scholarly journals Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity

1988 ◽  
Vol 249 (3) ◽  
pp. 865-870 ◽  
Author(s):  
C Mühlbacher ◽  
E Karnieli ◽  
P Schaff ◽  
B Obermaier ◽  
J Mushack ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Cytochalasin B ◽  
Phorbol Esters ◽  
Fat Cells ◽  
Transport Activity ◽  
Rat Adipocytes ◽  
Glucose Carrier ◽  
Stimulation Of

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.

scholarly journals Further evidence for a two-step model of glucose-transport regulation. Inositol phosphate-oligosaccharides regulate glucose-carrier activity

1989 ◽  
Vol 261 (3) ◽  
pp. 699-705 ◽  
Author(s):  
B Obermaier-Kusser ◽  
C Mühlbacher ◽  
J Mushack ◽  
E Seffer ◽  
B Ermel ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Phorbol Esters ◽  
Inositol Phosphate ◽  
Transport Activity ◽  
Insulin Effect ◽  
Step Model ◽  
Proposed Model ◽  
Glucose Carrier

The insulin effect on glucose uptake is not sufficiently explained by a simple glucose-carrier translocation model. Recent studies rather suggest a two-step model of carrier translocation and carrier activation. We used several pharmacological tools to characterize the proposed model further. We found that inositol phosphate (IP)-oligosaccharides isolated from the drug Actovegin, as well as the alkaloid vinblastine, show a partial insulin-like effect on glucose-transport activity of fat-cells (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 5.8%, insulin 59%, IP-oligosaccharides 30%, vinblastine 29%) without inducing carrier translocation. On the other hand, two newly developed anti-diabetic compounds (alpha-activated carbonic acids, BM 130795 and BM 13907) induced carrier translocation to the same extent as insulin and phorbol esters [cytochalasin-B-binding sites in plasma membranes: basal 5 pmol/mg of protein, insulin 13 pmol/mg of protein, TPA (12-O-tetradecanoylphorbol 13-acetate) 11.8 pmol/mg of protein, BM 130795 10.8 pmol/mg of protein], but produce also only 40-50% of the insulin effect on glucose-transport activity (basal 5.8%, insulin 59%, TPA 23%, BM 130795 35%). Almost the full insulin effect was mimicked by a combination of phorbol esters and IP-oligosaccharides (basal 7%, insulin 50%, IP-oligosaccharides 30%, TPA 23%, IP-oligosaccharides + TPA 45%). None of these substances stimulated insulin-receptor kinase in vitro or in vivo, suggesting a post-kinase site of action. The data confirm the following aspects of the proposed model: (1) carrier translocation and carrier activation are two independently regulated processes; (2) the full insulin effect is mimicked only by a simultaneous stimulation of carrier translocation and intrinsic carrier activity, suggesting that insulin acts through a synergism of both mechanisms; (3) IP-oligosaccharides might be involved in the transmission of a stimulatory signal on carrier activity.

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.

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

1990 ◽  
Vol 258 (4) ◽  
pp. C648-C653 ◽  
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.

Stimulation of glucose transport in skeletal muscle by hypoxia

1991 ◽  
Vol 70 (4) ◽  
pp. 1593-1600 ◽  
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.

Stimulatory effect of lithium on glucose transport in rat adipocytes is not mediated by elevation of IP1

1998 ◽  
Vol 275 (2) ◽  
pp. E272-E277 ◽  
Author(s):  
Xiaoli Chen ◽  
Ellen G. McMahon ◽  
Eric A. Gulve
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Transport Activity ◽  
Concentration Dependent Manner ◽  
Inositol 1 ◽  
Rat Adipocytes ◽  
Increase Glucose Uptake ◽  
Stimulation Of

Lithium has been shown to increase glucose uptake in skeletal muscle and adipose tissues. The therapeutic effect of lithium on bipolar disease is thought to be mediated by its inhibitory effect on myo-inositol-1-monophosphatase (IMPase). We tested the hypothesis that the stimulatory effect of lithium on glucose uptake results from inhibition of IMPase and the resultant accumulation of inositol monophosphates (IP1) by comparing the effects of lithium and a selective IMPase inhibitor, L-690,488, on isolated rat adipocytes. Insulin produced a concentration-dependent stimulation of 2-deoxy-d-[14C]glucose (2-DG) transport (10 μU/ml caused half-maximal activation). Acute exposure to lithium stimulated basal glucose transport activity in a concentration-dependent manner, with a threefold stimulation at 30 mM lithium. Lithium also potentiated insulin-stimulated 2-DG transport. Lithium produced a concomitant increase in IP1 accumulation. In contrast, L-690,488 increased IP1 to levels comparable to those of lithium without stimulatory effects on 2-DG transport. These results demonstrate that stimulatory effects of lithium on glucose transport are not mediated by the inhibition of IMPase and subsequent accumulation of IP1 in rat adipocytes.

scholarly journals Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland

1990 ◽  
Vol 272 (1) ◽  
pp. 99-105 ◽  
Author(s):  
R J Madon ◽  
S Martin ◽  
A Davies ◽  
H A C Fawcett ◽  
D J Flint ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammary Gland ◽  
Membrane Protein ◽  
Rat Brain ◽  
Glucose Transport ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Golgi Vesicle

Plasma membrane- and Golgi vesicle-enriched membrane fractions were prepared from day-10 lactating rat mammary glands. Each fraction was found to contain a single set of D-glucose-inhibitable cytochalasin B-binding sites: plasma membranes and Golgi vesicles bound 20 +/- 2 and 53 +/- 4 pmol of cytochalasin/mg of membrane protein (means +/- S.E.M.), with dissociation constants of 259 +/- 47 and 520 +/- 47 nM respectively. Anti-peptide antibodies against the C-terminal region (residues 477-492) of the rat brain/human erythrocyte glucose transporter labelled a sharp band of apparent Mr 50,000 on Western blots of both fractions. Treatment with endoglycosidase F before blotting decreased the apparent Mr of this band to 38,000, indicating that it corresponded to a glycoprotein. Confirmation that this immunologically cross-reactive band was a glucose transporter was provided by the demonstration that it could be photoaffinity-labelled, in a D-glucose-sensitive fashion, with cytochalasin B. Quantitative Western blotting studies yielded values of 28 +/- 5 and 23 +/- 3 pmol of immunologically cross-reactive glucose transporters/mg of membrane protein in the plasma membrane and Golgi vesicle fractions respectively. From comparison with the concentration of cytochalasin B-binding sites, it is concluded that a protein homologous to the rat brain glucose transporter constitutes the major glucose transport species in the plasma membranes of mammary gland epithelial cells. Glucose transporters are also found in the Golgi membranes of these cells, at least half of them being similar, if not identical, to the transporters of the plasma membrane. However, their function in this location remains unclear.

Calcium stimulates glucose transport in skeletal muscle by a pathway independent of contraction

1991 ◽  
Vol 260 (3) ◽  
pp. C555-C561 ◽  
Author(s):  
J. H. Youn ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Muscle Tension ◽  
Creatine Phosphate ◽  
Transport Activity ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Glucose Analogue ◽  
Stimulation Of

In this study we investigated the possibility that an increase in cytoplasmic Ca2+ concentration that is too low to cause muscle contraction can induce an increase in glucose transport activity in skeletal muscle. The compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which induces Ca2+ release from the sarcoplasmic reticulum (SR), caused a dose-dependent increase in tension in rat epitrochlearis muscles at concentrations more than approximately 200 microM. Although 100 microM W-7 did not increase muscle tension, it accelerated loss of preloaded 45Ca2+. Glucose transport activity, measured with the nonmetabolizable glucose analogue 3-O-methylglucose, increased sixfold in muscles treated for 100 min with 50 microM W-7 (P less than 0.001) and eightfold in response to 100 microM W-7 (P less than 0.001). The increase in glucose transport activity was completely blocked with 25 microM cytochalasin B. There was no decrease in ATP or creatine phosphate concentrations ([approximately P]) in muscles incubated with 50 microM W-7. Dantrolene (25 microM), which blocks Ca2+ release from the SR, blocked the effects of W-7 both on 45Ca2+ release and on glucose transport activity. 9-Aminoacridine, another inhibitor of Ca2+ release from the SR, also blocked the stimulation of hexose transport by W-7. Caffeine, a compound structurally unrelated to W-7 that also releases Ca2+ from the SR, also increased glucose transport activity. Incubation of muscles with 3 mM caffeine for 30 min, which did not cause contraction or lower [approximately P], induced a threefold increase in 3-O-methylglucose transport (P less than 0.001). These results provide evidence suggesting that an increase in cytoplasmic Ca2+ too low to cause contraction or [approximately P] depletion can bring about an increase in glucose transport activity in skeletal muscle.

scholarly journals The translocation of the glucose transporter sub-types GLUT1 and GLUT4 in isolated fat cells is differently regulated by phorbol esters

1991 ◽  
Vol 275 (3) ◽  
pp. 597-600 ◽  
Author(s):  
B Vogt ◽  
J Mushack ◽  
E Seffer ◽  
H U Häring
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Phorbol Esters ◽  
Fat Cells ◽  
Low Density ◽  
Isolated Fat Cells ◽  
Kinase C

Insulin stimulates glucose transport in isolated fat cells by activation of glucose transporters in the plasma membranes and through translocation of the glucose transporter sub-types GLUT4 (insulin-regulatable) and GLUT1 (HepG2 transporter). The protein kinase C-stimulating phorbol ester phorbol 12-myristate 13-acetate (PMA) is able to mimic partially the effect of insulin on glucose transport, apparently through stimulation of carrier translocation. In order to ascertain whether protein kinase C is involved in the translocation signal to both carrier sub-types, we determined the effect of PMA on the subcellular distribution of GLUT1 and GLUT4 by immunoblotting with specific antibodies directed against these transporters. Isolated rat fat cells (4 x 10(6) cells/ml) were stimulated for 20 min with insulin (6 nM) or PMA (1 nM). 3-O-Methylglucose transport was determined and plasma membranes and low-density microsomes were prepared for Western blotting. 3-O-Methylglucose transport was stimulated 8-9-fold by insulin, and 3-4-fold by PMA (basal, 5.6 +/- 2.3%; insulin, 43.6 +/- 7.3%; PMA, 18.4 +/- 4.9%, n = 9). PMA was able to increase the amount of GLUT4 in the plasma membrane fraction by 2.5(+/- 0.9)-fold (n = 6) whereas insulin stimulation was 4.4(+/- 1.7)-fold (n = 6), paralleled by a corresponding decrease of transport in the low-density microsomes (insulin, 50 +/- 5% of basal; PMA, 63 +/- 11% of basal, n = 6). Although PMA regulates the translocation of GLUT4, it has no effect on GLUT1 in the same cell fractions (increase in plasma membranes: insulin, 1.7 +/- 0.5-fold; PMA, 0.91 +/- 0.1-fold, n = 4; decrease in low-density microsomes: insulin, 53 +/- 11% of basal; PMA, 101 +/- 5% of basal, n = 4). These data are in favour of a role for protein kinase C in signal transduction to GLUT4 but not to GLUT1 in fat cells.

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