Insulin stimulation of glucose transport activity in rat skeletal muscle: increase in cell surface GLUT4 as assessed by photolabelling

We have used a photoaffinity label to quantify cell surface GLUT4 glucose transporters in isolated rat soleus muscles. In this system, insulin stimulated an 8.6-fold increase in 3-O-methylglucose glucose transport, while photolabelled GLUT4 increased 8-fold. These results demonstrate that the insulin-stimulated increase in glucose transport activity in skeletal muscle can be accounted for by an increase in surface-accessible GLUT4 content.

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Effects of phenylarsine oxide on stimulation of glucose transport in rat skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.4.c648 ◽

1990 ◽

Vol 258 (4) ◽

pp. C648-C653 ◽

Cited By ~ 32

Author(s):

E. J. Henriksen ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Glucose Transport ◽

Cytochalasin B ◽

Contractile Activity ◽

Transport Activity ◽

Rat Skeletal Muscle ◽

Phenylarsine Oxide ◽

Muscle Glucose Transport ◽

Glucose Analogue ◽

Stimulation Of

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.

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Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells

Biochemical Journal ◽

10.1042/bj2780235 ◽

1991 ◽

Vol 278 (1) ◽

pp. 235-241 ◽

Cited By ~ 40

Author(s):

A E Clark ◽

G D Holman ◽

I J Kozka

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Glucose Transport ◽

Time Course ◽

Transport Activity ◽

Insulin Stimulation ◽

Surface Labelling ◽

Lag Period ◽

Adipose Cells ◽

Stimulation Of

We have used an impermeant bis-mannose compound (2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos+ ++- 4-yloxy)-2- propylamine; ATB-BMPA) to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18 degrees C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t 1/2 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t 1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or less of cell-surface transporters over 60 min at 18 degrees C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

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Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane

Biochemical Journal ◽

10.1042/bj2320245 ◽

1985 ◽

Vol 232 (1) ◽

pp. 245-254 ◽

Cited By ~ 15

Author(s):

P A Hyslop ◽

C E Kuhn ◽

R D Sauerheber

Keyword(s):

Plasma Membrane ◽

Density Distribution ◽

Fold Increase ◽

Hexose Transport ◽

Transport Activity ◽

Insulin Stimulation ◽

Hexose Transporters ◽

Pre Treatment ◽

Isolated Rat ◽

Stimulation Of

We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone.

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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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Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins

Biochemical Journal ◽

10.1042/bj2810809 ◽

1992 ◽

Vol 281 (3) ◽

pp. 809-817 ◽

Cited By ~ 32

Author(s):

J Yang ◽

A E Clark ◽

R Harrison ◽

I J Kozka ◽

G D Holman

Keyword(s):

Cell Surface ◽

Glucose Transport ◽

Glucose Transporter ◽

Fluid Phase ◽

Transport Activity ◽

Phenylarsine Oxide ◽

Fluid Phase Endocytosis ◽

Surface Labelling ◽

Slow Dissociation ◽

Stimulation Of

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

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Effects of exercise training on glucose transport and cell surface GLUT-4 in isolated rat epitrochlearis muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.272.2.e320 ◽

1997 ◽

Vol 272 (2) ◽

pp. E320-E325 ◽

Cited By ~ 9

Author(s):

T. H. Reynolds ◽

J. T. Brozinick ◽

M. A. Rogers ◽

S. W. Cushman

Keyword(s):

Exercise Training ◽

Cell Surface ◽

Glucose Transport ◽

Muscle Glycogen ◽

Citrate Synthase ◽

Transport Activity ◽

Glut 4 ◽

Isolated Rat

The effects of exercise training on maximal glucose transport activity and cell surface GLUT-4 were examined in rat epitrochlearis muscle. Five days of swim training (2 x 3 h/day) produce a significant increase in citrate synthase activity (24.5 +/- 0.6 vs. 20.1 +/- 0.7 micromol x min(-1) x g(-1)), GLUT-4 content (22.9 +/- 0.8 vs. 17.4 +/- 0.4% GLUT-4 standard), and glycogen levels (54.3 +/- 9.4 vs. 28.6 +/- 9.4 micromol/g). Maximally, insulin-stimulated glucose transport activity and cell surface GLUT-4 are increased by 55 (1.50 +/- 0.11 vs. 0.97 +/- 0.10 micromol x ml(-1) x 20 min(-1)) and 48% [12.0 +/- 0.8 vs. 8.1 +/- 0.9 disintegrations x min(-1) (dpm) x mg(-1)], respectively, in exercise-trained epitrochlearis muscles. In contrast, hypoxia-stimulated glucose transport activity and cell surface GLUT-4 are reduced by 38 (0.78 +/- 0.08 vs.1.25 +/- 0.14 micromol x ml(-1) x 20 min(-1)) and 40% (5.7 +/- 0.9 vs. 9.4 +/- 1.2 dpm/mg), respectively, in exercise-trained epitrochlearis muscles. These results demonstrate that changes in insulin- and hypoxia-stimulated glucose transport activity after exercise training are fully accounted for by the appearance of cell surface GLUT-4 and support the concept of two intracellular pools of GLUT-4. Finally, we propose that high levels of muscle glycogen with exercise training may contribute to the decrease in hypoxia-stimulated glucose transport activity.

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Insulin-sensitive regulation of glucose transport and GLUT4 translocation in skeletal muscle of GLUT1 transgenic mice

Biochemical Journal ◽

10.1042/bj3370051 ◽

1998 ◽

Vol 337 (1) ◽

pp. 51-57 ◽

Cited By ~ 2

Author(s):

Garret J. ETGEN ◽

William J. ZAVADOSKI ◽

Geoffrey D. HOLMAN ◽

E. Michael GIBBS

Keyword(s):

Skeletal Muscle ◽

Cell Surface ◽

Transgenic Mice ◽

Glucose Transport ◽

Hind Limb ◽

Glucose Transporter ◽

Glut4 Translocation ◽

Transport Activity ◽

Fast Twitch Muscle ◽

Fast Twitch

Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased ∼ 3–8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased ∼ 4–14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity ∼ 2–3-fold in isolated muscles and to a much greater extent (∼ 7–20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to ∼ 50–75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 ∼ 2-fold in isolated EDL and ∼ 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a more robust responsiveness to insulin. Furthermore, the results demonstrate that muscle overexpressing GLUT1 has normal insulin-induced GLUT4 translocation and the ability to augment glucose-transport activity above the elevated basal rates.

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Stimulation of glucose transport in skeletal muscle by hypoxia

Journal of Applied Physiology ◽

10.1152/jappl.1991.70.4.1593 ◽

1991 ◽

Vol 70 (4) ◽

pp. 1593-1600 ◽

Cited By ~ 203

Author(s):

G. D. Cartee ◽

A. G. Douen ◽

T. Ramlal ◽

A. Klip ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Muscle Contraction ◽

Glucose Transport ◽

Glucose Transporter ◽

Cytochalasin B ◽

Sugar Transport ◽

Transport Activity ◽

Hindlimb Muscles ◽

Stimulation Of

Hypoxia caused a progressive cytochalasin B-inhibitable increase in the rate of 3-O-methylglucose transport in rat epitrochlearis muscles to a level approximately six-fold above basal. Muscle ATP concentration was well maintained during hypoxia, and increased glucose transport activity was still present after 15 min of reoxygenation despite repletion of phosphocreatine. However, the increase in glucose transport activity completely reversed during a 180-min-long recovery in oxygenated medium. In perfused rat hindlimb muscles, hypoxia caused an increase in glucose transporters in the plasma membrane, suggesting that glucose transporter translocation plays a role in the stimulation of glucose transport by hypoxia. The maximal effects of hypoxia and insulin on glucose transport activity were additive, whereas the effects of exercise and hypoxia were not, providing evidence suggesting that hypoxia and exercise stimulate glucose transport by the same mechanism. Caffeine, at a concentration too low to cause muscle contraction or an increase in glucose transport by itself, markedly potentiated the effect of a submaximal hypoxic stimulus on sugar transport. Dantrolene significantly inhibited the hypoxia-induced increase in 3-O-methylglucose transport. These effects of caffeine and dantrolene suggest that Ca2+ plays a role in the stimulation of glucose transport by hypoxia.

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Translocation of the brain-type glucose transporter largely accounts for insulin stimulation of glucose transport in BC3H-1 myocytes

Biochemical Journal ◽

10.1042/bj2690597 ◽

1990 ◽

Vol 269 (3) ◽

pp. 597-601 ◽

Cited By ~ 31

Author(s):

D M Calderhead ◽

K Kitagawa ◽

G E Lienhard ◽

G W Gould

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Fold Increase ◽

The Other ◽

Insulin Stimulation ◽

Glut 1 ◽

Glut 4 ◽

Rat Soleus Muscle ◽

The Brain ◽

Stimulation Of

Insulin-stimulated glucose transport was examined in BC3H-1 myocytes. Insulin treatment lead to a 2.7 +/- 0.3-fold increase in the rate of deoxyglucose transport and, under the same conditions, a 2.1 +/- 0.1-fold increase in the amount of the brain-type glucose transporter (GLUT 1) at the cell surface. It has been shown that some insulin-responsive tissues express a second, immunologically distinct, transporter, namely GLUT 4. We report here that BC3H-1 myocytes and C2 and G8 myotubes express only GLUT 1; in contrast, rat soleus muscle and heart express 3-4 times higher levels of GLUT 4 than GLUT 1. Thus translocation of GLUT 1 can account for most, if not all, of the insulin stimulation of glucose transport in BC3H-1 myocytes. On the other, hand, neither BC3H-1 myocytes nor the other muscle-cell lines are adequate as models for the study of insulin regulation of glucose transport in muscle tissue.

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