Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

A Constitutive Enzyme System for Glucose Transport by Chlorella sorokiniana

It was found that the transport system for glucose (as measured by deoxyglucose uptake) in the high temperature strain of Chlorella (strain 07-11-05 or C. sorokiniana) was constitutive and the rate of uptake did not increase upon incubation of autotrophically grown cells with either deoxyglucose or glucose. The uptake obeyed Michaelis-Menten type kinetics with a concentration of 200 micromolar for half-saturation. The maximum rate of uptake was nearly 10 times faster per cell (at 38 C) than that reported for any other Chlorella. This rapid accumulation of deoxyglucose causes the passive efflux to become significant compared to the pump-driven influx and nonlinear uptake appears even after only 3 to 4 minutes.     

Circadian rhythm of glucose uptake in cultures of skeletal muscle cells and adipocytes in Wistar-Kyoto, Wistar, Goto-Kakizaki, and spontaneously hypertensive rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-D-(1-3H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.     

Circadian Rhythm of Glucose Uptake in Cultures of Skeletal Muscle Cells and Adipocytes in Wistar-Kyoto,Wistar, Goto-Kakizaki,and Spontaneously Hypertensive Rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-d-(1-³H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.     

Ghrelin stimulates insulin-induced glucose uptake in adipocytes

The gastric and hypothalamic hormone ghrelin is the endogenous agonist of the growth hormone secretagogue receptor GHS-R1(a). Ghrelin stimulates growth hormone release and appetite via the hypothalamus. However, putative direct peripheral effects of ghrelin remain poorly understood. Rat adipose tissue expresses GHS-R1(a) mRNA, suggesting ghrelin may directly influence adipocyte function. We have investigated the effects of ghrelin on insulin-stimulated glucose uptake in isolated white adipocytes in vitro. RT-PCR confirmed the expression of GHS-R1(a) mRNA in epididymal adipose tissue. However, GHS-R1(a) expression was not detected in the peri-renal fat pads. Ghrelin increased insulin-stimulated deoxyglucose uptake in isolated white adipocytes extracted from the epididymal fat pads of male Wistar rats. Ghrelin 1000 nM significantly increased deoxyglucose uptake by 55% in the presence of 0.1 nM insulin. However, ghrelin administration in the absence of insulin had no effect on adipocyte deoxyglucose uptake, suggesting that ghrelin acts synergistically with insulin. Des-acyl ghrelin, a major circulating non-octanylated form of ghrelin, had no effect on insulin-stimulated glucose uptake. Furthermore, acylated ghrelin had no effect on deoxyglucose uptake in adipocytes from peri-renal fat pads suggesting that ghrelin may influence glucose uptake via the GHS-R1(a). Ghrelin therefore appears to directly potentiate adipocyte insulin-stimulated glucose uptake in selective adipocyte populations. Ghrelin may play a role in adipocyte regulation of glucose homeostasis.     

Signals in the activation of opioid mu-receptors by loperamide to enhance glucose uptake into cultured C2C12 cells.

In an attempt to understand the signal pathways of opioid mu-receptors for glucose metabolism, we used loperamide to investigate the glucose uptake into the myoblast C2C12 cells. Loperamide enhanced the uptake of radioactive deoxyglucose into C2C12 cells in a concentration-dependent manner that was abolished in cells pre-incubated with naloxone or naloxonazine at concentrations sufficient to block opioid mu-receptors. Pharmacological inhibition of phospholipase C (PLC) by U73122 resulted in a concentration-dependent decrease in loperamide-stimulated uptake of radioactive deoxyglucose into C2C12 cells. This inhibition of glucose uptake by U73122 was specific since the inactive congener, U73343, failed to modify loperamide-stimulated glucose uptake. Moreover, both chelerythrine and GF 109203X diminished the action of loperamide at concentrations sufficient to inhibit protein kinase C (PKC). The obtained data suggest that an activation of opioid mu-receptors in C2C12 cells by loperamide may increase glucose uptake via the PLC-PKC pathway.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Metabolic effects of insulin on cardiomyocytes from control and diabetic db/db mouse hearts

Diabetic db/db mice exhibit profound insulin resistance in vivo, but the specific degree of cardiac insensitivity to insulin has not been assessed. Therefore, the effect of insulin on cardiomyocytes from db/db hearts was assessed by measuring two metabolic responses (deoxyglucose uptake and fatty acid oxidation) and the phosphorylation of two enzymes in the insulin-signaling cascade [Akt and AMP-activated protein kinase (AMPK)]. Maximal insulin-stimulated deoxyglucose transport was reduced to 58 and 40% of control in cardiomyocytes from db/db mice at two ages (6 and 12 wk). Insulin-stimulated deoxyglucose uptake was also reduced in myocytes from transgenic db/db mice overexpressing the insulin-sensitive glucose transporter (db/db-hGLUT4). Treatment of db/db mice for 1 wk with an insulin-sensitizing peroxisome proliferator-activated receptor-gamma agonist (COOH) completely normalized insulin-stimulated deoxyglucose uptake. Insulin had no direct effect on palmitate oxidation by either control or db/db cardiomyocytes, but the combination of insulin and glucose reduced palmitate oxidation, likely an indirect effect secondary to increased glucose uptake. Insulin had no effect on AMPK phosphorylation from either control or db/db cardiomyocytes. Insulin increased the phosphorylation of Akt in all cardiomyocyte preparations (control, db/db, COOH-treated db/db) to the same extent. Thus insulin has selective metabolic actions in mouse cardiomyocytes; deoxyglucose uptake and Akt phosphorylation are increased, but fatty acid oxidation and AMPK phosphorylation are unchanged. Insulin resistance in db/db cardiomyocytes is manifested by reduced insulin-stimulated deoxyglucose uptake.     

THE KINETIC PROPERTIES OF HEXOSE TRANSPORT INTO SYNAPTOSOMES FROM GUINEA PIG CEREBRAL CORTEX

Abstract— Hexose uptake into synaptosomes has been shown to occur by a saturable mechanism with a relatively small component due to passive diffusion. Competition between glucose and deoxyglucose for entry was demonstrated and the kinetic properties of the process were studied by using glucose as a competitive inhibitor of deoxyglucose entry. During kinetic analysis of the transport process higher affinities for both hexoses were indicated by the results from the synaptosome preparation compared with those from cerebral cortex slices. The kinetic properties of glucose inhibition of deoxyglucose uptake into synaptosomes could not be interpreted completely in terms of a unimolecular transport model. The results appear to follow predictions for 'doubly competitive inhibition', and some evidence for polyvalency of the uptake process was obtained.     

Suppression of monocyte and neutrophil function by recombinant IL-2

Little IS known about the influence of IL-2 on phagocytes. We now describe the effects of human recombinant IL-2 on human neutrophil and monocyte functions related to mobility, phagocytosis, glucose uptake, respiration and degranulation. Neutrophil adherence and hexose monophosphate shunt activities were both suppressed after incubation with IL-2. IL-2 had no effect on neutrophil migration, phagocytosis, deoxyglucose uptake or degranulation, ionocytes demonstrated a greater sensitivity to IL-2 with suppression of monocyte adherence, random and stimulated migration, glucose uptake and hexose monophosphate shunt activity, even after addition of phorbol myristate acetate. Monocyte phagocytosis and degranulation were not affected. All of the effects observed were dose-dependent within a biologically active range for IL-2. These studies suggest that IL-2 may have an important down-regulatory role across a broad range of monocyte functions including movement, deoxyglucose uptake and respiration. However, its role in regulation of neutrophil function is limited to adherence and respiration. IL-2 may be a more versatile cytokine than has previously been appreciated.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Glucocorticoids enhance glucose uptake and affect differentiation and beta-adrenergic responsiveness in muscle cell cultures

The role of glucocorticoids (GLC) in liver glycogen metabolism is well characterized; its role in peripheral tissues is not as well understood (Baxter, 1976). GLC administration in vivo is associated with hyperglycemia, but it is not clear whether decreased glucose uptake in a peripheral tissue like muscle accounts, in part, for the effect. We investigated the relationship of glucose uptake to beta-adrenergic responsiveness in muscle cell cultures exposed to GLC. Under these conditions GLC and other serum factors are present in at least a tenfold dilution relative to in vivo conditions. We observed that the GLC dexamethasone (DEX) induces a significantly enhanced Vmax for deoxyglucose uptake in the rat muscle cell lines L8 (200--400%) and L6E9 (50--100%). DEX inhibits cell fusion and promotes epithelioid morphology within the effective dose range (L8 greater than L6E9). Growth is slightly enhanced (10--20%) at 0.1--1.0 microM. In these cells DEX also inhibits intracellular beta-adrenergic-sensitive cyclic AMP accumulation and reduces basal, catecholamine-sensitive and fluoride-sensitive adenylate cyclase in cell homogenates. The effects of DEX on deoxyglucose uptake and beta-adrenergic responsiveness are both dose (1 nM--0.1 nM) and time (1--3 days) dependent, and reversible. The degree of inhibition of the beta-adrenergic system seems to be directly related to the degree of enhancement of deoxyglucose uptake. These observations suggest that the action of DEX on muscle cell glucose uptake is related to its effect on the beta-adrenergic system.     

Preferential uptake and accumulation of oxidized vitamin C by THP-1 monocytic cells.

THP-1 cells preferentially accumulate vitamin C in its oxidized form. The uptake displays first-order kinetics and leads to a build-up of an outward concentration gradient which is stable in the absence of extracellular vitamin. The transport is faster than reduction by extracellular glutathione or by added cytosolic extract, and glutathione-depleted cells show the same uptake rates as control cells. In addition, energy depletion or oxidation of intracellular sulfhydryls does not inhibit accumulation of ascorbate. The accumulation, however, always occurs in the reduced form. The affinity for dehydroascorbate is lower (Km 450 microM vs 60 microM) than for reduced ascorbate, but the maximal rate is more than 30 times higher (581 compared to 19 pmol.min-1 per 106 cells), and it is independent of sodium, whereas the uptake of ascorbate is not. The sodium gradient also allows accumulation of reduced ascorbate. Inhibitors of glucose transport by the GLUT-1 transporter also inhibit uptake of dehydroascorbate (DHA), but there are some inconsistencies, because the Ki-values are higher than reported for the isolated transporter and one inhibitor (deoxyglucose) is noncompetitive. The preferential uptake of the dehydro-form of the vitamin may be useful for situations where this short-lived metabolite is formed by oxidation in the environment.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

An amino acid mixture enhances insulin-stimulated glucose uptake in isolated rat epitrochlearis muscle

Protein and certain amino acids (AA) have been found to lower blood glucose. Although these glucose-lowering AA are important modulators of skeletal muscle metabolism, their impact on muscle glucose uptake remains unclear. We therefore examined how an AA mixture consisting of 2 mM isoleucine, 0.012 mM cysteine, 0.006 mM methionine, 0.0016 mM valine, and 0.014 mM leucine impacts skeletal muscle glucose uptake in the absence or presence of a submaximal (sINS) or maximal insulin (mINS) concentration. The AA mixture, sINS, and mINS significantly increased 2-[(3)H]deoxyglucose (2-DG) uptake by 63, 79, and 298% above basal, respectively. When the AA mixture was combined with sINS and mINS, 2-DG uptake was further increased significantly by 26% (P = 0.028) and 14% (P = 0.032), respectively. Western blotting analysis revealed that the AA mixture increased basal and sINS Akt substrate of 160 kDa (AS160) phosphorylation, while AA mixture did not change phosphorylation of Akt or mammalian target of rapamycin (mTOR) under these conditions. Interestingly, addition of the AA mixture to mINS increased phosphorylation of mTOR, Akt as well as AS160, compared with mINS alone. These data suggest that certain AA increase glucose uptake in the absence of insulin and augment insulin-stimulated glucose uptake in an additive manner. Furthermore, these effects appear to be mediated via a pathway that is independent from the canonical insulin cascade and therefore may prove effective as an alternative therapeutic treatment for insulin resistance.     

Extension of the 2-Deoxyglucose Method to the Fetus in Utero: Theory and Normal Values for the Cerebral Glucose Consumption in Fetal Guinea Pigs

Abstract: Fetal cerebral metabolism changes during development. The normal fetal metabolic rate must be known to evaluate pathophysiological changes. Therefore, we determined the regional cerebral glucose consumption in the fetal guinea pig. This required the application of the 2-deoxyglucose method to this species. We measured both the transfer coefficients of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the lumped constant in chronically prepared undisturbed guinea pig dams using a three-compartment model. Furthermore, the ratio between the initial clearances of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the ratio between the phosphorylation coefficients of these substrates in the fetal brain were determined. The total cerebral glucose consumption measured by the deoxyglucose method (10 ± 1.2 µmol/100 g/min) was similar to that calculated from the glucose concentration and the phosphorylation coefficient of glucose in the cerebrum (10 ± 0.4 µmol/100 g/min). We conclude that the 2-deoxyglucose method is applicable to the guinea pig, and we further conclude that in the fetal guinea pig cerebral glucose consumption is 10 times lower than that in the adult.     

Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons

The influence of ACTH and some of its N-terminal related peptides was investigated on the uptake of (3H)-2-deoxy-D-glucose in pure cultures of neurons from chick embryo cerebral hemispheres. ACTH influences deoxyglucose uptake in a time and dose-dependent fashion. The stimulation of deoxyglucose uptake is observed after a delay of 6-8 h and requires active protein synthesis. ACTH does not affect deoxyglucose in non-neuronal cells (astroglial cells, hepatocytes, myoblasts, fibroblasts). The effect of various peptide hormones, neuropeptides and growth factors, active in the central nervous system or other tissues, has also been examined. None of these were able to stimulate deoxyglucose uptake, suggesting that the regulation of hexose uptake in neurons is specific for the ACTH-related peptides.     

The effect of 5 days of aspartate and asparagine supplementation on glucose transport activity in rat muscle

The consumption of protein supplements containing amino acids is increasing around the world. Aspartate (Asp) and asparagine (Asn) are amino acids metabolized by skeletal muscle. This metabolism involves biochemical pathways that are involved in increasing Krebs cycle activity via anaplerotic reactions, resulting in higher glutamine concentrations. A connection between amino acid supplementation, glycogen concentration, and glucose uptake has been previously demonstrated. The purpose of this study was to evaluate the effect of Asp and Asn supplementation on glucose uptake in rats using three different glycogen concentrations. The results indicate that Asp and Asn supplementation in rats with high glycogen concentrations (fed state) further increased the glycogen concentration in the muscle, and decreased in vitro 2‐deoxyglucose (a glucose analog) uptake by the muscle at maximal insulin concentrations. When animals had a medium glycogen concentration (consumed lard for 3 days), glucose uptake was higher in the supplemented group at sub‐maximal insulin concentrations. We conclude that supplementation of Asp and Asn reduced glucose transport in rat muscle only at higher levels of glycogen. The ingestion of lard for 3 days changed the responsiveness and sensitivity to insulin, and that group had higher levels of insulin sensitivity with Asp and Asn supplementation. Copyright © 2009 John Wiley & Sons, Ltd.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.