of GLUT4. Maximal in vitro insulin stimulation (2,400 pmol/l) resulted in a twofold increase compared with basal in both surface GLUT4 content (0.38 +/- 0.05 vs. 0.19 +/- 0.03 pmol/g wet muscle wt, P < 0.005) and 3-O-methylglucose transport (1.24 +/- 0.13 vs. 0.63 +/- 0.08 pmol x ml(-1) x h(-1), P < 0.005). The insulin-induced increment in 3-O-methylglucose transport was strongly correlated with the insulin-induced increase in cell surface GLUT4 content (r2 = 0.91; P < 0.005). The calculated turnover rate of human skeletal muscle GLUT4 amounted to approximately 8 x 10(4) min(-1) at 35 degrees C and was unaffected by insulin. In conclusion, maximal in vitro insulin stimulation of vastus lateralis muscle strips from healthy subjects resulted in a twofold rise in glucose transport as well as in cell surface content, whereas the turnover rate of GLUT4 was unaffected by insulin under the chosen experimental conditions.

3-O-Methylglucose↗
PubMed1996

Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein.

The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptors (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adipose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 11681-11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubrevin (VAMP 3) by using isoform-specific antisera. Additional antisera identify syntaxins 2 and 4 as the predominant target membrane SNAP receptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one-twentieth the level. Syntaxins 2 and 4 are enriched 5-10-fold in PM compared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (approx. 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular distributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes after the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fusion protein (NSF) and recombinant alpha-soluble NSF attachment protein (alpha-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, but not syntaxins 2 or 3. Complex formation requires ATP and is disrupted by ATP hydrolysis. When all membrane fractions are prepared from basal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, and participation of VAMPs 2 and 3 is enhanced 4-6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater than can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insulin-stimulated cells, SNARE complexes formed from PM alone contain similar levels of syntaxin 4 but 5-6-fold higher levels of VAMPs 2 and 3 compared with PM alone from basal cells. Addition of GLUT4 vesicle proteins to PM from insulin-treated cells results in a further 2-fold increase in VAMP 2 recovered in SNARE complexes. Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT4 vesicles so as to increase the efficiency of SNARE complex formation.

Adipose Tissue↗
PubMed1996

Characterisation of glucose transport in the hyperthermophilic Archaeon Sulfolobus solfataricus.

Sulfolobus solfiataricus is a hyperthermophilic Archaeon growing at 80 degrees C, pH 3. The glucose transport system of this organism has been characterised kinetically at this temperature and pH using 2-deoxy-D-glucose: the sugar analogue is transported into the cells with a Km = 1.8 +/- 0.3 microM and a Vmax = 3.6 +/- 0.1 nmol min(-1) (mg protein)(-1), with an intracellular accumulation of up to 200-fold over the extracellular concentration. Transport was significantly reduced at pH 5. Inhibition of 2-deoxy-D-glucose transport was investigated using a variety of sugars and sugar analogues; D-glucose, D-galactose and D-mannose showed the highest affinity for the transporter, with D-glucose possessing a Ki = 120 +/- 20 nM.

Biological Transport↗
PubMed1996

Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal.

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos -4-yloxy)-2-propylamine (ATM-BMPA). ATM-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-6 are mutated to alanine-N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min(-1). These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, while the distribution of the C-(LL_AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4 is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that the mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.

Amino Acid Sequence↗
PubMed1996

Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells.

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (approximately 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti(-)[insulin receptor substrate 1 (IRS1)] and anti-alpha-p85 antibodies show the same level of insulin-stimulated activity, approximately 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of alpha-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

3T3 Cells↗
PubMed1996

Contraction-independent effects of catecholamines on glucose transport in isolated rat cardiomyocytes.

The effects of catecholamines on glucose transport were studied in noncontracting isolated rat cardiomyocytes. alpha-Adrenergic treatment (phenylephrine, or norepinephrine + propranolol) led to an approximately fourfold stimulation of glucose transport in basal cells (no insulin). The effect of phenylephrine was suppressed by the alpha 2-antagonist yohimbine or the beta-antagonist propranolol. The beta-adrenergic agonist isoproterenol partially counteracted the action of phenylephrine (but not that of insulin). Phenylephrine increased glucose transport in two phases with apparent half times of 3.2 and 13.0 min, respectively. Correspondingly, different EC50 values were found after 10 and 45 min on phenylephrine addition (5.0 +/- 1.9 vs. 31.6 +/- 9.6 microM, respectively). Maximal stimulation by phenylephrine was at least partially additive to that of insulin and of other stimulators of glucose transport (e.g., H2O2, vanadate, lithium). Phenylephrine significantly increased the level of cell surface glucose carriers GLUT-1 (1.54-fold) and GLUT-4 (1.78-fold), as assessed by using the specific photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]- 1,3-bis(D-mannos-4-yloxy)propyl-2-amine. In conclusion, catecholamines stimulate cardiomyocyte glucose transport through alpha 1-adrenergic receptors independently or downstream of a contraction-evoked stimulus. This effect is at least partially explained by a recruitment of glucose transporters to the cell surface. The mechanism(s) and/or signals involved differ from those triggered by insulin and insulinomimetic agents.

Adrenergic Agents↗
PubMed1995

Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity.

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

Affinity Labels↗
PubMed1995

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

3-O-Methylglucose↗
PubMed1995

The effects of insulin on the level and activity of the GLUT4 present in human adipose cells.

Human adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propyl-amine, ATB-BMPA. The rates of 3-O-methyl-D-glucose transport and the cell-surface level of GLUT4 are very similar in the human and rat adipocyte in the basal state. The Vmax for 3-O-methyl-D-glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of GLUT4 suggests that this low transport activity is associated with a low GLUT4 abundance (39 x 10(4) sites/cell; 19.9 x 10(4) sites at the cell surface). The turnover number for human adipose cell GLUT4 (5.8 x 10(4) min-1) is similar to that observed for GLUT4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50% of the GLUT4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient GLUT4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗