Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity.

Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155±18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 °C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3,-bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.

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Divergent mechanisms for the insulin resistant and hyperresponsive glucose transport in adipose cells from fasted and refed rats. Alterations in both glucose transporter number and intrinsic activity.

Journal of Clinical Investigation ◽

10.1172/jci113649 ◽

1988 ◽

Vol 82 (2) ◽

pp. 691-699 ◽

Cited By ~ 36

Author(s):

B B Kahn ◽

I A Simpson ◽

S W Cushman

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Intrinsic Activity ◽

Insulin Resistant ◽

Adipose Cells

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

Biochemical Journal ◽

10.1042/bj2880325 ◽

1992 ◽

Vol 288 (1) ◽

pp. 325-330 ◽

Cited By ~ 81

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.

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Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin

Biochemical Journal ◽

10.1042/bj3150025 ◽

1996 ◽

Vol 315 (1) ◽

pp. 25-31 ◽

Cited By ~ 16

Author(s):

Mariko OMATSU-KANBE ◽

Mary Jane ZARNOWSKI ◽

Samuel W. CUSHMAN

Keyword(s):

Oxygen Consumption ◽

Glucose Transport ◽

Hormonal Regulation ◽

Glucose Transporter ◽

Subcellular Fractionation ◽

Transport Activity ◽

Brown Adipose ◽

Glucose Transporter Glut4 ◽

Adipose Cells ◽

Large Response

Isolated brown adipose cells from rats are prepared whose viability is indicated by the expected stimulation of oxygen consumption by noradrenaline and counter-regulation of this oxygen consumption response by insulin. Insulin stimulates 3-O-methyl-D-glucose transport by approx. 15-fold in the absence of adenosine, and adenosine augments this response at least 2-fold. The insulin-stimulated translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane is readily detected by subcellular fractionation and Western blotting, and the appearance of GLUT4 on the cell surface in response to insulin is demonstrated by bis-mannose photolabelling. Isoprenaline also stimulates glucose transport activity but only by approx. 3-fold; this effect is not altered by adenosine. Isoprenaline increases insulin-stimulated glucose transport activity in the absence of adenosine but decreases it in the presence of adenosine. These results demonstrate that although the regulation of glucose transport by insulin in brown adipose cells is qualitatively similar to that in white adipose cells, counter-regulation by adenosine and isoprenaline is at least quantitatively and may be qualitatively different. Isolated brown adipose cells from rats thus represent an excellent model for further examination of the mechanism by which multiple hormone signalling pathways interact to control glucose transport and GLUT4 subcellular trafficking.

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

Biochemical Journal ◽

10.1042/bj2490155 ◽

1988 ◽

Vol 249 (1) ◽

pp. 155-161 ◽

Cited By ~ 63

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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Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin-resistant glucose transport in adipose cells without restoring glucose transporter gene expression.

Journal of Clinical Investigation ◽

10.1172/jci115031 ◽

1991 ◽

Vol 87 (2) ◽

pp. 561-570 ◽

Cited By ~ 105

Author(s):

B B Kahn ◽

G I Shulman ◽

R A DeFronzo ◽

S W Cushman ◽

L Rossetti

Keyword(s):

Gene Expression ◽

Blood Glucose ◽

Glucose Transport ◽

Glucose Transporter ◽

Diabetic Rats ◽

Transporter Gene ◽

Insulin Resistant ◽

Adipose Cells

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

Diabetes ◽

10.2337/diabetes.41.5.592 ◽

1992 ◽

Vol 41 (5) ◽

pp. 592-597 ◽

Cited By ~ 4

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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Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells

Journal of Cell Science ◽

10.1242/jcs.113.23.4203 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4203-4210 ◽

Cited By ~ 1

Author(s):

D. Malide ◽

G. Ramm ◽

S.W. Cushman ◽

J.W. Slot

Keyword(s):

Adipose Tissue ◽

Glucose Transport ◽

Morphological Evidence ◽

Glut4 Translocation ◽

Transport Activity ◽

Brown Adipose ◽

Quantitative Immunoelectron Microscopy ◽

Adipose Cells ◽

Isolated Rat ◽

Stimulation Of

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

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Glucose transporters and glucose transport in skeletal muscles of 1- to 25-mo-old rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1993.264.3.e319 ◽

1993 ◽

Vol 264 (3) ◽

pp. E319-E327 ◽

Cited By ~ 8

Author(s):

E. A. Gulve ◽

E. J. Henriksen ◽

K. J. Rodnick ◽

J. H. Youn ◽

J. O. Holloszy

Keyword(s):

Glucose Transport ◽

Skeletal Muscles ◽

Glucose Transporter ◽

Contractile Activity ◽

Transport Activity ◽

Glut 4 ◽

Flexor Digitorum Brevis ◽

Old Rats ◽

Growth And Aging ◽

Flexor Digitorum

It is widely thought that aging results in development of insulin resistance in skeletal muscle. In this study, we examined the effects of growth and aging on the concentration of the GLUT-4 glucose transporter and on glucose transport activity in skeletal muscles of female Long-Evans rats. Relative amounts of immunoreactive GLUT-4 protein were measured in muscle homogenates of 1-, 10-, and 25-mo-old rats by immunoblotting with a polyclonal antibody directed against GLUT-4. In the epitrochlearis, plantaris, and the red and white regions of the quadriceps muscles, GLUT-4 immunoreactivity decreased by 14-33% between 1 and 10 mo of age and thereafter remained constant. In flexor digitorum brevis (FDB) and soleus muscles, GLUT-4 concentration was similar at all three ages studied. Glucose transport activity was assessed in epitrochlearis and FDB muscles by incubation with 2-deoxyglucose under the following conditions: basal, submaximal insulin, and either maximal insulin or maximal insulin combined with contractile activity. Glucose transport in the epitrochlearis muscle decreased by approximately 60% between 1 and 4 mo of age and then did not decline further between 4 and 25 mo of age. Transport activity in the FDB assessed with a maximally effective insulin concentration decreased only slightly (< 20%) between 1 and 7 mo of age. Aging, i.e., the transition from young adulthood to old age, was not associated with a decrease in glucose transport activity in either the epitrochlearis or the FDB.(ABSTRACT TRUNCATED AT 250 WORDS)

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Glucose transporter protein content and glucose transport capacity in rat skeletal muscles

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.259.4.e593 ◽

1990 ◽

Vol 259 (4) ◽

pp. E593-E598 ◽

Cited By ~ 97

Author(s):

E. J. Henriksen ◽

R. E. Bourey ◽

K. J. Rodnick ◽

L. Koranyi ◽

M. A. Permutt ◽

...

Keyword(s):

Protein Content ◽

Glucose Transport ◽

Glucose Transporter ◽

Contractile Activity ◽

Fiber Type ◽

Linear Regression Analysis ◽

Type I ◽

Fiber Types ◽

Transport Activity ◽

Glut 4

The relationships among fiber type, glucose transporter (GLUT-4) protein content, and glucose transport activity stimulated maximally with insulin and/or contractile activity were studied by use of the rat epitrochlearis (15% type I-20% type II2a-65% type IIb), soleus (84-16-0%), extensor digitorum longus (EDL, 3-57-40%), and flexor digitorum brevis (FDB, 7-92-1%) muscles. Insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake was greatest in the soleus, followed (in order) by the FDB, EDL, and epitrochlearis. On the other hand, contractile activity induced the greatest increase in 2-DG uptake in the FDB, followed by the EDL, soleus, and epitrochlearis. The effects of insulin and contractile activity on 2-DG uptake were additive in all the muscle preparations, with the relative rates being FDB greater than soleus greater than EDL greater than epitrochlearis. Quantitation of the GLUT-4 protein content with the antiserum R820 showed the following pattern: FDB greater than soleus greater than EDL greater than epitrochlearis. Linear regression analysis showed that whereas a relatively low and nonsignificant correlation existed between GLUT-4 protein content and 2-DG uptake stimulated by insulin alone, significant correlations existed between GLUT-4 protein content and 2-DG uptake stimulated either by contractions alone (r = 0.950) or by insulin and contractions in combination (r = 0.992). These results suggest that the differences in maximally stimulated glucose transport activity among the three fiber types may be related to differences in their content of GLUT-4 protein.

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