The Hemopoietic Growth Factor, Interleukin-3, Promotes Glucose Transport by Increasing the Specific Activity and Maintaining the Affinity for Glucose of Plasma Membrane Glucose Transporters*

(Received for publication, August 21, 1996, and in revised form, March 31, 1997)

Kathy D. McCoy , Nuzhat Ahmed , An S. Tan and Michael V. Berridge Dagger

From the Malaghan Institute of Medical Research, Wellington School of Medicine, P. O. Box 7060, Wellington South 6002, New Zealand

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 ± 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63-93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.


INTRODUCTION

The energy requirements of most mammalian cells are met through a continuous supply of glucose which circulates in the blood or is supplied in culture medium. Glucose enters cells through specific glucose transporter molecules (Gluts) present in the plasma membrane. A small family of facilitative glucose transporter molecules is known and the different transporter subtypes are expressed in a tissue-specific manner (1, 2). Glucose is transported into cells down its concentration gradient, the rate of transport being dependent on the absolute concentration of glucose, the steepness of the gradient and the level of expression of transporters at the cell surface. The extent to which the intrinsic glucose transporting ability of individual transporters is regulated is not known although evidence suggests that this level of regulation is also important. For example, excessive Glut-1 expression on human erythrocytes implies negative control, while in pigeon erythrocytes, sugar transport via Glut-1 appears to be negatively regulated in that sugar transport can be derepressed by metabolic poisons like cyanide (3). Similarly, acute stimulation of rat liver clone 9 cells with azide markedly increased glucose uptake without increasing Glut-1 expression at the plasma membrane or transporter affinity for glucose (4). In another study, depriving 3T3-L1 adipocytes of glucose increased glucose transport without increasing transporter expression or translocation (5).

In insulin-responsive tissues in the basal state, glucose transport occurs primarily through Glut-1, but when stimulated by insulin, the majority of transport occurs through Glut-4. In adipose and muscle cells, increased transport in response to insulin occurs in the short-term by translocation of Glut-4 from an intracellular pool to the plasma membrane (6-8). The Glut-1 transporter subtype may also be recruited to the plasma membrane in response to insulin although this occurs to a much lesser extent than with Glut-4 (9, 10). However, in addition to recruitment, there is also evidence for modulation of the intrinsic activity of both Glut-1 and Glut-4 (11-14).

Despite the importance of glucose uptake for normal cell metabolism, regulation of glucose transport in systems responding to signals other than insulin has not been widely studied. Increased glucose transport in response to growth factors has been shown previously in fibroblasts (15), retinal epithelial cells (16), developing myocytes (17, 18), lymphoid cells (19), and other hemopoietic cells (20-22). With some cells, increased transport has been shown to occur through increased glucose transporter expression or translocation of stored transporters to the plasma membrane (15-19). However, in other studies intrinsic activation of transporters already expressed at the cell surface may be involved in the stimulation of glucose transport (23, 24).

In the pro-B lymphocyte cell line, Ba/F3, treatment with IL-31 or insulin-like growth factor-1 led to decreased transporter Km for glucose compared with untreated cells, whereas no change in Vmax was observed (23). This suggests that growth factors can regulate the affinity of glucose transporters for glucose without altering the number of transporters present. These studies were confirmed and extended with the growth factor-dependent myeloid cell line, 32D. IL-3 was shown to maintain the affinity of transporters for glucose without altering Vmax, whereas IL-3 withdrawal resulted in an increase in Km and these changes were associated with protein phosphorylation (24). In these studies, both Ba/F3 and 32D cells are presumed to express predominantly Glut-1 as the major transporter subtype as has been shown for other cultured hemopoietic cells. These studies suggest that growth factors may act to regulate glucose transport in hemopoietic cells by modulating the activation state of glucose transporters.

The present study extends these findings by showing that IL-3 stimulates 2-DOG uptake into 32D cells without a corresponding increase in transporter expression in crude cell membranes or translocation of intracellularly stored transporters to the plasma membrane. Furthermore, withdrawal of IL-3 leads to a significant decline in 2-DOG uptake that is associated with a decline in transporter affinity for glucose. Changes in transporter expression may also contribute to this effect. These results provide strong independent evidence that IL-3 acts through its receptor to modulate the intrinsic transport function of glucose transporters on hemopoietic cells, and raise the possibility that cell survival, growth, proliferation, and function may be regulated by growth factors and cytokines at the level of glucose transport in addition to other levels of metabolic and cell cycle control.


EXPERIMENTAL PROCEDURES

Cells and Cell Culture

32D clone 23 cells (32D), originally derived from long-term bone marrow cultures of C3H/HeJ mice (25), were obtained from Prof. J. D. Watson (Genesis Research and Development Corp., Auckland, NZ) and were maintained in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, 10% Wehi-3-conditioned medium as a source of IL-3, 25 µg/ml penicillin, and 25 µg/ml streptomycin and cultured at 37 °C in a humidified incubator maintained at 5% CO2.

Measurement of 2-Deoxyglucose Uptake

32D cells (4 × 105 cells/ml) were cultured in serum-free RPMI containing 2 ng/ml IL-3 (recombinant murine IL-3 obtained from Prof. J. D. Watson, Genesis Research and Development Corp.) for 16 h. Cells were washed three times, resuspended to 5 × 106 cells/ml in serum-free RPMI and treated with or without 20 ng/ml IL-3 for the times indicated. Cells were collected by centrifugation, counted, and cell viability assessed by trypan blue exclusion. 1 × 106 viable cells were removed in duplicate at each time point for 2-deoxyglucose (2-DOG) uptake measurement and the remaining cells put on ice for membrane preparation. Cells were resuspended in 250 µl of glucose-free RPMI (Sigma) and preincubated for 3 min at 37 °C. 2-[3H]DOG (1 µCi, Amersham) and 2-DOG (Fluka, Buchs, Switzerland) were added to a final concentration of 100 µM, or for kinetic analysis, 0.2-2.5 mM. Glucose uptake was measured for 3 min at 37 °C. Uptake was stopped by adding 250 µl of ice-cold glucose-free RPMI containing 0.6 mM phloretin and the cells placed on ice. Cells were centrifuged through a cushion of 10% ice-cold bovine serum albumin for 30 s at 8,800 × g in a Microfuge, washed with ice-cold glucose-free RPMI and the pellet lysed with 1% Triton X-100 and radioactivity determined in a liquid scintillation counter (Beckman).

Measurement of 3-O-Methylglucose Uptake

3-O-[3H]Methylglucose (Amersham) uptake was determined by a similar procedure to that described above for 2-DOG uptake except that cells were starved of serum for 2 h in the absence of IL-3 prior to treatment with and without IL-3. The reaction was performed at 37 °C and was terminated after 30 s at which time uptake was in the linear range.

Crude Membrane Preparation

Total cell membranes were isolated by the method of Nagamatsu et al. (26) with slight modifications. Briefly, cells were homogenized in 1 ml of homogenization buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM sucrose, 1 mM phenylmethylsulfonyl fluoride). The nuclei and cell debris were removed from the homogenate by centrifugation at 900 × g for 10 min at 4 °C. The resulting supernatant was centrifuged at 110,000 × g for 75 min at 4 °C (SW40 rotor, Beckman ultracentrifuge). The membrane pellet was solubilized in buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) for a minimum of 1 h at 4 °C. Insoluble material was removed by centrifugation at 14,000 × g for 10 min at 4 °C and 1 µg/ml aprotinin added to solubilized membrane samples prior to storage at -70 °C.

Cell Fractionation

Cells were incubated with or without 20 ng/ml IL-3 for 6 h, then were harvested and resuspended in Hepes-sucrose buffer (10 mM Hepes pH 7.4, 250 mM sucrose, 1 mM EGTA, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin, pepstatin A, and aprotinin) at 3.0 × 107 cells/ml. The cell suspension was then added to an equal volume of 1 mM Hepes and incubated for 30 min on ice to allow cells to swell. Cells were fragmented by passing 10-15 times through a ball bearing homogenizer (8.020-mm bore, EMBL, Heidelberg, Germany) with a 8.008-mm ball bearing until approximately 95% of cells were broken. The cell homogenate was centrifuged at 900 × g for 10 min to remove nuclei and unbroken cells. The postnuclear homogenate was then layered over a continuous linear gradient of 0-45% Nycodenz (Nycomed Pharma, Oslo, Norway) in Hepes sucrose buffer containing protease inhibitors and the gradient centrifuged at 101,000 × g (SW40 rotor, Beckman ultracentrifuge) at 4 °C for 2 h. Gradient fractions (0.4 ml) were collected from the bottom of the gradient using a peristaltic pump.

Alkaline phosphodiesterase I (EC 3.1.4.1) was used as a marker enzyme for plasma membrane. Membrane fractions were added to reaction buffer (20 mM Tris-HCl, pH 9.0, 0.1% Triton X-100, 2 mM sodium thymidine 5'-monophosphate p-nitrophenyl ester) and absorbance read at 405 nm after 2 h incubation at 37 °C.

NADPH-cytochrome c reductase (EC 1.6.2.4) was used to monitor endoplasmic reticulum. Membrane fractions were added to reaction buffer (50 mM Tris-HCl, pH 7.5, 0.33 mM KCN, 0.1 mM NADPH, and 0.05 mM cytochrome c) and absorbance read at 550 nm after 15 min incubation at room temperature.

Protein Estimation

The concentration of protein in the membrane samples was determined by a modified Lowry protein assay (27).

SDS-PAGE and Western Blot Analysis

15 µg of total membrane protein or 20 µl of each gradient fraction were added to Laemmli sample buffer, incubated for 10 min at 37 °C, and proteins separated on 8% SDS-polyacrylamide gels using a Mini-Protean II apparatus (Bio-Rad). Proteins were electrophoretically transferred to supported nitrocellulose membranes (Hybond-C Super, Amersham) at 15 V for 30 min. Nonspecific binding to membranes was blocked by incubating in Tris-buffered saline, 0.05% Tween 20 (TBS/T) containing 5% non-fat dried milk and 5% bovine serum albumin for 1 h at room temperature. Membranes were then incubated with primary antisera diluted in TBS/T containing 1% bovine serum albumin for 2 h at room temperature. Rabbit polyclonal antiserum against the COOH terminus of rat Glut-1 or Glut-4 (1/500 dilution, EastAcres Biologicals, Southbridge, MA) and affinity-purified antibodies against the COOH terminus of mouse Glut-3 (1/50 dilution, gift from G. W. Gould, University of Glasgow, Scotland) were employed. Membranes were washed with TBS/T, then incubated for 1 h with affinity-purified swine Ig anti-rabbit IgG-conjugated to horseradish peroxidase (DAKO Corp., Carpinteria, CA) diluted 1/4000 in TBS/T. Membranes were washed and developed using enhanced chemiluminescence (Amersham).

Nitrocellulose membranes were stripped of bound antibodies by submerging in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubating at 50 °C for 30 min. Membranes were washed with TBS/T, then blocked before reprobing with another antibody.

Densitometric analysis used a Macintosh LC 630 computer and the public domain NIH image program. Density of autoradiographic bands was linear over the range used in the experiments described.


RESULTS

Effects of IL-3 Addition and Withdrawal on 2-DOG Uptake by 32D Cells

To determine the effects of IL-3 on glucose uptake, 32D cells were preincubated with 2 ng/ml IL-3 in the absence of serum for 16 h. This preconditioning regime has been shown to maintain the viability of cells in the absence of cell proliferation (24). Cells were then treated in the presence or absence of 20 ng/ml IL-3 for times up to 16 h and 2-[3H]DOG uptake determined. IL-3 significantly stimulated glucose uptake with the greatest increase occurring after 6 h (Fig. 1). Cells incubated for the same period of time without IL-3 showed reduced 2-DOG uptake. Cell viability as determined by trypan blue exclusion was greater than 91% in the presence of IL-3, and at times up to 12 h in the absence of IL-3 but dropped to 74% after 16 h incubation without IL-3.


Fig. 1. Effect of IL-3 addition and withdrawal on 2-DOG uptake by 32D cells. 32D cells were incubated in the presence (black-square) or absence (square ) of 20 ng/ml IL-3 and [3H]2-DOG uptake was determined at various times. Values are the mean ± S.E. of a minimum of three separate experiments involving duplicate determinations. Where error bars are not shown they fall within the symbol. Compared with the zero time point all results are significant to p < 0.02 by the non-parametric paired Wilcoxon Signed-Rank test.
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Effects of IL-3 on Glucose Transporter Expression

To determine whether increased glucose uptake in response to IL-3 was associated with a corresponding increase in transporter expression, crude membranes were prepared from 32D cells treated with or without IL-3 for times up to 16 h. Membrane proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes for immunoblotting with subtype-specific antisera against Glut-1 and Glut-3, both of which were known to be present on 32D cells.2 Fig. 2 shows that there was little change in the expression of Glut-1 and Glut-3 in the presence of IL-3 at times up to 16 h, whereas IL-3 withdrawal for 16 h caused Glut-1 and Glut-3 expression to decline sharply. The ratio of Glut-1:Glut-3 did not alter significantly during the course of the experiment. Densitometric analysis of Glut-1 and Glut-3 expression is summarized in Fig. 3 and shows no significant changes in the expression of Glut-1 and Glut-3 prior to 16 h.


Fig. 2. Effect of IL-3 addition and withdrawal on the expression of total membrane Glut-1 and Glut-3 in 32D cells. 32D cells were serum-starved for 16 h in the presence of 2 ng/ml IL-3 after which they were treated with or without 20 ng/ml IL-3 for the times indicated. Crude membranes were prepared from the cells as indicated under "Experimental Procedures." Membrane proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for Glut-1 (A), Glut-3 (B), and Glut-4 (C) using subtype-specific antisera or affinity-purified antibodies as described under "Experimental Procedures." The positions of molecular mass standards are indicated.
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Fig. 3. Quantitation of glucose transporters on total membranes from 32D cells. Glut-1 and Glut-3 were quantitated by densitometric analysis on 32D cells treated with (closed bars) or without (dotted bars) IL-3 and changes compared with the zero time point. A, Glut-1; B, Glut-3; C, Glut-1 + Glut-3. Values are the mean from two separate experiments.
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Effects of IL-3 on the Subcellular Distribution of Glut-1 and Glut-3

To assess whether changes in 2-DOG uptake in response to IL-3 addition and withdrawal were associated with corresponding changes in the subcellular distribution of glucose transporters, membranes and organelles from 32D cells treated with or without IL-3 for 6 h were separated by density gradient centrifugation on Nycodenz gradients, and the presence of Glut-1 and Glut-3 in each cell fraction was determined. Cell fractions containing plasma membrane were identified using alkaline phosphodiesterase I, while fractions containing endoplasmic reticulum were identified using NADPH-dependent cytochrome c reductase. Figs. 4 and 5 show that a single immunoreactive peak of Glut-1 and Glut-3 was observed in each gradient, and that most immunoreactivity was associated with the plasma membrane marker. No obvious changes in the distribution of Glut-1 or Glut-3 were observed in the presence or absence of IL-3. A consistent tendency for Glut-3 to be associated with less dense fractions than Glut-1 is evident in both gradients.


Fig. 4. Distribution of glucose transporters on membranes from 32D cells treated for 6 h with IL-3. 32D cells were treated for 16 h in serum-free media supplemented with 2 ng/ml IL-3, washed, then treated with 20 ng/ml IL-3 for 6 h. Membranes from cell homogenates were separated into Nycodenz gradients and the presence of Glut-1, Glut-3, and Glut-4 in each fraction determined by Western blotting. Fractions containing the plasma membrane were identified by alkaline phosphodiesterase I activity (black-square) and fractions containing endoplasmic reticulum identified by NADPH-cytochrome c reductase activity (square ).
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Fig. 5. Distribution of glucose transporters on membranes from 32D cells cultured for 6 h in the absence of IL-3. 32D cells were treated for 16 h in serum-free media supplemented with 2 ng/ml IL-3, washed, then incubated in the absence of IL-3 for 6 h. Membranes from cell homogenates were separated into Nycodenz gradients and the presence of Glut-1, Glut-3, and Glut-4 in each fraction determined by Western blotting. Fractions containing the plasma membrane were identified by alkaline phosphodiesterase I activity (black-square) and fractions containing endoplasmic reticulum identified by NADPH cytochrome c reductase activity (square ).
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Quantitation of Glut-1 and Glut-3 immunoreactivity in each fraction by densitometric analysis permitted transporters in fractions containing plasma membrane to be compared with those containing endoplasmic reticulum (Table I). Most Glut-1 (63-77%) and Glut-3 (82-96%) was associated with the plasma membrane, and the distribution showed only small (<20%) differences between IL-3 treatment and withdrawal and when compared with exponentially growing cells.

Table I. Quantification of glucose transporters in the plasma membrane and endoplasmic reticulum-associated fractions of 32D cells

Quantitative densitometry showing the distribution of glucose transporter subtypes in the plasma membrane (PM) and microsomal (ER) fractions from experiments shown in Figs. 4 and 5 and from exponentially growing cells. The balance of transporters was in other gradient fractions. Determinations were carried out twice with each cell treatment.
Glut-1
Glut-3
I (ER) II (PM) I (ER) II (PM)

% total transporters % total transporters
Exponentially growing cells 33 65 3 93
 -IL-3 35 63 18 82
+IL-3 23 77 4 96

Effects of IL-3 on the Kinetics of 2-DOG Uptake by 32D Cells

We have previously shown that withdrawal of IL-3 from serum-starved 32D cells for 1 h reduces transporter affinity for glucose, or conversely, that IL-3 maintains transporter affinity for glucose (24). Table II compares the kinetics of 2-DOG uptake following IL-3 treatment and withdrawal for 1, 2, and 6 h with that of exponentially growing cells and serum-starved cells. A similar 1.6-2.2-fold increases in transporter Km for glucose were observed at each time point following IL-3 withdrawal. In contrast, Vmax showed no significant change following IL-3 withdrawal for 1 and 2 h, but was elevated at 6 h.

Table II. Effects of IL-3 on the kinetics of 2-DOG uptake

Data on 1-h IL-3 treatment from Ref. 24. All data are the mean of n experiments each performed in duplicate or triplicate.
Treatment
Kinetic parameters
Time IL-3 n Km Vmax

h 20 ng/ml mM nmol/106 cells/min
Exponentially growing cells in WEHI 3-CM
 -  - 8 1.24  ± 0.07 5.45  ± 0.45
Serum-starved cells treated with 2 ng/ml IL-3 for 16 h
0 + 3 1.48  ± 0.14 2.81  ± 0.59
1 + 4 1.35  ± 0.15 3.15  ± 0.34
2 + 3 1.54  ± 0.02 2.60  ± 0.17
6 + 3 1.57  ± 0.09 2.64  ± 0.03
1  - 4 2.96  ± 0.28a 3.42  ± 0.53
2  - 3 2.55  ± 0.32a 2.93  ± 0.27
6  - 3 2.67  ± 0.47a 4.51  ± 0.43

a Significantly different from cells treated in the presence of IL-3, p < 0.05.

Effects of IL-3 on the Kinetics of 3-O-Methylglucose Uptake by 32D Cells

Because 2-DOG is rapidly phosphorylated following uptake into the cell, it is possible that phosphorylation may become rate-limiting for 2-DOG uptake. Involvement of post-transport phosphorylation in uptake kinetics was determined using 3-O-methylglucose, a glucose analogue which is not phosphorylated. Table III shows that IL-3 withdrawal resulted in a similar 2-fold increase in transporter Km for 3-O-methylglucose without altering Vmax. These results indicate that phosphorylation is not rate-limiting for 2-DOG uptake under these conditions.

Table III. Effects of IL-3 on the kinetics of 3-O-methylglucose uptake


Cell treatmenta Km Vmax

mM nmol/106 cells/min
WEHI-3 CMb 1.80  ± 0.38c 2.58  ± 0.33
+IL-3 1.70  ± 0.06c 2.38  ± 0.60
 -IL-3 3.60  ± 0.09 3.82  ± 1.20

a 32D cells were starved of serum for 2 h before being treated with or without IL-3 for a further 2 h. All data are the mean ± S.E. of three separate experiments done in triplicate.
b Exponentially growing cells without any pretreatment.
c Significantly different from cells in the absence of IL-3, p < 0.005.

Effects of Protein Kinase and Tyrosine Phosphatase Inhibitors on the Regulation of 2-DOG Uptake by IL-3

To determine whether the changes in affinity of glucose transporters for glucose observed following IL-3 withdrawal were mediated by protein kinases that have been associated with growth factor signaling, 32D cells starved of serum for 2 h were treated with 20 ng/ml IL-3 for a further 2 h in the presence of the tyrosine kinase inhibitor, genistein, the protein kinase C inhibitor, staurosporine, or the tyrosine phosphatase inhibitor, vanadate. Fig. 6 shows that genistein and staurosporine extensively inhibited IL-3-stimulated 2-DOG uptake while vanadate stimulated uptake. The stimulatory effect of vanadate was also observed in the absence of IL-3.


Fig. 6. Effects of inhibitors of protein kinases and tyrosine phosphatase on 2-DOG uptake by 32D cells. Inhibitor concentrations are presented in Table IV. Cells were serum-starved for 2 h before being treated with or without IL-3 and inhibitors for a further 2 h. Data are the mean ± S.E. of two experiments involving triplicate determinations.
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To establish that the effects of these inhibitors on 2-DOG uptake were being mediated at the level of the transporters, the kinetics of uptake was determined in the presence and absence of each inhibitor. Table IV shows that genistein and staurosporine reversed the IL-3-mediated increase in affinity of glucose transporters for glucose. In contrast, vanadate had a much lesser effect on the IL-3-mediated affinity change. Furthermore, in the absence of IL-3, vanadate acted like IL-3 to increase transporter affinity for glucose to a level indistinguishable from that seen with IL-3.

Table IV. Effects of inhibitors of protein kinases and tyrosine phosphatase on the kinetics of IL-3-stimulated 2-DOG uptake in 32D cells

Cells were serum-starved for 2 h before being treated with or without IL-3 and inhibitors for a further 2 h. All data are the mean ± S.E. of at least three separate experiments, each performed in triplicate.
Inhibitor IL-3 Km Vmax

20 ng/ml mM nmol/106 cells/min
None  - 4.73  ± 0.35 3.62  ± 0.38
None + 1.40  ± 0.01a 2.58  ± 0.20
Genistein (500 µM) + 6.32  ± 0.11b 4.64  ± 0.19
Staurosporine (2 × 10-6 M) + 5.24  ± 0.20b 4.34  ± 0.64
Vanadate (50 µM)  - 1.36  ± 0.45b 3.14  ± 0.24
Vanadate (50 µM) + 2.40  ± 0.51 3.80  ± 0.15

a Significantly different from cells treated in the absence of IL-3, p < 0.01.
b Significantly different from cells treated in the absence of inhibitor, p < 0.02.

These results are consistent with the hemopoietic growth factor, IL-3, regulating glucose transporter activity by a mechanism involving protein kinases and tyrosine phosphatase.


DISCUSSION

In this study we show that treatment of serum-starved 32D cells with IL-3 promotes 2-DOG uptake by a mechanism that cannot be fully explained in terms of increased glucose transporter expression or translocation of stored transporters to the plasma membrane. Withdrawal of IL-3 resulted in reduced 2-DOG transport without a corresponding reduction in glucose transporter expression or change in the subcellular distribution of transporters. An approximate 2-fold difference in glucose transporter specific activity (2-DOG transported per min/immunoreactive glucose transporter molecule) was evident between cells treated with and without IL-3 for 6 h. These results support previous kinetic analysis of 2-DOG uptake by 32D and Ba/F3 cells treated in the presence and absence of IL-3 (23, 24). In those studies, an approximate 2-2.5-fold difference in affinity of glucose transporters for glucose was observed between treatments at 0.5 and 1 h, but no changes in Vmax were evident. Similar changes in Km were observed in the present study following treatment with and without IL-3 for 2 and 6 h (Table II). In these experiments, no changes in Vmax were observed at 2 h but a small increase was evident in cells deprived of IL-3 for 6 h. Thus, changes in glucose uptake following IL-3 treatment and withdrawal appear to be largely explained by growth factor-induced changes in the affinity of transporters for glucose rather than transporter expression and translocation.

Switching serum-starved 32D cells maintained on 2 ng/ml IL-3 for 16 h to 20 ng/ml IL-3 stimulated 2-DOG uptake by 73% (Fig. 1), but no increases in the affinity of glucose transporters for glucose or in Vmax were observed prior to 6 h (24) (Table II). Furthermore, at times up to 12 h, overall glucose transporter expression increased by less than 20% (Fig. 3), and translocation to the plasma membrane was within 20% (Table I). Furthermore, previous studies have shown that cytochalasin B binding to total membranes and plasma membrane-enriched fractions was unaltered following exposure to saturating concentrations of IL-3 for 1 h (24). Thus, the increased 2-DOG uptake observed does not correspond with the apparent ability of cells to bind and transport 2-DOG. Most kinetic analyses involved 0.2-2.5 mM 2-DOG, whereas 2-[3H]DOG uptake studies were carried out at 0.1 mM 2-DOG. It is also possible that 2-DOG uptake may depart from ideal enzyme kinetics at low 2-DOG concentrations. Nevertheless, significant effects of IL-3 on 2-DOG uptake were observed at physiological 2-DOG concentrations of 1-5 mM (Ref. 24 and data used to derive kinetic parameters in Table II) and therefore the results are likely to be of physiological significance.

Reduced 2-DOG uptake following IL-3 withdrawal is more easily explained since the lower affinity for glucose observed is associated with lower glucose transport in the absence of changes in Vmax (Table II), cytochalasin B binding (24), or transporter expression at the plasma membrane (Table I, Fig. 3). However, the 25% reduction in 2-DOG at 6 h (Fig. 1) is within the level of significance of the glucose transporter expression data presented in Fig. 3 making it difficult to exclude changes in expression contributing to the reduced glucose transport observed. What is clear is that changes in glucose transporter expression and translocation following IL-3 treatment and withdrawal do not fully explain the differences in 2-DOG uptake observed.

32D cells were shown to express both Glut-1 and Glut-3 glucose transporter subtypes with Glut-3, which has a higher affinity for glucose than Glut-1, being more exclusively localized to the plasma membrane than Glut-1. The relatively high affinity of glucose transporters for 2-DOG observed with exponentially growing and IL-3-treated 32D cells (1.2-1.6 mM) suggests that Glut-3 may largely dominate transport in these cells. However, the fact that Glut-1 and Glut-3 expression and subcellular localization differed by less than 20% in serum-starved 32D cells treated with and without IL-3 for 6 h and the fact that the ratio of Glut-1:Glut-3 did not change markedly, suggests that differential effects of IL-3 on these transporter subtypes cannot be invoked to explain the differences in 2-DOG uptake and affinity for glucose observed.

These results can be contrasted with azide stimulation of rat liver Clone 9 cells (5, 28) where increased glucose transport is associated with an increase in Vmax and cytochalasin B binding without increased plasma membrane Glut-1 or transporter Km for glucose. Clearly, regulation of the specific activity of Gut-1 in this system differs from IL-3-dependent activation of glucose transport in 32D cells. With Clone 9 cells, an azide-sensitive negative regulator that binds to the carboxyl terminus of Glut-1 was proposed, but with IL-3-dependent 32D cells, the situation appears to be more complex, involving a 0.7-fold increase in glucose transport without apparent changes in Vmax or Km (Fig. 1, Table II), or cytochalasin B binding (24) and relatively small (<20%) changes in plasma membrane glucose transporters (Fig. 3).

Changes in the intrinsic activity of glucose transporters have also been observed in insulin-stimulated Chinese hamster ovary K1 fibroblasts and 3T3-L1 adipocytes (5, 29, 30), while treatment of 3T3-L1 cells with cadmium (32) or the protein synthesis inhibitor, anisomycin (33, 34) enhanced the intrinsic catalytic activity of Glut-1 and Glut-4. This evidence was used to support a model where the intrinsic catalytic activity of glucose transporters was suppressed in the basal state and could be activated by insulin, cadmium, and inhibitors of protein synthesis. However, growth factors do not always increase glucose transport by intrinsic activation of transporters, increased transporter expression having been shown to be responsible in quiescent Swiss 3T3 fibroblasts stimulated by serum, platelet-derived growth factor, or fibroblast growth factor (35).

Regulation of the intrinsic activity of glucose transporters is not well understood in any system. With insulin-stimulated cells where increased glucose transport exceeds that due to transporter translocation by severalfold, transporter phosphorylation has been inversely associated with increased intrinsic activity (36, 37), but with other cell systems regulatory proteins that associate with Glut-1 have been postulated to explain increased transport in the absence of increased transporter expression (5).

32D cells were found to express both the universal erythrocyte/brain glucose transporter subtype, Glut-1, and the brain/neuronal cell transporter subtype, Glut-3. Glut-1 protein is expressed in many different tissues and cells and has been shown to be present in all cell culture lines examined (1). In the mouse, Glut-3 protein has been shown to be expressed in brain and neural cell lines but has not been detected in mouse liver or fat membranes (1). However, in addition to brain and neural cell expression, Glut-3 protein has also been shown to be expressed in rat L6 muscle cells at both the myoblast and myotube stages (17), in myogenic cells during differentiation (38), and in human peripheral blood lymphocytes enriched in T cells (19). In addition, studies from our laboratory show that the Glut-3 transporter subtype is expressed in many mouse hemopoietic cells.3

Both Glut-1 and Glut-3 were found to be largely associated with the plasma membrane of exponentially growing 32D cells rather than intracellular vesicles and the endoplasmic reticulum, and this localization did not change markedly in serum-starved cells stimulated with or deprived of IL-3. However, it was observed that Glut-3 expression was shifted toward the less dense region of the Nycodenz gradients compared with Glut-1, demonstrating that the membrane expression of Glut-3 and Glut-1 is not identical. Glut-3 protein has previously been shown to be targeted to the cell surface in primary neurones (39), and when expressed in Chinese hamster ovary cells (40) and Xenopus oocytes (31). These results are consistent with the increased association of Glut-3 protein with plasma membranes in 32D cells.

In conclusion, we have provided evidence that the growth factor-dependent myeloid cell line, 32D, which expresses both Glut-1 and Glut-3 transporter subtypes, responds to IL-3 in serum-free conditions by regulating the transport activity of plasma membrane glucose transporters under conditions where changes in transporter expression and translocation contribute little to the observed changes in glucose transport.


FOOTNOTES

*   This work was supported by the Cancer Society of New Zealand and its Wellington Division, the Health Research Council of New Zealand, and the Wellington Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Malaghan Institute of Medical Research, P. O. Box 7060, Wellington South, New Zealand. Tel.: 64-4-389-5096 (ext. 825); Fax: 64-4-389-5095; E-mail: mimrmb{at}wnmeds.ac.nz.
1   The abbreviations used are: IL-3, interleukin-3; 2-DOG, 2-deoxyglucose; PAGE, polyacrylamide gel electrophoresis.
2   M. Kansara and M. Berridge, unpublished results.
3   M. Kansara, A. S. Tan, G. W. Gould, B. L. Bennett, and M. V. Berridge, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Dr. Gwyn Gould for the generous gift of antiserum and affinity-purified antibodies against mouse Glut-3 and Professor Jim Watson for providing the recombinant IL-3.


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