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

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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Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice

Biochemical Journal ◽

10.1042/bj3210075 ◽

1997 ◽

Vol 321 (1) ◽

pp. 75-81 ◽

Cited By ~ 17

Author(s):

Joseph T. BROZINICK ◽

Scott C. McCOID ◽

Thomas H. REYNOLDS ◽

Cindy M. WILSON ◽

Ralph W. STEVENSON ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Surface ◽

Glucose Transport ◽

Glucose Transporter ◽

Subcellular Fractionation ◽

Muscle Membrane ◽

Transport Activity ◽

Hindlimb Muscles ◽

Subcellular Membrane ◽

Glucose Transporter Glut4

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 µ-units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

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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.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)61162-7 ◽

1987 ◽

Vol 262 (11) ◽

pp. 5118-5124 ◽

Cited By ~ 4

Author(s):

B.B. Kahn ◽

S.W. Cushman

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Diabetic Rats ◽

Intrinsic Activity ◽

Transport Activity ◽

Streptozotocin Diabetic Rats ◽

Adipose Cells

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

Biochemical Journal ◽

10.1042/bj2850223 ◽

1992 ◽

Vol 285 (1) ◽

pp. 223-228 ◽

Cited By ~ 11

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.

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Noradrenaline increases glucose transport into brown adipocytes in culture by a mechanism different from that of insulin

Biochemical Journal ◽

10.1042/bj3140485 ◽

1996 ◽

Vol 314 (2) ◽

pp. 485-490 ◽

Cited By ~ 29

Author(s):

Yasutake SHIMIZU ◽

Danuta KIELAR ◽

Yasuhiko MINOKOSHI ◽

Takashi SHIMAZU

Keyword(s):

Cyclic Amp ◽

Glucose Uptake ◽

Glucose Transport ◽

Glucose Transporter ◽

Sympathetic Nerves ◽

Intrinsic Activity ◽

Brown Adipocytes ◽

Brown Adipose ◽

Km Value ◽

Glucose Transporter Glut4

Glucose uptake into brown adipose tissue has been shown to be enhanced directly by noradrenaline (norepinephrine) released from sympathetic nerves. In this study we characterized the glucose transport system in cultured brown adipocytes, which responds to noradrenaline as well as insulin, and analysed the mechanism underlying the noradrenaline-induced increase in glucose transport. Insulin increased 2-deoxyglucose (dGlc) uptake progressively at concentrations from 10-11 to 10-6 M, with maximal stimulation at 10-7 M. Noradrenaline concentrations ranging from 10-8 to 10-6 M also enhanced dGlc uptake, even in the absence of insulin. The effects of noradrenaline and insulin on dGlc uptake were additive. The stimulatory effect of noradrenaline was mimicked by the β3-adrenergic agonist, BRL37344, at concentrations two orders lower than noradrenaline. Dibutyryl cyclic AMP also mimicked the stimulatory effect of noradrenaline, and the antagonist of cyclic AMP, cyclic AMP-S Rp-isomer, blocked the enhancement of glucose uptake due to noradrenaline. Furthermore Western blot analysis with an anti-phosphotyrosine antibody revealed that, in contrast with insulin, noradrenaline apparently does not stimulate intracellular phosphorylation of tyrosine, suggesting that the noradrenaline-induced increase in dGlc uptake depends on elevation of the intracellular cyclic AMP level and not on the signal chain common to insulin. When cells were incubated with insulin, the content of the muscle/adipocyte type of glucose transporter (GLUT4) in the plasma membrane increased, with a corresponding decrease in the amount in the microsomal membrane. In contrast, noradrenaline did not affect the subcellular distribution of GLUT4 or that of the HepG2/erythrocyte type of glucose transporter. Although insulin increased Vmax. and decreased the Km value for glucose uptake, the effect of noradrenaline was restricted to a pronounced decrease in Km. These results suggest that the mechanism by which noradrenaline stimulates glucose transport into brown adipocytes is not due to translocation of GLUT but is probably due to an increase in the intrinsic activity of GLUT, which is mediated by a cyclic AMP-dependent pathway.

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Thrombin-induced translocation of GLUT3 glucose transporters in human platelets

Biochemical Journal ◽

10.1042/bj3280511 ◽

1997 ◽

Vol 328 (2) ◽

pp. 511-516 ◽

Cited By ~ 27

Author(s):

R. Lynn SORBARA ◽

Theresa M. DAVIES-HILL ◽

Ellen M. KOEHLER-STEC ◽

J. Susan VANNUCCI ◽

K. McDonald HORNE ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Glucose Transport ◽

Glucose Transporter ◽

Glucose Transporters ◽

Human Platelets ◽

Half Time ◽

Transport Activity ◽

Kinase C ◽

Adipose Cells

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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Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes

Biochemical Journal ◽

10.1042/bj2900707 ◽

1993 ◽

Vol 290 (3) ◽

pp. 707-715 ◽

Cited By ~ 10

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.

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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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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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