scholarly journals Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-0-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose

1993 ◽  
Vol 290 (2) ◽  
pp. 497-501 ◽  
Author(s):  
A Schürmann ◽  
K Keller ◽  
I Monden ◽  
F M Brown ◽  
S Wandel ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Binding Domains ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Phosphorylation of the adipose/muscle-type glucose transporter (GLUT4) and its relationship to glucose transport activity

1992 ◽  
Vol 285 (1) ◽  
pp. 223-228 ◽  
Author(s):  
A Schürmann ◽  
G Mieskes ◽  
H G Joost
Keyword(s):  
Protein Kinase ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Low Density ◽  
Transport Activity ◽  
Muscle Type ◽  
Kinase C ◽  
Glucose Transporter Glut4

The effects of protein phosphorylation and dephosphorylation on glucose transport activity reconstituted from adipocyte membrane fractions and its relationship to the phosphorylation state of the adipose/muscle-type glucose transporter (GLUT4) were studied. In vitro phosphorylation of membranes in the presence of ATP and protein kinase A produced a stimulation of the reconstituted glucose transport activity in plasma membranes and low-density microsomes (51% and 65% stimulation respectively), provided that the cells had been treated with insulin prior to isolation of the membranes. Conversely, treatment of membrane fractions with alkaline phosphatase produced an inhibition of reconstituted transport activity. However, in vitro phosphorylation catalysed by protein kinase C failed to alter reconstituted glucose transport activity in membrane fractions from both basal and insulin-treated cells. In experiments run under identical conditions, the phosphorylation state of GLUT4 was investigated by immunoprecipitation of glucose transporters from membrane fractions incubated with [32P]ATP and protein kinases A and C. Protein kinase C stimulated a marked phosphate incorporation into GLUT4 in both plasma membranes and low-density microsomes. Protein kinase A, in contrast to its effect on reconstituted glucose transport activity, produced a much smaller phosphorylation of the GLUT4 in plasma membranes than in low-density microsomes. The present data suggest that glucose transport activity can be modified by protein phosphorylation via an insulin-dependent mechanism. However, the phosphorylation of the GLUT4 itself was not correlated with changes in its reconstituted transport activity.

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

2000 ◽  
Vol 164 (2) ◽  
pp. 187-195 ◽  
Author(s):  
R Romero ◽  
B Casanova ◽  
N Pulido ◽  
AI Suarez ◽  
E Rodriguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Total Protein Content ◽  
Glucose Transporter Proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.

scholarly journals Development of the hormone-sensitive glucose transport activity in differentiating 3T3-L1 murine fibroblasts. Role of the two transporter species and their subcellular localization

1990 ◽  
Vol 270 (2) ◽  
pp. 331-336 ◽  
Author(s):  
M Weiland ◽  
A Schürmann ◽  
W E Schmidt ◽  
H G Joost
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Transport Activity ◽  
Intracellular Compartment ◽  
Differentiated Cells ◽  
Murine Fibroblasts ◽  
Igf I ◽  
Undifferentiated Cells ◽  
Hormone Sensitive

The development of a hormone-responsive glucose transport activity during differentiation of 3T3-L1 murine fibroblasts to an insulin-sensitive adipocyte-like phenotype was studied. Glucose transport activity was insensitive to insulin or insulin-like growth factor I (IGF-I) before differentiation, and was increased by 8-10-fold after differentiation by both insulin and IGF-I via their own respective receptors. In contrast, in undifferentiated cells insulin and IGF-I stimulated a large increase of [3H]thymidine incorporation into DNA via IGF-I receptors, indicating that undifferentiated 3T3-L1 cells are equipped with fully functioning hormone (IGF-I) receptors. Thus the previously described increase in expression of insulin receptors during differentiation cannot solely account for the development of hormone-sensitive glucose transport in the 3T3-L1 cell. The total glucose transport activity reconstituted from membrane fractions was increased by about 3-fold during differentiation. In differentiated cells, more than 80% of the total reconstitutable glucose transport activity was detected in an intracellular compartment (200,000 g microsomes) as compared with about 20% in undifferentiated cells. Immunoblots with specific antiserum confirmed previous reports indicating that the adipose tissue/muscle glucose transporter (GT3) was exclusively present in the differentiated cells, whereas the erythrocyte/brain glucose transporter (GT1) was detected in both differentiated and undifferentiated cells. Upon differentiation, GT1 was redistributed from plasma membranes to the intracellular compartment. In addition, the newly formed GT3 was predominantly found (greater than 80% of total) in the microsomal fraction of differentiated cells. Both GT1 and GT3 appeared to be hormone-sensitive, since in differentiated cells insulin as well as IGF-I gave rise to their translocation from the intracellular compartment to the plasma membrane. These data suggest that, in addition to the specific expression of the GT3 transporter, the formation of a large pool of intracellular glucose transporters comprising both GT1 and GT3 contributes to the development of insulin sensitivity in the 3T3-L1 cell.

scholarly journals Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 707-715 ◽  
Author(s):  
H M Thomas ◽  
J Takeda ◽  
G W Gould
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Transport Activity ◽  
Glut 4 ◽  
Transporter Family ◽  
Native Cell

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5′-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

scholarly journals Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin

1993 ◽  
Vol 291 (3) ◽  
pp. 861-867 ◽  
Author(s):  
H Katagiri ◽  
T Asano ◽  
H Ishihara ◽  
J L Lin ◽  
K Inukai ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Competitive Inhibitor ◽  
Tryptophan Residue ◽  
Wild Type ◽  
Turnover Number ◽  
Glut1 Glucose Transporter

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

scholarly journals Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells. Modulation by isoprenaline and adenosine

1992 ◽  
Vol 288 (1) ◽  
pp. 325-330 ◽  
Author(s):  
S J Vannucci ◽  
H Nishimura ◽  
S Satoh ◽  
S W Cushman ◽  
G D Holman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Transport Activity ◽  
Surface Accessibility ◽  
Adipose Cells ◽  
Membrane Concentration

Insulin-stimulated glucose transport activity in rat adipocytes is inhibited by isoprenaline and enhanced by adenosine. Both of these effects occur without corresponding changes in the subcellular distribution of the GLUT4 glucose transporter isoform. In this paper, we have utilized the impermeant, exofacial bis-mannose glucose transporter-specific photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) [Clark & Holman (1990) Biochem. J. 269, 615-622], to examine the cell surface accessibility of GLUT4 glucose transporters under these conditions. Compared with cells treated with insulin alone, adenosine in the presence of insulin increased the accessibility of GLUT4 to the extracellular photolabel by approximately 25%, consistent with its enhancement of insulin-stimulated glucose transport activity; the plasma membrane concentration of GLUT4 as assessed by Western blotting was unchanged. Conversely, isoprenaline, in the absence of adenosine, promoted a time-dependent (t1/2 approximately 2 min) decrease in the accessibility of insulin-stimulated cell surface GLUT4 of > 50%, which directly correlated with the observed inhibition of transport activity; the plasma membrane concentration of GLUT4 decreased by 0-15%. Photolabelling the corresponding plasma membranes revealed that these alterations in the ability of the photolabel to bind to GLUT4 are transient, as the levels of both photolabel incorporation and plasma membrane glucose transport activity were consistent with the observed GLUT4 concentration. These data suggest that insulin-stimulated GLUT4 glucose transporters can exist in two distinct states within the adipocyte plasma membrane, one which is functional and accessible to extracellular substrate, and one which is non-functional and unable to bind extracellular substrate. These effects are only observed in the intact adipocyte and are not retained in plasma membranes isolated from these cells when analysed for their ability to transport glucose or bind photolabel.

scholarly journals Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells

1988 ◽  
Vol 249 (1) ◽  
pp. 155-161 ◽  
Author(s):  
H G Joost ◽  
T M Weber ◽  
S W Cushman
Keyword(s):  
Activation Energy ◽  
Plasma Membrane ◽  
Membrane Transport ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transport Activity ◽  
Adipose Cells ◽  
Stimulation Of

Conditions are described which allow the isolation of rat adipose-cell plasma membranes retaining a large part of the stimulatory effect of insulin in intact cells. In these membranes, the magnitude of glucose-transport stimulation in response to insulin was compared with the concentration of transporters as measured with the cytochalasin-B-binding assay or by immunoblotting with an antiserum against the human erythrocyte glucose transporter. Further, the substrate- and temperature-dependencies of the basal and insulin-stimulated states were compared. Under carefully controlled homogenization conditions, insulin-treated adipose cells yielded plasma membranes with a glucose transport activity 10-15-fold higher than that in membranes from basal cells. Insulin increased the transport Vmax. (from 1,400 +/- 300 to 15,300 +/- 3,400 pmol/s per mg of protein; means +/- S.E.M.; assayed at 22 degrees C) without any significant change in Km (from 17.8 +/- 4.4 to 18.9 +/- 1.4 nM). Arrhenius plots of plasma-membrane transport exhibited a break at 21 degrees C, with a higher activation energy over the lower temperature range. The activation energy over the higher temperature range was significantly lower in membranes from basal than from insulin-stimulated cells [27.7 +/- 5.0 kJ/mol (6.6 +/- 1.2 kcal/mol) and 45.3 +/- 2.1 kJ/mol (10.8 +/- 0.5 kcal/mol) respectively], giving rise to a larger relative response to insulin when transport was assayed at 37 degrees C as compared with 22 degrees C. The stimulation of transport activity at 22 degrees C was fully accounted for by an increase in the concentration of transporters measured by cytochalasin B binding, if a 5% contamination of plasma membranes with low-density microsomes was assumed. However, this 10-fold stimulation of transport activity contrasted with an only 2-fold increase in transporter immunoreactivity in membranes from insulin-stimulated cells. These data suggest that, in addition to stimulating the translocation of glucose transporters to the plasma membrane, insulin appears to induce a structural or conformational change in the transporter, manifested in an altered activation energy for plasma-membrane transport and possibly in an altered immunoreactivity as assessed by Western blotting.

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