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.

beta-Adrenergic stimulation has been reported to inhibit insulin-stimulated glucose transport in adipocytes. This effect has been attributed to a decrease in the intrinsic activity of the GLUT-4 isoform of the glucose transporter that is mediated by phosphorylation of GLUT-4. Early studies showed no inhibition of insulin-stimulated glucose transport by epinephrine in skeletal muscle. The purpose of this study was to determine the effect of epinephrine on GLUT-4 phosphorylation, and reevaluate the effect of beta-adrenergic stimulation on insulin-activated glucose transport, in skeletal muscle. We found that 1 microM epinephrine, which raised adenosine 3',5'-cyclic monophosphate approximately ninefold, resulted in GLUT-4 phosphorylation in rat skeletal muscle but had no inhibitory effect on insulin-stimulated 3-O-methyl-D-glucose (3-MG) transport. In contrast to 3-MG transport, the uptakes of 2-deoxyglucose and glucose were markedly inhibited by epinephrine treatment. This inhibitory effect was presumably mediated by stimulation of glycogenolysis, which resulted in an increase in glucose 6-phosphate concentration to levels known to severely inhibit hexokinase. We conclude that 1) beta-adrenergic stimulation decreases glucose uptake by raising glucose 6-phosphate concentration, thus inhibiting hexokinase, but does not inhibit insulin-stimulated glucose transport and 2) phosphorylation of GLUT-4 has no effect on glucose transport in skeletal muscle.

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Discrepancy between glucose transport and transporters in human femoral adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1989.256.1.e179 ◽

1989 ◽

Vol 256 (1) ◽

pp. E179-E185 ◽

Cited By ~ 1

Author(s):

E. Karnieli ◽

R. Moscona ◽

R. Rafaeloff ◽

Y. G. Illouz ◽

M. Armoni

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Cytochalasin B ◽

Intact Cell ◽

Transport Activity ◽

Intact Cells ◽

Insulin Resistant ◽

Large Size ◽

Microsomal Membranes ◽

Subcutaneous Adipose

Obesity is known to be associated with insulin resistance in human and rat adipocytes. However, it is not known what are the perturbations in insulin action that contribute to disproportional femoral obesity. Thus femoral subcutaneous adipose tissue was obtained from lean women with various degrees of disproportional obesity, by liposuction. 3-O-methylglucose (3-O-methyl-D-glucopyranose) transport was measured in intact cells, and glucose transporter levels in plasma and low-density microsomal membranes were assessed using the cytochalasin B binding assay. A sixfold cellular enlargement was associated with increase in both basal and insulin-stimulated glucose transport activity in the intact cell, and a 300-600% increase in insulin stimulating effect per se. However, when glucose transporter levels were assessed, this cellular enlargement was accompanied by a 40-70% transporter depletion (in largest cells compared with smallest ones) in both subcellular fractions examined, from either basal or insulin-stimulated cells. This discrepancy, between increasing cellular glucose transport rates and relative depletion of transporter levels, suggests that these cells are not insulin resistant, as could be expected from their large size. A role for other factor(s), additional to glucose transporter levels, in the regulation of cellular glucose uptake rate is thus suggested.

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Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport

Biochemical Journal ◽

10.1042/bj2510491 ◽

1988 ◽

Vol 251 (2) ◽

pp. 491-497 ◽

Cited By ~ 12

Author(s):

S Matthaei ◽

J M Olefsky ◽

E Karnieli

Keyword(s):

Protein Synthesis ◽

Glucose Transport ◽

Plasma Membranes ◽

Glucose Transporter ◽

Cytochalasin B ◽

Glucose Transporters ◽

Electrophoretic Analysis ◽

Adipocyte Plasma Membranes ◽

Inhibitor Of Protein Synthesis ◽

Adipose Cells

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.

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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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The sulfonylurea drug, glimepiride, stimulates glucose transport, glucose transporter translocation, and dephosphorylation in insulin-resistant rat adipocytes in vitro

Diabetes ◽

10.2337/diabetes.42.12.1852 ◽

1993 ◽

Vol 42 (12) ◽

pp. 1852-1867 ◽

Cited By ~ 20

Author(s):

G. Muller ◽

S. Wied

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Rat Adipocytes ◽

Insulin Resistant

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Decreased expression of glucose transporter in muscle from insulin-resistant patients

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1991.260.3.e459 ◽

1991 ◽

Vol 260 (3) ◽

pp. E459-E463 ◽

Cited By ~ 6

Author(s):

G. L. Dohm ◽

C. W. Elton ◽

J. E. Friedman ◽

P. F. Pilch ◽

W. J. Pories ◽

...

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Vastus Lateralis ◽

Insulin Dependent Diabetes Mellitus ◽

Dependent Diabetes Mellitus ◽

Obese Patients ◽

Insulin Resistant ◽

Glut 4 ◽

Obesity And Diabetes

We have observed that in vitro incubated human muscle fiber strips from obese patients with or without non-insulin-dependent diabetes mellitus (NIDDM) have reduced insulin-stimulated glucose transport rates compared with nonobese control patients. To investigate if the decrease in glucose transport is associated with a depletion of glucose transport protein, we performed Western blot analysis of muscle samples from nonobese control, obese nondiabetic, and obese NIDDM patients to measure the levels of the muscle-adipose tissue glucose transporter (GLUT-4) protein. Glucose transporter protein was depressed by 23% in the obese nondiabetic and 18% in the obese NIDDM group. The results were essentially the same in the rectus abdominus and vastus lateralis muscles. These data suggest that the decreased glucose transport rate observed in muscle of these obese patients with or without NIDDM may be due, at least in part, to a decreased expression of the "insulin-sensitive" (GLUT-4) glucose transporter. This alteration may play a role in the insulin resistance seen in obesity and diabetes.

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Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise

Journal of Applied Physiology ◽

10.1152/jappl.1990.68.1.193 ◽

1990 ◽

Vol 68 (1) ◽

pp. 193-198 ◽

Cited By ~ 96

Author(s):

L. J. Goodyear ◽

M. F. Hirshman ◽

P. A. King ◽

E. D. Horton ◽

C. M. Thompson ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transport ◽

Time Course ◽

Plasma Membranes ◽

Glucose Transporter ◽

Male Rats ◽

Intrinsic Activity ◽

Plasma Membrane Vesicles

Recent reports have shown that immediately after an acute bout of exercise the glucose transport system of rat skeletal muscle plasma membranes is characterized by an increase in both glucose transporter number and intrinsic activity. To determine the duration of the exercise response we examined the time course of these changes after completion of a single bout of exercise. Male rats were exercised on a treadmill for 1 h (20 m/min, 10% grade) or allowed to remain sedentary. Rats were killed either immediately or 0.5 or 2 h after exercise, and red gastrocnemius muscle was used for the preparation of plasma membranes. Plasma membrane glucose transporter number was elevated 1.8- and 1.6-fold immediately and 30 min after exercise, although facilitated D-glucose transport in plasma membrane vesicles was elevated 4- and 1.8-fold immediately and 30 min after exercise, respectively. By 2 h after exercise both glucose transporter number and transport activity had returned to nonexercised control values. Additional experiments measuring glucose uptake in perfused hindquarter muscle produced similar results. We conclude that the reversal of the increase in glucose uptake by hindquarter skeletal muscle after exercise is correlated with a reversal of the increase in the glucose transporter number and activity in the plasma membrane. The time course of the transport-to-transporter ratio suggests that the intrinsic activity response reverses more rapidly than that involving transporter number.

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