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 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.

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 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 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.

Effect of insulin and glucocorticoids on glucose transporters in rat adipocytes

1987 ◽  
Vol 252 (4) ◽  
pp. E441-E453 ◽  
Author(s):  
C. Carter-Su ◽  
K. Okamoto
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Maximal Response ◽  
Transport Activity ◽  
Rat Adipocytes ◽  
Dose Dependent Fashion

The ability of glucocorticoids to modify the effect of insulin on glucose transport was investigated in both intact isolated rat adipocytes and in membranes isolated from hormone-treated adipocytes. In intact adipocytes, dexamethasone, a potent synthetic glucocorticoid, inhibited insulin-stimulated 3-O-methylglucose transport at all concentrations of insulin tested (0-2,000 microU/ml). Insulin sensitivity, as well as the maximal response to insulin, was decreased by dexamethasone in the absence of a change in insulin binding. The inhibition was observed regardless of which hormone acted first, was blocked by actinomycin D, and resulted from a decrease in Vmax rather than an increase in Kt of transport. In plasma membranes isolated from insulin-treated adipocytes, glucose transport activity and the amount of glucose transporter covalently labeled with [3H]cytochalasin B were increased in parallel in a dose-dependent fashion. The amount of labeled transporter in a low-density microsomal fraction (LDMF) was decreased in a reciprocal fashion. In contrast, addition of dexamethasone to insulin-stimulated cells caused decreases in both transport activity and amount of labeled transporter in the plasma membranes. This was accompanied by a small increase in the amount of [3H]cytochalasin B incorporated into the glucose transporter in the LDMF. These results are consistent with both insulin and glucocorticoids altering the distribution of glucose transporters between the plasma membrane and LDMF, in opposite directions.

scholarly journals Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments

1992 ◽  
Vol 281 (3) ◽  
pp. 829-834 ◽  
Author(s):  
Y Shibasaki ◽  
T Asano ◽  
J L Lin ◽  
K Tsukuda ◽  
H Katagiri ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Cell Lines ◽  
Cho Cells ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Chinese Hamster ◽  
Transport Activity ◽  
Fibroblastic Cell ◽  
Cellular Environment

Rat GLUT4 (adipocyte/muscle-type glucose transporter) was expressed in two fibroblastic cell lines, Chinese hamster ovary (CHO) cells and 3T3-L1 fibroblasts, under the control of the methallothionein I promoter. Although immunoblotting with a GLUT4-specific anti-peptide antibody demonstrated that the amount of GLUT4 expressed was comparable with that in 3T3-L1 adipocytes and rat adipose tissues, no increase in 2-deoxy-D-glucose uptake was observed in the basal state in fibroblasts. Immunocytochemical studies showed that the expressed GLUT4 appeared to be localized in a specific region in the cytoplasm. These results were in marked contrast to those obtained in CHO cells expressing GLUT1 (HepG2/erythrocyte-type glucose transporter) using the same expression vector. In this case the expressed GLUT1 protein appeared to reside mainly on the plasma membranes, and a significant increase in glucose uptake was observed. Although insulin increased glucose uptake in CHO cells and 3T3-L1 fibroblasts as well as in the cells expressing rat GLUT4, an increment due to insulin above basal values was small, at most 2-fold, and no significant differences were observed in insulin-stimulated glucose uptake between transfected and parental cells. In addition, no apparent differences in the subcellular distribution of expressed GLUT4 were observed between the insulin-stimulated and the basal state. These results indicate that in fibroblastic cell lines GLUT1 and GLUT4 proteins are sorted in a different fashion, and the expression of GLUT4 protein per se is not enough to produce a large insulin-induced increase in glucose transport activity such as that observed in rat adipocytes and 3T3-L1 adipocytes. Thus unidentified aspects of the cellular environment which are present in the adipocytes but not in fibroblastic cell lines may be required for a large insulin-induced increase in glucose transport activity to be observed.

Glucose as regulator of glucose transport activity and glucose-transporter mRNA in hamster beta-cell line

Diabetes ◽  
1992 ◽  
Vol 41 (5) ◽  
pp. 592-597 ◽  
Author(s):  
N. Inagaki ◽  
K. Yasuda ◽  
G. Inoue ◽  
Y. Okamoto ◽  
H. Yano ◽  
...  
Keyword(s):  
Beta Cell ◽  
Cell Line ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Transport Activity ◽  
Beta Cell Line ◽  
Glucose Transporter Mrna
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